Quantitative and Qualitative Proteomic Investigations of the Α7 Nicotinic Acetylcholine Receptor by William J. Brucker, III

Quantitative and Qualitative Proteomic Investigations of the α7 Nicotinic Acetylcholine Receptor

By William J. Brucker, III Sc.B., Brown University, 2004

Thesis

Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the Department of Molecular Pharmacology, Physiology & Biotechnology at Brown University

PROVIDENCE, RHODE ISLAND MAY 2013

Signature Page This dissertation by William J. Brucker, III is accepted in its present form by the Department of Molecular Pharmacology, Physiology & Biotechnology as satisfying the dissertation requirement for the degree of Doctor of Philosophy.

Date ______Edward Hawrot, Ph.D, Advisor

Recommended to the Graduate Council

Date ______Wayne Bowen, Ph.D, Reader

Date ______James Clifton, Ph.D, Reader

Date ______Nadine Kabbani, Ph.D, Reader

Date ______Julie Kauer, Ph.D, Reader

Approved by the Graduate Council

Date ______Peter M. Weber, Dean of the Graduate School

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CURRICULUM VITA

William J. Brucker, III

Dr. Brucker was born in Bethel Park, Pennsylvania in May 1982. He graduated from

Brown University in 2004 with a Bachelor of Science with Honors degree in

Chemistry. He then matriculated to the MD/PhD program at Brown Medical School.

Dr. Brucker also served as an adjunct professor of biology at Rhode Island College from 2007 to 2011. He founded a non-profit organization, Providence Alliance of

Clinical Educators, dedicated to improving the quality and applicability of high school science curriculum for students across the country. Dr. Brucker’s distinguished honors include: 2001 Howard Hughes Medical Institute Fellowship, 2002 Brown University

Royce Fellowship, 2003 Undergraduate Award for Excellence in Chemistry/ Rhode

Island American Chemical Society, 2003 Barry M. Goldwater Scholarship and

Excellence in Education Foundation Scholar Award, 2004 Sigma Xi National

Scientific Research Honors Society, 2004 “Ivy 50” award for All-time Ivy League

Scholar, 2011 Peterson Fund for Educational Enhancement Award. Dr. Brucker has co-authored several peer-reviewed publications as well as anthologies of science parables for educational use. Dr. Brucker is pursuing additional training in Pediatrics in the University of Connecticut Pediatric Residency program.

ACKNOWLEDGEMENTS

To my wife, Stacy, thank you for supporting me through the writing process

To Joao Paulo, who aided greatly in the preparation of this dissertation

To Dr. Hawrot, thank you for your constant support and guidance.

To Dr. Clifton, thanks for being mass spec mentor all of these years.

To members of the Hawrot lab past and present, thank you for your company and friendship.

TABLE OF CONTENTS

CURRICULUM VITA ...... iv ACKNOWLEDGEMENTS ...... v LIST OF TABLES ...... x LIST OF FIGURES ...... xi LIST OF ABBREVIATIONS ...... xiii CHAPTER 1: GENERAL INTRODUCTION ...... 1 Acetylcholine receptors ...... 2 Nicotinic acetylcholine receptors: subunit stoichiometry and assembly ...... 2 Nicotinic acetylcholine receptor subunit domains and properties ...... 4 The cytoplasmic loop and signaling ...... 5 Function of neuronal nicotinic acetylcholine receptors ...... 8 α7 Nicotinic Acetylcholine Receptors ...... 10 Role in the Hippocampus ...... 13 Intracellular Signaling ...... 14 Knockout Observations: Cognition, Anxiety, Baroreceptor Response ...... 17 Non-Neuronal Expression ...... 19 Nicotine and Nicotinic Acetylcholine Receptors...... 20 Burden of Smoking ...... 20 Nicotine Abuse and Psychiatric Disease ...... 22 Pharmokinetics of Nicotine ...... 23 Sensitization and Up-regulation of nAChRs ...... 23 Nicotine and Reinforcement ...... 24 α7 nAChRs and Nicotine Reinforcement Pathways ...... 25 Withdrawal ...... 27 Withdrawal Phenotypes and nAChR Subunits ...... 27 Oral Nicotine Exposure in a Mouse Model ...... 28 Metabolism of Nicotine ...... 29 α7 nAChR targeted smoking cessation therapies ...... 30 Nicotine, Proliferation, and Malignancy ...... 30 Nicotine, Arteriogenesis, and Vasculature ...... 31 Nicotine, α7 nAChRS and Pathologic Vasculogenesis ...... 32 α7 nAChR and Inflammation ...... 35 Studies with α7 Specific Agonists and Positive Allosteric Modulators (PAMS)... 37 Effects on Cognition ...... 38 Effects on Sensory Gating ...... 39 Effects on Nociception ...... 41 Neuropathic Pain ...... 41

Visceral Pain ...... 42 α7 nAChR and Development ...... 43 Consequences of Prenatal Nicotine Exposure...... 43 Nicotinic AChRs and Development ...... 44 α7 nAChR Effects on Glutaminergic Synapse Formation ...... 44 α7 nAChR Effect on Chloride Gradient ...... 45 α7 nAChR and Neuropathology...... 46 Nicotine, α7 nAChR and Anxiety ...... 46 Alzheimer’s Disease ...... 48 α7 and Schizophrenia ...... 50 Proteomics ...... 51 Mass Spectrometry Based Proteomics ...... 53 Peptide Ionization Techniques ...... 57 Tandem Mass Spectrometry ...... 59 Mass Analyzers ...... 60 Orbitrap...... 62 SEQUEST ...... 63 Quantitative Mass Spectrometry ...... 66 Label Free Mass Spec with Spectral Counts ...... 67 Limitations of Spectral Counting ...... 68 QSPEC...... 70 PANTHER ...... 71 References ...... 73 CHAPTER 2: FURTHER ANALYSIS OF POTENTIAL INTERACTING PROTEINS OF THE α7 NACHR ...... 78 Introduction ...... 78 Materials ...... 91 DSME preparation ...... 91 Western Blotting ...... 91 Proteomics ...... 92 Production of α-Bungarotoxin Conjugated Affinity Beads ...... 92 Preparation of Detergent Solubilized Membrane Extracts (DSME) ...... 93 Isolation of Bgtx Sensitive Complexes with Bgtx Conjugated Affinity Beads ...... 94 Fractionation with SDS-PAGE ...... 95 Protein Visualization and In-Gel Tryptic Digestion ...... 95 Mass Spectrometry ...... 96 Data Processing ...... 97 Western Blotting ...... 98 Results ...... 99 Mass Spectrometry Studies ...... 99 Western Blot Studies ...... 107 Discussion ...... 114 References ...... 121

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CHAPTER 3: 125 I-α-BUNGAROTOXIN BINDING AS A PROBE FOR METHOD OPTIMIZATION AND QUANTIFICATION ...... 126 Introduction ...... 126 Materials ...... 127 Methods ...... 127 Bgtx Conjugated Affinity Bead Positive Control ...... 130 KO DSME Negative Control ...... 130 Methyllycaconitine (MLA) Negative Control ...... 131 Glycine Conjugated Sepharose Beads Negative Control ...... 131 Use of on Bead 125 I-Bgtx Binding Assays to Validate and Quantify Elution Conditions from Bgtx Affinity Beads ...... 132 Elution with Solubilization Buffer ...... 134 Elution with Carbachol in Solubilization Buffer ...... 134 High Salt Elutions ...... 134 Boiling Beads ...... 134 Discussion ...... 137 References ...... 139 CHAPTER 4: QUANTITATIVE AND QUALITATIVE APPROACHES TO UNCOVER THE ROLE OF α7 NACHR IN THE NEUROBIOLOGY OF NICOTINE ADDICTION ...... 140 Role of Proteomics in the Study of Addiction ...... 142 Previous Neuroproteomic Studies of Nicotine ...... 142 Materials ...... 147 Nicotine Exposure ...... 147 Rationale for Oral Nicotine Exposure in a Mouse Model ...... 148 Low Nicotine Affinity of the α7 nAChR ...... 149 Nicotine exposure ...... 150 Tissue Preparation ...... 150 Isolation of Bgtx Sensitive Proteins ...... 151 Gel Washing Steps, Reduction, Alkylation, In-Gel Tryptic Digestion, and Peptide Extraction ...... 153 Qualitative Mass Spectrometry ...... 154 Venn diagrams ...... 155 Protein Networks and Functional Analysis of Protein ...... 155 QSPEC spectral counting analysis ...... 156 Results ...... 156 Overview of Datasets ...... 156 Analysis of Consistency between Data sets ...... 157 Quantitative Results ...... 158 Qualitative Analysis of Proteins Unique to Nicotine and Control Datasets ...... 158 Discussion ...... 213 Comparison of Current Proteomic Studies toPreviousRelated Proteomic Studies 213

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Correlation between Previous β-Containing nAChR Proteomic Study ...... 214 Correlation between Previous α7 nAChR based Proteomic Studies ...... 214 Quantitative Proteomic Studies...... 216 Proteins Downregulated by Nicotine Exposure ...... 216 Proteins Up-regulated by Nicotine Exposure ...... 218 Structural Proteins ...... 221 Proteins involved in Ion and SmallMolecule Transport ...... 224 Protein Synthesis and Gene Regulation ...... 224 Analysis of Qualitative Data ...... 226 Pathways with Participating Proteins Identified only in Nicotine Exposed Datasets ...... 226 Pathways with Participating Proteins Identified only in Control Datasets ...... 227 Pathways with Participating Proteins Identified in both Nicotine Exposed and Control Datasets ...... 228 Analysis of α7 nAChR related proteins appearing in multiple pathways ...... 229 Interaction with other signaling pathways ...... 232 The endocrine system and α7 nAChRs ...... 232 Vascular proliferation and α7 nAChRs ...... 233 Immune System and α7 nAChRs...... 234 Opioid Signaling and the α7 nAChR ...... 235 References ...... 236 CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS ...... 242 Challenges in Detection of α7 nAChR Peptides ...... 242 Refining the α7 nAChR interactome by sample selection ...... 247 Utility of Qualitative Proteomic Data ...... 250 Quantitative Proteomic Studies...... 253 Inflammation, Neuropathic Pain, and Future α7 nAChR Experiments...... 256 Generation of Negative Control Models ...... 257 References ...... 259 APPENDICES ...... 261

LIST OF TABLES

Table 1.1: Functional nAChR pharmacology (Sharma & Vijayaraghavan, 2008)...... 6 Table 2.1: Location of molecular interactions on the cytoplasmic loop of the α7 nAChR ...... 86 Table 2.2: Proteins from Wild-Type Mouse Brain Tissue (Paulo et al., 2009) ...... 102 Table 2.3: Proteins seen in 3 of 4 WT datasets and never in a KO dataset...... 103 Table 2.4: Proteins previously identified in at least one WT dataset that were also identified in the current WT dataset but not in a KO dataset...... 104 Table 2.5: Proteins Ruled out of Interactome by Appearing in New KO Dataset ..... 105 Table 4.1: Effect of nicotine exposure on protein expression in different regions of rat brain...... 145 Table 4.2: Parameters for MaxQuant Andromeda ...... 155 Table 4.3: Proteins with less than 1.5 fold decrease in response to nicotine exposure ...... 166 Table 4.4: Proteins with greater than 1.5 fold increase in response to nicotine exposure ...... 167 Table 4.5: Summary of pathways participated in by proteins unique to the nicotine- exposed datasets. The pathways in bold are unique to nicotine-exposed datasets...... 170 Table 4.6: Pathways participated in by proteins identified only in the control datasets. The pathways in bold are unique to the control datasets...... 177 Table 4.7: Pathways participated in by proteins identified in both nicotine-exposed and control datasets...... 186 Table A.1: Proteins Identified Only in Nicotine-Exposed Samples ...... 262 Table A.2: Proteins Identified Only in Control Samples ...... 274 Table A.3: Proteins Identified in Both the Nicotine-Exposed and Control Samples . 288

LIST OF FIGURES

Figure 1.1: Nicotinic acetylcholine receptor transmembrane topology (Picciotto et al., 2001)...... 7 Figure 1.2: Distribution and assembly of neuronal nAChR subtype (Gotti, Zoli, & Clementi, 2006) ...... 11 Figure 1.3: Potential intracellular signaling pathways of the α7 nAChR (Picciotto et al., 2001)...... 16 Figure 1.4: Distribution of neuronal nAChRs on non-neuronal cells (Egleton et al. 2009) ...... 21 Figure 1.5: The role of α7-nAChR signaling in cell proliferation and vasculogenesis (Egleton et al. 2009) ...... 34 Figure 1.6: Flow chart of botton up proteomic experiment (Steen & Mann, 2004) .... 56 Figure 1.7: Types of peptide product ions that are formed by fragmentation (Steen & Mann, 2004) ...... 61 Figure 1.8: SEQUEST Algorithm (Steen & Mann, 2004) ...... 65 Figure 1.9: Flow Chart Illustrating Spectral Count Based Quantitative Mass Spectrometry (Zhu et al., 2010) ...... 69 Figure 2.1: The cytoplasmic loop of the α7 nAChR as a proposed site for protein interactions ...... 85 Figure 2.2: Western Blot Probing for the α7 nAChR with antibody ab23832...... 110 Figure 2.3: Western Blot Probing for GNB3 ...... 111 Figure 2.4: Western Blot Probing for GAP- 43 (neuromodulin) and NMDA zeta from the DSME of WT or KO mice ...... 112 Figure 2.5: Western Blot Probing for GLUR2 ...... 113 125 Figure 3.1: On-Bead I-Bgtx Binding Assay Protocol ...... 129 Figure 3.2: Specific Bgtx Binding in Negative Control Models for On-Bead Binding Assay ...... 133 Figure 3.3: Use of On Bead 125 I-Bgtx Binding Assays to Validate and Quantify Elution Conditions from Bgtx Affinity Beads ...... 136 Figure 4.1: Venn diagram of proteins exclusive to and in common with nicotine exposed and control groups...... 160 Figure 4.2: Venn diagrams of the distribution of unique identified proteins in nicotine exposed and control data sets...... 161 Figure 4.3: Reproducibility of protein identifications in the same dataset...... 162

Figure 4.4: Comparison of Spectral Count consistency in control datasets (C1, C2, C3) ...... 163 Figure 4.5: Comparison of Spectral Count consistency in nicotine-exposed datasets (N1, N2, N3) ...... 164 Figure 4.6: The statistical basis for assigning significance to fold change of +/- 1.5. 165 Figure 4.7: Analysis of functionalities (A) and intracellular roles (B) of proteins identified in nicotine-exposed and control datasets...... 168 Figure 4.8: Further analysis of intracellular roles and functionalities of proteins identified in nicotine exposed and control datasets...... 169

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LIST OF ABBREVIATIONS

xiii

CHAPTER 1: GENERAL INTRODUCTION

Nicotine, a chemical compound found in tobacco products, plays a role in several physiologic phenomena including: reinforcement, inflammation reduction, analgesia, improved cognition and attention, vasculogenesis, cell proliferation and survival. Individuals suffering from psychopathology employ nicotine as a means of self-medication. In addition to the well-known effects of nicotine in the central nervous system (CNS), other organ systems such as the gastrointestinal tract are also becoming better recognized as a target for nicotine modulation. Nicotine acts through several varieties of nicotinic acetylcholine receptors (nAChRs) variably expressed throughout the body. A refined understanding of which receptor subtypes modulate different physiological effects in response to nicotine is important in order to better target therapies to ameliorate conditions such as addiction, inflammatory bowel disease and neurodegenerative disorders. One nAChR that has been implicated in several pathways is the α7 subtype. The quantitative and qualitative proteomic description of the α7 nAChR is the focus of this dissertation. The identification of proteins that interact or associate with the α7 nAChRa may provide a key to improve the understanding of the mechanisms by which nicotine and endogenous agonists alter neuronal plasticity and impact several devastating and highly prevelent disorders that are explored here in the introduction.

Acetylcholine receptors

There are two distinct types of receptors that are activated by the endogenous ligand, acetylcholine and they are differentiated by the exogenous ligands that activate them. Muscinarinic acetylcholine receptors are G-protein coupled receptors activated by muscarine of which there are five types (Egleton, Brown, & Dasgupta, 2009).

Nicotinic acetylcholine receptors are integral membrane proteins and prototypic members of the ligand gated ion channel superfamily. They are cation channels with varying permeability to calcium and mediate fast synaptic transmission in the central and peripheral nervous systems as well as being essential to muscle contraction

(Kalamida et al., 2007).

Nicotinic acetylcholine receptors: subunit stoichiometry and assembly

Nicotinic acetylcholine receptors are cation channels within the cys-loop superfamily of ligand gated ion channels (Pandya & Yakel, 2013). There are two main subtypes, muscle type nAChRs and neuronal type nAChRs (Kalamida et al., 2007).

Both subtypes are pentameric ion channels with the subunits arranged around a central pore lined by charged amino acids that affects the selection of ions permitted to pass through (Egleton et al., 2009; Fowler, Arends, & Kenny, 2008). Upon activation by an agonist like acetylcholine or nicotine, the domains of each of the five nAChR subunits rearrange such that the central pore opens and permits cationic trafficking (Fowler et al., 2008). Through cDNA studies seventeen acetylcholine receptor subunits have been discovered to exist, although they are not all expressed in humans. These subunits include ( α1- α10), ( β1- β4), γ, δ, and ε (Egleton et al., 2009). The α subunit carries the

2 binding site for acetylcholine (Egleton et al., 2009). Muscle type nAChRs are composed of ( α1) 2(β1)( δ)( ε) in mature animals and ( α1) 2(β1)( γ)(ε) during development (Kalamida et al., 2007). The subunit stoichiometry and composition of muscle type nAChRs is the same in every neuromuscular junction throughout the body and is conserved in different species as well.

This consistency is lost in neuronal nAChRs whose subunit composition and stoichiometry vary considerably throughout the central and peripheral nervous systems. Neuronal nAChR subunits co-assemble in many possible combinations of α and β subunits and can exist as a collection of homopentameric α subunits ( α7, α8, and

α9) or a heteropentameric assembly of α and β subunits in an a ( α)2(β)3 stoichiometry

(Egleton et al., 2009; Fowler et al., 2008). Because the α subunit contains the agonist- binding site for acetylcholine, heteromeric receptors can have two binding sites while homomeric receptors have 5 binding sites (Egleton et al., 2009; Fowler et al., 2008).

Different subunit compositions allow the formed receptor to have diverse biological and physiological properties including cation selectivity and pharmacological profile

(Fowler et al., 2008). For example, the α7 nAChR is more permeable to calcium than other nAChRs. The calcium to sodium permeability ratio of the α7 nAChR is 10:1 compared to 4:1 described for other nAChR subtypes (Gahring & Rogers, 2005).

Although many different subunit combinations are possible, the assembly of nAChRs is a heavily regulated process, requiring appropriate subunit interactions to form fully functional receptors (Fowler et al., 2008). This regulation results in a limited number of observed nAChR combinations (Fowler 2008). Other than indirect studies of subunit detection like mRNA assays, the only way to confirm the expression of

3 neuronal nAChRs is through binding studies with subtype specific ligands.

Unfortunately, the lack of subtype specific ligands available for investigative purposes restricts the detectable range of subunit compositions leaving the full possible in vivo range a mystery (Fowler et al., 2008). Αlpha-Bungarotoxin (Bgtx) has a high and exclusive affinity for α7 nAChRs (1 nM), the primary homomeric neuronal nAChR found in mammalian systems, and has been an invaluable tool for detecting and quantifying the receptor in vivo , Table 1.1 (Sharma & Vijayaraghavan, 2008).

Nicotinic acetylcholine receptor subunit domains and properties

Despite the diversity in physiology and pharmacology among nAChR subunits, all subunits share a common topology, Figure 1.1 (Picciotto et al., 2001). Each subunit contains a long N-terminal extracellular domain that carries multiple sites for glycosylation, a conserved disulfide bond between two cysteine residues separated by

15 amino acids and a region called the main immunogenic region (Picciotto et al.,

2001). This large extracellular domain contains important elements for agonist binding and is crucial to surface expression (Castelan et al., 2007; Castillo et al., 2009).

Deletion in this region of the α7 nAChR subunit prevents surface expression in

Xenopus oocyte expression studies (Castillo et al., 2009). Following the long N- terminal extracellular domain are four transmembrane sequences called M1-M4

(Picciotto et al., 2001). The transmembrane regions are arranged in concentric layers around a central aqueous pore with the M2 domain lining the pore. The M1 and M3 domains shield M2 from the surrounding lipid bilayer and the M4 domain is the outermost and most lipid exposed region (Fowler et al., 2008). There is also a short extracellular C-terminal domain (Egleton et al., 2009). Between the M3 and M4

4 transmembrane regions is a large cytoplasmic loop made of 100-150 amino acids that links M3 and M4. The composition of the cytoplasmic loop is highly divergent between subunits and is critical to many aspects of cellular targeting and trafficking from the endoplasmic reticulum and targeting to axonal or dendritic compartments

(Kabbani, Woll, Levenson, Lindstrom, & Changeux, 2007; Picciotto et al., 2001).

Deletions in regions of the cytoplasmic loop of the α7 nAChR can increase channel conductance and either increase or decrease surface receptor expression indicating that this region plays a large regulatory role in the function of the receptor (Castelan et al.,

2007; Valor et al., 2002).

The cytoplasmic loop and signaling

The different physiological and pharmacological properties observed among nAChRs are hypothesized to be attributable to variations in the cytoplasmic loop sequences. There is otherwise a high degree of homology between the receptor subunits (Picciotto et al., 2001). The structure of the cytoplasmic loop sequence has been shown to affect kinetic properties and subcellular localization of nAChRs

(Picciotto et al., 2001). The cytoplasmic loop is critical for appropriate receptor targeting to different cellular localizations. For example chick ganglion the α7 receptor localizes perisynaptically; however, when an α3 cytoplasmic loop region is inserted mutant α7 receptor is expressed at the synapse.

Table 1.1: Functional nAChR pharmacology (Sharma & Vijayaraghavan, 2008 ).

Figure 1.1: Nicotinic acetylcholine receptor transmembrane topology (Picciotto et al., 2001). A) Transmembrane topology. There are four transmembrane sequences (M1-M4) in each receptor subunit. These regions are arranged to form an aqueous pore with the M2 domain lining the pore. B) Comparison of structural difference between muscle- type and neuronal-type nAChRs. Conserved amino acids are indicated by superimposed shapes.

The cytoplasmic loop also has multiple phosphorylation sites. Number and position of these phosphorylation sites may account for subunit functional differences.

Kinases (protein kinase A, protein kinase C and SRC) alter channel properties of muscle-type receptor by phosphorylation (Picciotto et al., 2001). Phosphorylation alters channel properties, expression, assembly and turnover. Studies have demonstrated by protein kinase A phosphorylation of the cytoplasmic loop of α4 in response activation by nicotine (Picciotto et al., 2001). Calmodulin kinase is believed to negatively regulate the channel activity of the α7 subunits; mutations in the cytoplasmic loop can lead to α7 nAChR mediated excitotoxicity (Picciotto et al.,

2001). The α7 nAChR boasts a particularly long cytoplasmic loop length (Kabbani et al., 2007). It is logical that this cytoplasmic loop is a site of protein interaction for downstream signaling molecules associated with the α7 nAChR because calcium ions passing through the channel would be in immediate proximity to interact with calcium activated proteins like protein kinase C. Calcineurin is a protein phosphatase that regulates calmodulin kinase activity. Calcineurin’s substrates have two consensus sequences PxIxIT and LxVP neither of which are present in the α7 nAChR (Li, Rao,

& Hogan, 2011; Roy & Cyert, 2009; Rusnak & Mertz, 2000).

Function of neuronal nicotinic acetylcholine receptors

Neuronal nAChRs are found throughout the central and peripheral nervous systems as well as a variety of non-neuronal locations, Figure 1.2. In mammalian systems the most abundant nAChRs in the peripheral nervous system are α3β4 nAChRs and in the central nervous system are α4β2 and α7 nAChRs (Fowler et al.,

2008). The neuronal nAChRs are implicated in physiological functions like cognition,

8 reward, motor activity, emotion and analgesia (Fowler et al., 2008; Pandya & Yakel,

2013). These receptors are also believed to play large roles in pathological conditions like Parkinson’s disease, Alzheimer’s disease, anxiety, epilepsy, schizophrenia, depression, and autism (Pandya & Yakel, 2013). Throughout the central and peripheral nervous systems they mediate fast synaptic transmission and regulate the activity of several neurotransmitter pathways through the central nervous systems

(Fowler et al., 2008). One of the predominant roles of the nAChRs in the CNS is the modulation of release of other neurotransmitters. Nicotine has been shown to stimulate the release of most neurotransmitters in the brain including dopamine, glutamate,

GABA, norepinephrine and serotonin (Fowler et al., 2008). Neuronal nAChR subtypes are ubiquitous within the nervous system making their individual functions challenging to ascertain. Achieving this task is a source of intense research. The nAChRs are expressed on both excitatory and inhibitory circuits and can modulate and potentiate the release of excitatory and inhibitory neurotransmitters (Picciotto &

Kenny, 2013). This enables them to increase inhibition of circuits when activity is high and increase the activity of less active circuits (Picciotto & Kenny, 2013). The overall effect of this circuit level integration is that nicotine can modulate behavioral function in paradoxical ways (Picciotto & Kenny, 2013). At low doses nicotine can act as an anxiolytic but at higher doses can be anxiogenic, suggesting modulation of plasticity and activity at a network level more than linear modulations of individual synaptic pathways (Sharma & Vijayaraghavan, 2008).

α7 Nicotinic Acetylcholine Receptors

Two nAChR subtypes predominate in the mammalian brain, α4β2 and α7

(Pandya & Yakel, 2013; Sharma & Vijayaraghavan, 2008). The α4β2 receptor is the most abundant subtype in the brain and the α7 is second (Pandya & Yakel, 2013;

Sharma & Vijayaraghavan, 2008). The α7 subtype has much lower affinity for nicotine than the α4β2 containing receptors (Govind, Vezina, & Green, 2009; Sharma

& Vijayaraghavan, 2008). Compared to the α4β2 receptor, the α7 nAChR desensitizes rapidly after activation (Govind et al., 2009). In addition to the α4β2 receptors there are a wide variety of nAChRs that have a high affinity for nicotine but are less well characterized, α4, α5, and α6 (Govind et al., 2009). The α7 nAChR has several endogenous activating ligands: acetylcholine and Ly6/urokinase plasminogen-type activator receptor related protein-1 and -2 (SLURP-1) and (SLURP-2) (Egleton et al.,

2009). SLURP-1 binds to α7 and enhances the amplitude of acetylcholine induced currents and can lead to apoptosis from excitatoxicity (Egleton et al., 2009).

The α7 nAChR is located in virtually all brain regions but is localized most densely in the hippocampus on GABAergic interneurons (Sharma & Vijayaraghavan,

2008). The hippocampus is the region of the brain associated with learning and memory (Sharma & Vijayaraghavan, 2008). The only region of the brain deficient in

α7 nAChRs is the thalamus (Picciotto et al., 2001). The ventral tegmental area (VTA) is the area of the brain associated with reward and drug addiction. Roughly half of the neurons in the VTA possess α7 subtypes of nAChRs, a density two fold less that found

Figure 1.2: Distribution and assembly of neuronal nAChR subtype (Gotti, Zoli, & Clementi, 2006) A. Distribution of neuronal nAChR subtypes in the murine brain B. Diversity of neuronal nAChR assembly and location of ACh binding sites

in the hippocampus (Grabus, Martin, & Imad Damaj, 2005; Picciotto & Kenny, 2013;

Sharma & Vijayaraghavan, 2008). The α7 nAChR is believed to play a role in the re- enforcement affects of drugs of abuse. Both α4β2 and α7 nAChRs are activated in the ventral striatum and are involved in cocaine induced dopamine release (Levin et al.,

2009). The involvement of both subtypes is essential for the sensitization of cocaine effects on dopamine release in this area of the brain (Levin et al., 2009).

A major challenge to understanding the physiology of neuronal nAChRs is the diversity of their localization. These receptors are not only localized to different anatomical locations within the nervous system like the VTA and the hippocampus but they are also found in different parts of the neuron as well (Sharma & Vijayaraghavan,

2008). Nicotinic AChRs are found on the soma, dendrites, and synaptic terminals

(Sharma & Vijayaraghavan, 2008). In the CA1 area of the hippocampus the α7 nAChRs are located both pre and post synaptically (Hu, Liu, Chang, & Berg, 2002). In other presynaptic locations the α7 subtype influences the release of glutamate, GABA, and norepinephrine, and dopamine (Picciotto & Kenny, 2013; Sharma &

Vijayaraghavan, 2008). Postsynaptic α7 nAChRs mediate excitatory impulses

(Campbell, Fernandes, John, Lozada, & Berg, 2011; Hu et al., 2002; Pandya & Yakel,

2013). The high calcium permeability of the α7 nAChR and its ability to cause calcium induced calcium release (CICR) and activate calcium dependent second messengers gives it metabotropic properties as well as the typical depolarizing properties of an ion channel (Conroy, Liu, Nai, Margiotta, & Berg, 2003; Gahring &

Rogers, 2005). The α7 receptor is also expressed on a variety of non-neuronal cells in

CNS like astrocytes and microglia cells. These cell types outnumber neurons

12 significantly and serve to modulate some of their activities (Campbell et al., 2011;

Pandya & Yakel, 2013). Over the last decade evidence that astrocytes have an active role in modulation of synaptic activity has been building. Astrocytes have a form of excitability not mediated by fast synaptic potentials but by slowly propagating calcium signaling. They can communicate back and forth to neurons by the release of multiple vesicular and nonvesicular signals (Sharma & Vijayaraghavan, 2008).

Role in the Hippocampus

Activation of α7 nAChRs at the mossy terminals induces a long lasting slow calcium gradient at nerve terminals resulting at least partially from the release of calcium from internal endoplasmic reticulum stores (Sharma & Vijayaraghavan,

2008). This burst of transmitter release is sufficient to drive the postsynaptic neuron above its firing threshold. This is the first instance of a presynaptic action potential independent transmission in the CNS (Sharma & Vijayaraghavan, 2008). This leads to an increase in glutamate release frequencies as well as CAMKII mediated concerted release of multiple quanta at these terminals (Sharma & Vijayaraghavan, 2008). This is particularly interesting because it causes an action potential independent of information coming down the presynaptic axon. This signaling mechanism is important because it can be “hijacked” by the exogenous ligand nicotine that activates the α7 nAChR resulting in strengthening the synapses in nonphysiological contexts. It can be triggered by serum nicotine concentrations seen in smokers. This can lead to an altering of signal strength that is dependent on the presence of nicotine to maintain rather than normal physiological context, setting into effect a new equilibrium or nicotine influenced allostasis (Sharma & Vijayaraghavan, 2008). The withdrawal of the drug [nicotine] disrupts this equilibrium causing instability and distress. The 13 activation of α7 nAChRs on these mossy fibers by nicotine induces slow inward calcium currents with decay times that vary directly with agonist exposure length

(Sharma & Vijayaraghavan, 2008). The ability of addictive drugs to usurp brain reward circuits relevant to natural rewards and establish addictive behaviors is hypothesized to be due in part to changes in synaptic plasticity of the mesolimbic dopamine system triggered by drugs of abuse (Niehaus, Murali, & Kauer, 2010).

Long-term potentiation (LTP) of excitatory synapses is a mechanism that underlies the ability of neuronal circuits to form memories in addiction or other brain functions

(Niehaus et al., 2010). Addictive drugs may take over brain reward circuitry causing the value of the drug to the organism to be overlearned (Niehaus et al., 2010).

Intracellular Signaling

The α7 receptor is viewed as having metabotropic properties due to its high calcium permeability that rivals an NMDA receptor (Gahring & Rogers, 2005), Figure

1.3. The activation of the α7 nAChR causes it to increase intracellular calcium and activate second messenger systems like PI3-kinase/AKT pathway, activate transcriptional systems like CREB, and initiate proteolytic processes like the cleaving of beta-amyloid into the nontoxic variant (Gahring & Rogers, 2005). Activation of α7 nAChRs leads to the activation of the transcription factor, cAMP response element binding protein (CREB). CREB is a transcription factor that binds both calcium and camp. It controls the expression of numerous genes and has been implicated in having a role in learning and memory (Hu et al., 2002). Depending on the method of CREB activation it can either encourage or impede synapse formation. One gene transcribed in response to CREB activation is c-fos, a protein found to increase in neurons in response to increased synaptic activity and frequently exposure to drugs of abuse (Hu 14 et al., 2002). Αlpha-7 nAChR activation has been directly linked to increases in c-fos expression. Calcium influx through the α7 nAChR raises the intracellular calcium concentration to a critical level. Calcium is released from internal stores leading to the activation of both Calmodulin (CaM) and MAPK. The activation of MAPK and CAM lead to the production of a long lasting phosphorylated form of CREB that affects transcriptional changes (Hu et al., 2002). These transcriptional changes can be achieved by low but sustained levels of nicotine are consistent with those likely to be achievable by normal cholinergic tone or sustained nicotine use (Hu et al., 2002).

Interestingly in vitro studies demonstrated that activation of α7 nAChRs by nicotine at

1 µM could cause CREB phosphorylation but incubation with 10 µM of nicotine was far less efficient. This could have to do with the desensitization of the receptors meaning that chronic low level activation could be more effective at inducing transcriptional changes than higher levels of nicotine (Hu et al., 2002). The EC50 of the α7 nAChR as evidenced by channel conductance is estimated to be 27-55 µM

(Papke & Porter Papke, 2002; Sharma & Vijayaraghavan, 2008). 1µM of nicotine is far below these EC50 values indicating that the calcium influx through the receptors at this low level of agonist concentration may be too low to detect as a current but may be sufficient to induce the CICR necessary for the activation of the rest of the cascade, one of the hall marks of metabotropic signaling is amplification (Gahring & Rogers,

2005).

Figure 1.3: Potential intracellular signaling pathways of the α7 nAChR (Picciotto et al., 2001)

Knockout Observations: Cognition, Anxiety, Baroreceptor Response

The development of α7 nAChR knockout mice provided investigators the opportunity to observe scenarios where an animal model can exist without the α7 nAChRs. Behavioral and biochemical studies have improved the understanding of its role in physiology, pathology, and addiction. These observations are not without flaws. This knockout animal has been without the gene of interest for its entire life.

Development and physiological compensation may have occurred which is known as masking (Fowler et al., 2008). Αlpha-7 knockout mice have increased expression of

α3 and α4 subunits; the β3 knockout mice display decreased α6 subunit expression

(Fowler et al., 2008). Αlpha-7 knockout mice appear and behave like their wild type counterparts in most respects; however, performance evaluations in behavioral and nicotine administration studies are often inconsistent.

Activation of the α7 nAChR is associated with increased performance in attentive and working memory tasks. Consistent with these results the α7 nAChR partial agonist SSR180711 enhances episodic memory in wild type but not α7 nAChR knockout mice (Fowler et al., 2008). Virally mediated over-expression of the α7 nAChR in the murine hippocampus improves spatial memory. Αlpha-7 knockout mice display deficits in both attention and working memory. They also have marked deficits in the acquisition of an operant response to obtain gustatory rewards, suggesting that they have difficulty remembering which choices give them reward (Fowler et al.,

2008). Similar cognitive defects are noted in rats following antisense mediated knockdown of α7 nAChR subunits (Fowler et al., 2008). The defects in memory and learning are not consistent from task to task. The α7 knockout mice have not shown significant differences in the ability to learn from their wild type counterparts in 17 contextual or cued fear-conditioning, passive avoidance learning, or in tests of motor learning (Picciotto et al., 2001).

In response to decreased blood pressure the vascular system constricts and the heart rate increases, decreases in blood pressure are rapidly met by increases in heart rate through detection by the baroreceptors in the carotid bodies, the carotid bodies send afferent signals through cranial nerve 9 to the medulla where sympathetic tone is increased. The α7 knockout mice display a decreased baroreceptor reflex compared to wild type controls which may reflect defects in any step of this pathway (Picciotto et al., 2001). This baroreceptor-mediated deficit was demonstrated by decreased response to vasodilatory agents. Paradoxically, these mice also demonstrated supersensitivity to adrenergic agonists indicating that while responsive to adrenergic signaling they have difficulty producing it endogenously (Picciotto et al., 2001).

Possibly related to this decrease in endogenous sympathetic tone, α7 nAChR knockout mice demonstrate decreased susceptibility in response to anxiogenic behavioral stimuli like the open field cage (Picciotto et al., 2001). Αlpha-7 knockout mice spent more time in the center of an open field cage than their wild type counterparts. Being in an open field puts mice at risk for attack and is therefore a well- described anxiogenic stimuli in mice. This resistance to stress did not persist during light-dark tests of anxiety mediated behavior or in fear associated cue conditioning

(Picciotto et al., 2001). Some of these differences may reflect a decreased response to anxiogenic stimuli rather than a lack of one. Stress responses in other rodent models have been linked to epigenetic changes in glucocortocoid receptor expression in the

CNS as a result of maternal licking and subsequent changes in stress response

(Hellstrom, Dhir, Diorio, & Meaney, 2012). The decreased stress response seen in α7 nAChR knockout mice could be linked to changes in rearing or α7 mediated signaling defect in the hypothalamic pituitary axis. Interestingly, a side effect of α7 specific agonists is to induce anxiety and nicotine induces anxiogenic effects at higher doses in mice that may serve to activate a greater population of the less nicotine sensitive α7 nAChRs.

The α7 knockout mice are more sensitive to the behavioral actions of ethanol than wild type mice are (Fowler et al., 2008). This includes ethanol induced locomotor stimulation and loss of righting reflex. Little is known about the reinforcing effects of ethanol in α7 knockout mice and other strains (Fowler et al., 2008). During fetal development the cholinergic system develops before the glutaminergic system. The α7 nAChR has a large role in mediating excitatory impulses in that state (Lozada et al.,

2012). Activation of α7 nAChRs during this period is directly related to the formation of excitatory glutaminergic synapses (Lozada et al., 2012). It is possible that fewer glutaminergic synapses are formed in the α7 knockout mice giving their basal circuitry a more inhibitory tone. A CNS depressant that enhances inhibitory signaling, like ethanol, may have enhance effect in a system whose equilibrium already has an inhibitory skew.

Non-Neuronal Expression

Besides expression in the nervous system the α7 nAChRs are expressed on a wide variety of non-neuronal cells, Figure 1.4. Macrophages, microglia, astrocytes, B- cells, T-cells, neutrophils, and natural killer cells, Langerhans cells, epithelial cells of the gut, skin, and bronchioles, adipocytes, and endothelial cells have all been demonstrated to express the α7 nAChR (Egleton et al., 2009; Gahring & Rogers, 2005; 19

Sharma & Vijayaraghavan, 2008). The activation of the α7 nAChRs on these cells by nicotine may be responsible for many of its observed physiological effects like anti- inflammatory properties, ability to change or exacerbate the course of inflammatory bowel disease, and its ability to induce vasculogenesis (Egleton et al., 2009). Nicotine has been shown to increase the expression of leukocyte adhesion molecules like the integrin VCAM-1 through an α7 activated mechanism (Egleton et al., 2009).

Lymphocytes express some proteins typically associated with neurons like choline acetyltransferase. It is believed that lymphocytes make and secrete acetylcholine as part of an autocrine function (Egleton et al., 2009; Gahring & Rogers, 2005).

Nicotine and Nicotinic Acetylcholine Receptors

Burden of Smoking

Cigarette smoking has become one of the largest global public health problems and is one of the largest preventable causes of death and disease in the developed world (Di, Yan, Zhao, Chang, & Zhao, 2012; Fowler et al., 2008). Tobacco related disease is responsible for 440,000 deaths annually and results in $160 billion in health care costs in the Unites States. By 2020, tobacco related disease is predicted to be the largest single health problem worldwide (Fowler et al., 2008). In addition to the commonly associated risks of lung cancer and chronic obstructive pulmonary disease, smoking is a predisposing factor for insulin resistance, the cause of type II diabetes

(Gahring & Rogers, 2005). Smokers are both insulin resistant and hyperinsulinemic.

Men who smoke are 4 times more likely to develop diabetes (Gahring & Rogers,

2005). Although multiple campaigns have been developed for smoking cessation,

Figure 1.4: Distribution of neuronal nAChRs on non-neuronal cells (Egleton et al. 2009)

success is extremely difficult due to the addictive nature of nicotine (Di et al., 2012).

Roughly 25% of adults in the United States have been smokers in the past (Fowler et al., 2008). Despite known negative health consequences associated with smoking only

10% of those who try to quit can remain abstinent after one year (Fowler et al., 2008).

Smokers that do successfully quit before the onset of smoking related illness can largely avoid the increased mortality risk. Unfortunately, most smokers who do quit relapse within the first month (Fowler & Kenny, 2011).

Nicotine Abuse and Psychiatric Disease

Human smoking behavior is far more complicated than just short-term reward.

In many instances it is motivated by secondary gain, such as weight control or management of psychiatric illness (Picciotto & Kenny, 2013). It is estimated that as many as 44% of cigarettes are purchased by people with co-morbid psychiatric illness.

Importantly, the rates of tobacco dependence are far higher in patients suffering from psychiatric illnesses compared to the general population. This may reflect altered signaling resulting from psychiatric disease pathology (Fowler et al., 2008; Picciotto

& Kenny, 2013). The ability of smoking to improve attention in individuals with schizophrenia is likely to contribute to high smoking rates in this population. A large proportion of smokers also report that they smoke to control symptoms of anxiety and depression due to the positive affective state induced by nicotine (Di et al., 2012;

Picciotto & Kenny, 2013). The rate of smoking in people with affective disorders is nearly double the rate in the general population (Picciotto & Kenny, 2013). An understanding of the role on nAChRs in psychiatric disorders associated with high rates of tobacco addiction may reveal novel insights into nicotine addiction (Fowler et 22 al., 2008). Disordered eating is a recognized psychopathology with a mortality rate higher than clinical depression. Negative body image and desire to maintain low weight is a prevalent problem in young women. Disordered eating prevalence is estimated to be 3-6% of adolescent females and a lifetime prevalence of 3% (McBride,

McManus, Thompson, Palmer, & Brugha, 2013). To an individual with disordered eating the long term effects of smoking are not as significant as the short-term gain of appetite suppression. The appetite suppressing effects of nicotine may be mediated at least in part by the desensitization of α7 nAChRs. Blocking α7 nAChRs in the VTA with MLA can decrease food seeking in animal models (Picciotto & Kenny, 2013).

Pharmokinetics of Nicotine

The concentration of nicotine in the average smoker’s blood is between (1 nM-

1 µM) (Egleton et al., 2009). Unlike the endogenous ligand acetylcholine, which is either rapidly degraded or removed from the receptor vicinity, nicotine is not degraded or removed and has a half-life of 2-3 hours (Gahring & Rogers, 2005; Govind et al.,

2009). Because nicotine is lipophilic it accumulates in certain tissues in excess of maximum serum concentrations. It can reach concentrations of up to 10 µM in the brain while it is typically in the high nanomolar range in the blood (Gahring & Rogers,

2005). With nicotine levels achieved by smokers, the acute effect of nicotine following smoking is to activate and then desensitize receptors (Govind et al., 2009).

Sensitization and Up-regulation of nAChRs

Chronic exposure to nicotine in vivo robustly up-regulates α4β2 nAChRs receptors while α7 and α3β4 nAChRs are more resistant to up-regulation (Fowler et al., 2008; Govind et al., 2009). Repeated locomotor studies in rats demonstrate that sensitization occurs with repeated nicotine administration. This sensitization is the

23 result of the VTA neurons being more responsive to nicotine believed to be due to an up-regulation of nAChRs on the neuronal surfaces (Govind et al., 2009). The up- regulation of these receptors is due to their agonist-mediated desensitization. There are several proposed mechanisms of up-regulation: decreased cell surface turnover, increase in receptor trafficking from the endoplsmic reticulum, increased subunit maturation and assembly, changes in subunit stoichiometry, blocking degradation of subunits in the ER, and conformational changes of subunits (Govind et al., 2009).

Changes of subunit stoichiometry in sensitization could include the α5 subunit combining with α4β2, α3β2, and α3β4 receptors to produce a receptor that has a higher affinity for nicotine but a more rapid desensitization rate (Fowler et al., 2008).

Nicotine and Reinforcement

Nicotine is the primary chemical that mediates the reinforcing properties of tobacco product consumption (Fowler et al., 2008; Fowler & Kenny, 2011). Nicotine is a molecule whose effect on nAChRs has been the primarily focused on its physiological impact within the confines of the brain and the peripheral nervous system (Gahring & Rogers, 2005). Nicotine has proven to have reinforcing properties in both humans and animals (Besson et al., 2006; Fowler et al., 2008). Many other components of tobacco smoke may contribute to nicotine addiction perhaps by increasing the reinforcing effects of nicotine (Fowler et al., 2008). Nicotine acts in the brain by activating neuronal nAChRs and engaging numerous intracellular signaling cascades that likely relate to nicotine mediated neuroplasticity and ultimately the development of tobacco dependence (Fowler & Kenny, 2011). The precise molecular mechanisms mediating the reinforcement and withdrawal from nicotine are largely unknown as are the exact way that nAChR activation leads to them. 24

α7 nAChRs and Nicotine Reinforcement Pathways

Nicotine mediates its reinforcing effects by activating α4β2 and α7 nAChRs in the mesostriatal dopamine pathway. The mesostriatal dopamine reward circuit is primarily composed of the VTA, prefrontal cortex and nucleus accumbens. The neurons in all of these areas express high levels of nAChRS (Di et al., 2012). In animal studies the VTA has been shown to be of central importance in mediating the reinforcing activities in rat models (Besson et al., 2006; Govind et al., 2009). Nicotine self-administration is decreased in animal models by creating lesions in the mesolimbic pathway. Infusing nicotinic antagonists directly into the VTA has been shown to decrease nicotine reinforcement as well. Chronic nicotine exposure can lead to alterations in the activity of the mesolimbic dopaminergic neurons, the desensitization of multiple nAChR subtypes, and rearrangement of subunit stoichiometry (Besson et al., 2006).

The α7 nAChR is present in the VTA and thought to have a modulatory role rather than a direct role in nicotine reinforcement(Picciotto & Kenny, 2013). Several animal studies have demonstrated a decrease in nicotine self-administration with methyllycaconitine MLA injection into the VTA; however, this result has not been uniformly demonstrated across multiple animal studies (Fowler et al., 2008; Levin et al., 2009; Picciotto & Kenny, 2013). Despite the lack of a dominant effect on reinforcement the α7 subunit is believed to be involved in nicotine dependent plasticity within the VTA. Knockout of the α7 subunit does not affect conditioned place preference or self-administration of nicotine. However, antagonizing the α7 in the nucleus accumbens or anterior cingulate increases the motivation to self- administer nicotine (Picciotto & Kenny, 2013). Interestingly, the infusion of selective 25

α7 agonist decreases the need to self administer nicotine. Given the likelihood of a more modulatory role in nicotine reinforcement it is possible that the effects of the α7 nAChRs absence in α7 knockout mouse may produce more subtle symptoms

(Picciotto & Kenny, 2013). Another hypothesis is that many of the animal models are poor models of nicotine administration because nicotine exposure only lasts a few weeks where in human smokers it is many years. Some studies have indicated a role in β2 containing nAChRs in establishing nicotine reinforcement but indicate that the

α7 containing nAChRs may have a role in maintaining reinforcement over time (Levin et al., 2009).

Studies of wild type and β2 nAChR knockout mice have demonstrated the central role of the α4β2 containing nAChRs in nicotine reinforcement and self- administration (Levin et al., 2009). Previous studies have shown β2 knockout mice were not susceptible to the reinforcing effects of nicotine injected directly into the

VTA. Although β2 knockout mice do no self-administer nicotine like wild type mice do, they are still susceptible to the development of a mecamylamine, nonspecific nAChR antagonist, induced withdrawal. This would indicate that although the β2 receptor is involved in the reinforcing effect of nicotine there are other nAChR subtypes responsible for the mediation of its withdrawal effects (Besson et al., 2006).

Multiple receptor subtypes involved in reinforcement and withdrawal is a pattern also seen in opiate addiction (Besson et al., 2006). With better and more thorough understanding of the neural basis of nicotine reinforcement and withdrawal, it may be possible to develop more targeted approaches to smoking cessation therapy (Levin et al., 2009).

Withdrawal

Nicotine addiction is characterized by chronic compulsive self-administration and the induction of a withdrawal syndrome on cessation of nicotine use (Besson et al., 2006). The reinforcing effect of nicotine is linked to more than just the reward stimuli but also an escape from the aversive consequences of nicotine withdrawal

(Fowler et al., 2008; Picciotto & Kenny, 2013). The duration and severity of nicotine withdrawal may predict relapse in abstinent human smokers. The efficacy of nicotine replacement therapy for smoking cessation may be related to the reduction of nicotine withdrawal in abstinent smokers (Fowler et al., 2008; Picciotto & Kenny, 2013).

Withdrawal Phenotypes and nAChR Subunits

Among animal models nicotine withdrawal shares many common behavioral features: abdominal constriction, rearing, grooming, jumping and body scratching and forelimb shaking. The severity of these aforementioned activities is usually scored in order to determine the severity of withdrawal (Besson et al., 2006). The β4 subunit is believed to be necessary for the physical expressions of nicotine withdrawal like scratching and rearing and shaking (Grabus et al., 2005). The role of the α7 receptor in this somatic withdrawal is subject to debate. Methyllycaconitine (MLA) administration in mice chronically exposed to nicotine has been shown to produce the somatic symptoms of nicotine withdrawal but these results are not consistently reproduced (Grabus et al., 2005). The ability of MLA to increase somatic withdrawal signs in WT and β2 KO mice after chronic exposure by osmotic mini-pump has been seen in two studies (Salas, Pieri, & De Biasi, 2004) (Jackson, Walters, & Damaj,

2009). Somatic withdrawal is likely related to a combination of subunits and α7, α5 and β4 but not β2 (Fowler et al., 2008). The affective components of withdrawal

27 reflect a negative change in the visible mood (affect). Accumulating evidence suggests that the affective components of withdrawal may play a more important role than the somatic aspects in the severity of withdrawal from drugs of abuse (Fowler et al.,

2008). The α7 is believed to be involved in the hyperalgesia that accompanies nicotine withdrawal (Grabus et al., 2005).

Oral Nicotine Exposure in a Mouse Model

Animal studies of nicotine administration are not perfect analogs of human nicotine administration. Inhalation (smoking) is the most common route of nicotine administration in humans, but this is not a method that can be easily and directly modeled in animals. Besides the approximations of routes of administration, the concept of length of administration is also an issue. Many human smokers smoke for several years, far longer duration than an animal study can approximate. Most rodent studies last only a period of weeks (Levin et al., 2009).

Murine models are used to study the behavioral, physiological, and biochemical effects of nicotine; rats are most commonly used (Matta et al., 2007). In general mice are less sensitive than rats to nicotine and require higher dosages to yield a similar physiological response. The nicotine induced seizure threshold in rats is

(0.5-2 mg/kg); in mice it is 2-6 mg/kg (Matta et al., 2007). The nicotine binding properties of high affinity nAChRs do not differ among animal models. In terms of mouse strains the susceptibility of different mice to nicotine varies widely which can account for the variability and inconsistency of results seen in many of the nicotine exposure studies. The amount of nicotine consumed by drinking water is strain dependent. C3H mice consume 4mg/kg per day while C57/Bl/6 mice can consume as much as 12 mg/kg/day (Matta et al., 2007). 28

Chronic nicotine administration is frequently modeled by oral nicotine administration in drinking water (Grabus et al., 2005; Matta et al., 2007). Tolerance to nicotine following oral exposure was maximized if the exposure period lasted longer than 4 weeks. Mice will consume pure water or nicotine containing solutions with little discriminative preference (Matta et al., 2007). Consumption of nicotine varies with sex and age, with adolescent female mice drinking higher concentrations of nicotine and consuming more water. The oral route is pharmacologically relevant with serum nicotine concentrations reaching levels as high as 15.85 µM and 538 µM in different experiments. These are vastly higher than what would be seen in human smokers (Matta et al., 2007). Chronic oral nicotine intake was able to precipitate signs of withdrawal after its cessation like somatic signs and hyperalgesia. Chronic nicotine also promoted tolerance as indicated by reduced analgesia and hypothermia (Matta et al., 2007). Abrupt cessation of chronic oral nicotine leads to an increased number of somatic withdrawal signs and hyperalgesia in mice. Most of these behaviors peaked within 2-3 days of nicotine cessation. Chronic oral nicotine also increases plasma nicotine and cotinine levels, produces hyperactivity, up-regulates α4β2 nAChRs

(Grabus et al., 2005).

Metabolism of Nicotine

Nicotine undergoes rapid first-pass hepatic metabolism. Nicotine is first converted into its imminium form. The conversion is catalyzed by cytochrome P450

(CYP2A6) (Sharma & Vijayaraghavan, 2008). The imminuim form is rapidly metabolized into the major metabolite cotinine. CYP2A6 is the key enzyme in this pathway and is also responsible for the metabolism of cotinine to t-3-hydroxy- cotinine. CYP2A6 is a highly polymorphic protein with at least 23 detectable variants 29 in the population each with varying degrees of efficiency (Sharma & Vijayaraghavan,

2008). Individuals with less effective CYP2A6 have decreased clearance of nicotine and maintain higher blood levels leading to an increased interval between desires for their next cigarette. Trials tested administration of methoxalen, a CYP2A6 inhibitor, demonstrated a reduction in subject’s need to smoke (Frishman, 2009; Sharma &

Vijayaraghavan, 2008). CYP2A6 is responsible for activating procarcinogens from tobacco components; inhibiting the CYP2A6 enzyme may cause people to smoke fewer cigarettes with mildly safer results (Frishman, 2009).

α7 nAChR targeted smoking cessation therapies

Neuronal nAChRs are the primary site of action of nicotine in the brain, as such they are considered to be important targets for the development of therapeutic agents that may facilitate smoking cessation efforts (Fowler et al., 2008). Varenicline, a drug based on the alkaloid cytosine, is a partial agonist of α4β2 nAChRs and a full agonist of α7 nAChRs. It has been approved for smoking cessation (Fowler et al.,

2008; Sharma & Vijayaraghavan, 2008; Zaniewska, McCreary, Stefanski,

Przegalinski, & Filip, 2008). Varenicline has a proposed dual mechanism for relieving nicotine withdrawal symptoms and inhibiting rewarding properties during smoking. In the presence of nicotine, varenicline reduces nicotine uptake and accumbal nicotine evoked dopamine response in rats indicating that nicotine exposure in the presence of nicotine leads to a reduced reward. Varenicline’s efficacy is comparable to nicotine patches or bupropion for the therapy of nicotine addiction (Zaniewska et al., 2008).

Nicotine, Proliferation, and Malignancy

The α7 nAChR is expressed in normal human small airway epithelial cells, small cell lung cancer, and non-small cell lung cancer cells (Shen et al., 2012).

Previous studies reveal that cigarette smoking-induced tumor cell invasion and metastasis may result, at least in part, from activation of PKC signal transduction pathway (Shen et al., 2012). The intracellular calcium pathway is known to be a major mitogenic pathway in human neuroendocrine tumors and in small cell lung cancers (Egleton et al., 2009; Hung et al., 2008; Shen et al., 2012). Nicotine has been shown to induce the proliferation of small cell lung cancers by activation of the α7 nAChRs. The activation of α7 nAChRs led to an increase in intracellular calcium and

L-type voltage operated calcium channels that then in turn increase proliferation of small cell lung cancer cells (Egleton et al., 2009). Nitrosamine 4-(methylnitrosamino)-

1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen of tobacco products. It induces malignancy and tissue invasion through an α7 dependent pathway (Shen et al., 2012) (Egleton et al., 2009). After NNK activates the α7 nAChR it activates a protein kinase cascade including c-Src, PKCi and FAK, resulting in the proliferation and migration of lung cancer cells. These cascades are prevented by bungarotoxin. The c-Src, PKCi and

FAK proteins are major regulators of cell migration and invasion (Shen et al., 2012).

Nicotine, Arteriogenesis, and Vasculature

Possibly one of the more important, but publicly under-appreciated, morbidities of long-term smoking is the deleterious effect on vasculature. Occlusion of peripheral arteries (claudication) presents as pain and restricted range of motion of a limb. This most commonly develops in the lower extremities due to narrowing of the femoral, tibial, or popliteal arteries. In other settings such as ischemic limb models of mice nicotine has been demonstrated the ability to promote arteriogenesis (Egleton et

31 al., 2009). In the setting of an ischemic insult nicotine seems to have a proliferative effect on many cell types including endothelial cells through α7 nAChR dependent mechanisms that lead to activation of calcium signaling pathways (Egleton et al.,

2009), Figure 1.5. Multiple convergent studies have demonstrated that α7 nAChRs are responsible for mediating endothelial proliferation and invasion necessary for angiogenesis. It has been demonstrated that transfection of α7 siRNA ablates the proliferative and proangiogenic effects of nicotine in both aortic and human lung microvasculature endothelial cells, suggesting that the α7 nAChRs are the dominant subtype responsible for these effects (Egleton et al., 2009).

The nicotine-induced arteriogenesis has the potential to be protective in ischemic brain diseases and may underlie some of nicotine’s proposed neuroprotective effects in physiological and pathological vasculogenesis (Egleton et al., 2009).

Nicotine has been found to promote angiogenesis and tumor invasion in lung and gastric cancers. The proangiogenic activity of nicotine may promote pathologic arteriogenesis raising concerns that prolonged use of nicotine patches and gums may be associated with increased risk of neoplasia (Egleton et al., 2009).

Nicotine, α7 nAChRS and Pathologic Vasculogenesis

Macular degeneration is the leading cause of blindness in the elderly. Smoking is the biggest risk factor for its development. The key pathology of this disease is the proliferation of vascular tissue in the choroid. Levels of VEGF, TNF-α, prostaglandins, and MMP’s are elevated (Egleton et al., 2009). Smoking is also a major modifiable risk factor for diabetic retinopathy whose pathophysiology includes harmful neo-angiogenesis in the retina. This abnormal vascular proliferation is in part driven by ischemia caused by microvascular changes associated with chronic 32 hyperglycemia. It is also hypothesized that chronically elevated nicotine levels activating α7 nAChRs also contributes to this pathology (Egleton et al., 2009).

Microglia play a large role in the regulation of vasculogenesis; they express the α7 nAChRs. Both choroidal and retinal endothelial cells express nAChR subytpes including α7 and α4β2. Smooth muscle cells selectively express nAChR subtypes dependent on their localization, α3 and α5 can be found among arteries but are absent on intrapulmonary and renal vessels. The thoracic aorta expresses all α subunits except

α9 (Egleton et al., 2009). The α7 receptor is expressed on the majority of smooth muscle cells and intrapulmonary arteries with the exception of renal circulation.

Nicotine exposure was found to change lung morphology in Wistar rats. These changes were characterized by increased angiogenesis, infiltration by monocytes and irregular collapse. The α7 receptors have been found in arteries devoid of cholinergic innervations (Egleton et al., 2009). Monocytes have been known to play a role in atherogenesis and angiogenesis. Infiltrating monocytes produce survival factors which in turn activate endothelial cells and promote neo-vascularization. Alpha- 7 nAChRs have been detected on monocytes and may be mechanism by which promotes atherogenesis (Egleton et al., 2009).

Figure 1.5: The role of α7-nAChR signaling in cell proliferation and vasculogenesis (Egleton et al. 2009)

α7 nAChR and Inflammation

Nicotine has long been associated with anti-inflammatory properties. It has been hypothesized that the pro-inflammatory elements in cigarettes smoke are countered to some degree by the anti-inflammatory effects of nicotine (Gahring &

Rogers, 2005). This may offer a rational explanation for why few smokers develop diseases of chronic inflammation such as pulmonary Langerhans cell histiocytosis. It has also been established that there is a lower incidence of sarcoidosis in smokers

(Gahring & Rogers, 2005). Inflammatory bowel disease (Ulcerative colitis and

Crohn’s disease) is characterized by damage of intestinal mucosa. Nicotine has been previously associated with interesting but contradictory effects on these two disease forms. Nicotine has been shown to aggravate Crohn’s disease but alleviate symptoms associated with Ulcerative colitis (Campbell et al., 2011; Gahring & Rogers, 2005).

Even nicotine patches have proven effective in decreasing symptoms of ulcerative colitis in clinical trials (Campbell et al., 2011; Gahring & Rogers, 2005). Interestingly, when individuals with ulcerative colitis stopped smoking they developed exacerbation of their disease symptoms (Gahring & Rogers, 2005). Nicotine patches have unfavorable side effect profiles as they are nonspecific nicotinic agonists and can activate a wide variety of nAChRs causing a host of unwanted autonomic effects.

The α7 nAChR is believed to have a principal role in mediating the anti- inflammatory properties of nicotine (Campbell et al., 2011). In the absence of exogenous activation by nicotine it is believed that the parasympathetic division of the autonomic nervous system acts as a cholinergic anti-inflammatory pathway that regulates systemic and local inflammation through the activation of α7 nAChRs on

35 immune cells (Campbell et al., 2011; Costantini et al., 2012; Freitas, Ghosh, Ivy

Carroll, Lichtman, & Imad Damaj, 2013). The α7 nAChRs are expressed on macrophages, key mediators of inflammation. They induce, modulate, and eventually resolve inflammation. Acetylcholine mediated activation of α7 nAChRs on the surface of macrophages is important to downregulate the immune system by reducing the synthesis of proinflammatory cytokines and preventing tissue damage (Freitas et al.,

2013). The binding of the α7 nAChR on inflammatory cells alters intracellular inflammatory signaling resulting in a decreased activation and translocation c-myc and

NF-κB, which leads to the suppression of cytokine formation (Costantini et al., 2012;

Sharma & Vijayaraghavan, 2008). The anti-inflammatory role of the α7 nAChRs has been demonstrated in numerous physiological models of sepsis, ischemia, postoperative ileus, pancreatitis and lung injury (Campbell et al., 2011). These effects are not observed in α7 nAChR knockout mice and can be abrogated by α7 nAChR antagonists. Nicotine inhibits lipopolysaccharide induced TNF α release in rat microglia and ironically has been shown to stimulate purinergic receptor P2X7 TNF α release, low levels of TNF α release by P2X7 are associated with neuroprotection while high levels of TNF α as elicited by lipopolysaccaride secretion are neurotoxic

(Egleton et al., 2009). The activation of α7 leads to a suppression of JNK and p38

MAPK that regulate the posttranscriptional steps of TNF release. The microglial α7 nAChRs are coupled to phospholipase C activation and CICR from IP3 sensitive calcium stores (Suzuki et al., 2006).

Alpha- 7 nAChRs are also believed to play a role in reducing stress-induced free radicals in the brain. Stress-induced free radicals are often produced by ischemia,

36 trauma, glutaminergic excitatoxicity and hypoglycemia (Parada et al., 2013). Previous studies suggest that the activation of α7 nAChRs on microglia initiates anti- inflammatory processes and neuroprotection by activating nuclear factor erythroid-2 related factor (Nrf2) and the expression of its target cytoprotective gene, heme oxygenase (HO-1) to combat oxidative stress and inflammation generated under ischemic conditions (Parada et al., 2013). Nrf2 is a transcription factor that is the master regulator of redox homeostasis. This transcription factor controls the expression of phase II enzymes that act in a cytoprotective manner against oxidative stress including HO-1 and the catalytic subunit of glutamate cysteine ligase. HO-1 is the rate-limiting step in the degradation of heme into carbon monoxide and biliverdin.

Carbon monoxide negatively regulates macrophages, preventing inflammatory damage of tissues. PNU-282987, an α7 specific agonist, was shown to induce HO-1 expression at a level that supports protection against brain ischemia (Parada et al.,

2013).

Studies with α7 Specific Agonists and Positive Allosteric Modulators (PAMS)

Investigation of nAChRs is hampered by a lack of subtype specific ligands.

Many physiological properties of the nAChRs have been inferred through nonspecific activation by nicotine in subunit knockout mice. For receptors like α7 nAChRs, hypothesized to act primarily as a modulator, it can be difficult to ascertain physiological function in a system where a broad activating ligand like nicotine is used. Fortunately, specific ligands are available for the α7 nAChR subtype permitting more targeted observations of the α7 nAChR’s role in physiology (Pandya & Yakel,

2013). The α7 specific agonists are designed to activate only the α7 nAChR; however,

37 many are also activate β2 containing nAChRs to varying degrees. Positive allosteric modulators, in contrast, do not activate the receptor directly but rather make the receptor more susceptible to activation (Pandya & Yakel, 2013). PNU-120596, a

PAM, when co-administered with nicotine desensitized α7 nAChRs and reduced the current through them to non-detectable levels. When PNU-120596 was applied alone and agonist activity was left to general cholinergic tone, desensitization did not occur and peak current through the receptor was greater than with activation of nicotine alone (Callahan, Hutchings, Kille, Chapman, & Terry, 2013).

Effects on Cognition

Aplha-7 nAChRs are strongly believed to play a large role in attention, learning and memory. Infusion of the α7 specific antagonist MLA into the murine hippocampus decreases working memory on radial arm mazes. Infusion of MLA into the ventral and Dorsal Hippocampus significantly impairs spatial and working memory in the radial arm maze as well (Levin et al., 2009). Specific activation of the

α7 nAChR seems to have significant precognitive effects. To some extent both PAMs and α7 nAChR specific agonists have been shown to increase performance on tests of cognition although the results are frequently inconsistent among studies (Callahan et al., 2013; Pandya & Yakel, 2013). Studies that employ PAMs as a rescue from drug induced deficits are most likely to demonstrate effectiveness. PAMs have reversed scopolamine, phencyclidine, MK-801, and amphetamine induced cognitive deficits to basal levels (Callahan et al., 2013). PNU-120596, PAM, and PNU-282987, α7 specific agonist did not significantly improve spatial learning and memory in rats as monotherapy, but they were independently able to reverse scopolamine induced cognitive impairments to normal levels (Pandya & Yakel, 2013). In rats the type I 38

PAM, NS1738, attenuated scopolamine-induced deficits in a water maze and improved the performance of social recognition tasks to the same extent as nicotine

(Callahan et al., 2013).

The α7 nAChR PAMs have been shown to be more effective at inducing the cognitive enhancing properties of the α7 nAChR than the specific agonists, especially when coupled with an acetylcholinesterase (Callahan et al., 2013; Pandya & Yakel,

2013). This may have to do with the rapid desensitization kinetics of the α7 nAChR.

Galantamine, which has activity as an acetylcholinesterase inhibitor, acts as a positive allosteric modulator of α7 nAChRs and α4β2 nAChRs. The efficacy of galantamine as a positive allosteric modulator of the α7 nAChR is fairly modest in comparison to the more recently designed PAMs. It is unclear if galantamine offers any additional clinical advantages over other acetylcholinesterase inhibitors, like donezepil (Callahan et al., 2013). There is some future interest in combining PAMs with acetylcholinesterase inhibitors for the purpose of treating Alzheimer’s disease.

Effects on Sensory Gating

Abnormal sensory gating is one of the hallmarks of schizophrenia and may be related to polymorphisms in the α7 gene (Picciotto et al., 2001). Auditory gating is measured as changes in specific peak of EEG recordings (Sharma & Vijayaraghavan,

2008). This response, known as the P50 auditory evoked response, is typically seen

40-80ms after the presentation of the auditory stimulus. In normal individuals, presentation of two stimuli closely spaced in time (less than 500ms) results in the attenuation of the P50 response to the second stimulus. This relative suppression of the

P50 response is an indicator of sensory gating (Sharma & Vijayaraghavan, 2008). In schizophrenic patients there is much less suppression of the P50 evoked response 39 leading to the idea that defects in this process may contribute to schizophrenic symptoms manifested by marked distractibility and poor attention. Nicotine restores the P50 ratios of schizophrenic patients to normal levels. This implies that nAChRs may play a large role in this process. The most implicated nAChR is the α7 nAChR

(Sharma & Vijayaraghavan, 2008).

Postmortem analysis of brain tissue from schizophrenic patients demonstrates decreased abundance of α7 nAChRs. A large number of correlations have shown that reduced levels of α7 nAChRS in the brain correlate to defects in P50 gating (Sharma

& Vijayaraghavan, 2008). Infusion of α7 nAChR antagonists decreases P50 ratios, while α7 nAChR specific agonists increase them. The physiological output is that

MLA prevents the ability to block a second closely spaced signal. Alpha-7 nAChR agonists promote the ability to block it out. As a result of these findings the α7 nAChR has become an increasingly popular target to improve the symptoms of schizophrenia

(Sharma & Vijayaraghavan, 2008). Two Benzylidine derivatives of anabasine, a marine worm toxin, are being developed as α7 specific agonists for schizophrenia,

GTS-21 and 4-OH GTS-21. The α7 nAChR PAM, compound 6, normalized sensory gating deficits in the schizophrenia mouse model (DBA/2) (Callahan et al., 2013). The

DBA mouse strain demonstrates decreased levels of Bgtx binding in their hippocampi compared to controls (Picciotto et al., 2001). Another PAM, JNJ-1930942, was able to improve sensory gating deficits in DBA/2 mice (Callahan et al., 2013). Amphetamines in animal models can induce sensory gating deficits. These deficits are improved with the α7 nAChR PAM, PNU-120596. The results of these studies reflect that α7 nAChR

PAMs have a potential role in schizophrenia therapeutic applications by improving deficits in information processing and cognitive flexibility (Callahan et al., 2013).

Effects on Nociception

One of the withdrawal effects of nicotine mediated by the α7 nAChRs is hyperalgesia. This indicates the α7 receptor has anti-nociceptive properties when activated (Costa, Motta, Manjavachi, Cola, & Calixto, 2012). Anti-nociceptive effects of nicotine can be completely abrogated by MLA administration supporting the idea that the anti-nociceptive properties of nicotine are mediated by α7 receptors. To date several drugs used to treat chronic pain (opioids, anti-epileptics, and antidepressants) have been shown to have significant side effects and provide incomplete pain relief for patients. Development of drugs possessing increased efficacy and safety is necessary; nAChR agonists have been explored as potential therapeutic targets (Freitas et al.,

2013).

Neuropathic Pain

The anti-nociceptive properties of the α7 nAChR make it a potentially interesting target for treating neuropathic pain (Freitas et al., 2013). Systemic α7 nAChRs agonists may be effective in treating neuropathic pain by reducing neuronal injury and peripheral immune cell activation. Treatments for neuropathic pain continue to be developed targeting neuroimmune interactions (Loram et al., 2012).

Intraperitoneal injection of a selective α7 nAChR agonist PNU 282987 reduced referred mechanical hyperalgesia at all periods of evaluation. The α7 nicotinic acetylcholine receptor has been associated with anti-nociceptive effects against acute inflammatory and neuropathic pain models (Campbell et al., 2011). Chronic neuropathic pain arises due to long-term plasticity in the somatosensory pathway to

41 the cortex. These alterations in plasticity often occur after nerve injury or dysfunction in the central nervous system leading to enhanced pain sensation even in the presence of otherwise non-noxious stimuli (Freitas et al., 2013). Drugs that attenuate glial activation and their pro-inflammatory products have been shown to resolve neuropathic pain. Following nerve injury or inflammation, peripheral immune cells are recruited to the site of injury and resident satellite cells within the affected dorsal root ganglia (DRG) become activated (Loram et al., 2012). These activated immune cells produce proinflammatory cytokines and may be critical in the development of chronic pain. Research is now recognizing the complex integration of both peripheral and central immune cells and neurons in the development and maintenance of neuropathic pain. Activation of glial cells in the spinal cord subsequently led to increased cytokine production and inflammation and damage of neural tissues, TC-7020 an α7 nAChR agonist was able to reverse neuropathic allodynia (Loram et al., 2012).

Visceral Pain

Visceral pain is extremely difficult to treat, It is opiate resistant and poorly controlled with acetaminophen. Recently, the potential use of α7 nAChR agonists to control the colonic afferent hypersensitivity induced by colitis was explored

(Campbell et al., 2011). This could have important clinical relevance to abdominal pain and discomfort are reported in both IBD and IBS are a consequence of visceral hypersensitivity. In a study of a colonic inflammation mouse model oral nicotine administration inhibited pre-established referred hyperalgesia induced by colitis through the activation of the α7 nAChR. Treatment with PNU 282987 widely replicated the results of nicotine. The mechanism of hyperalgesia is distinct from its mechanism of anti-inflammatory properties (Campbell et al., 2011). 42

α7 nAChR and Development

Consequences of Prenatal Nicotine Exposure

Approximately 15% of pregnant women in the US smoke tobacco. In addition to negative effects on maternal health, smoking during pregnancy can lead to low birth weights and increases in prenatal and postnatal mortality (Horst et al., 2012). The off- spring of smokers are at increased risk for the development of psychiatric disorders during childhood like attention deficit hyperactivity disorder (ADHD). This suggests that tobacco exposure during gestation alters processes critical to normal neurodevelopment and have persistent consequences. The processing of sensory information, especially auditory processing, is extremely susceptible to disruption by in utero nicotine exposure (Horst et al., 2012). Nicotinic receptors in adult born olfactory bulbs are primarily expressed β2 containing nAChRS. Acute nicotine exposure increases the proliferation of neurons in the SVZ through FGF-2 signaling.

The same treatment has no effect on the proliferation of neurons in the dentate

(Campbell et al., 2011). Prenatal nicotine exposure studies in mouse models yield inconsistent results. In some cases prenatal nicotine exposure leads to neuronal cell apoptosis; in others there is no observed defect. These observations may indicate that the timing of nicotine exposure is critical to its effect. Nicotine exposure prior to and during neurogenesis considerably diminishes generation and early survival of adult born neurons in the dentate. Chronic nicotine infusion starting a week after generation of adult born neurons markedly enhances survival throughout dendritic development

(Campbell et al., 2011).

Nicotinic AChRs and Development

Nicotinic acetylcholine receptors are particularly important during early prenatal and peri-natal circuit formation and in pathophysiology of age-related neuronal degeneration (Gotti et al., 2006). The dentate gyrus of the hippocampus is the home of the adult born neurons; it receives extensive cholinergic innervation. Newly generated neurons in the adult hippocampus express both α7 and β2 nAChRs

(Campbell et al., 2011). Nicotinic signaling is important for neuronal development.

During early stages of development repeated exposure to nicotine decreased the long- term survivability of these neurons. Studies in both α7 and β2 knockout mice demonstrate that fewer neurons survive the critical period and become integrated into neural networks. High doses of nicotine administered by osmotic minipump or intraperitoneal injection were found to decrease neurogenesis in the dentate. Studies of

α7 KO mice demonstrate that they have fewer surviving neurons than their WT counter parts (Campbell et al., 2011). The α7 nAChR is believed to be the essential nAChR in mediating the appropriate maturation of neurons and ensuring their appropriate integration into adult circuits. Loss of adult born neurons during development may lead to a predisposition to drug seeking behaviors (Campbell et al.,

2011).

α7 nAChR Effects on Glutaminergic Synapse Formation

Although glutamate is the primary excitatory transmitter in the adult brain, early in development this role may belong to the α7 nAChR. In early development glutaminergic synapses are only beginning to form while cholinergic signaling is already widespread (Lozada et al., 2012). Activation of the α7 nAChR promotes glutaminergic synapse formation during development (Lozada et al., 2012; Risso et

44 al., 2004). This dependence becomes clear when comparing WT mice to α7 KO mice

(Lozada 2012). Ultrastructural analysis, immunostaining, and patch clamp recording all reveal synaptic deficits when the α7 nAChR is absent (Lozada et al., 2012).

Nicotinic cholinergic receptors reach their highest levels early in development when glutaminergic synapses are initially forming. Early nicotinic signaling is known to influence specific aspects of local circuit formation and path finding. Activation of α7 nAChRs early in life leads to an increase in excitatory signaling and glutaminergic synapse formation. Αlpha-7 nAChR KO mice that have fewer glutaminergic synapses compared to their WT counterparts (Lozada et al., 2012). A possible consequence of prenatal nicotine exposure could be to encourage the formation of an excess of excitatory glutaminergic synapses that could lead to net increases in excitatory circuitry, possibly presenting as an ADHD phenotype. Nicotine exposure in vitro was shown to enhance glutaminergic synapse formation in doses as low as 1 µM (Lozada et al., 2012).

α7 nAChR Effect on Chloride Gradient

One of the α7 nAChR’s most established roles during development is the reversal of the chloride gradient that causes GABAergic activity to switch from excitatory to inhibitory (Liu, Neff, & Berg, 2006). The excitatory GABAergic period is critical to neuronal maturation and integration into circuits during embryonic development and after adult neurogenesis. In rats this conversion occurs in the first week of post-natal life, a time when the α7 nAChRs reach their peak levels in tissues.

During the early stages of development GABAergic signaling is excitatory because the intracellular chloride gradient is reversed. Activation of α7 nAChRs drives the maturation of the nervous system by expressing mature chloride channels that lead to 45 the reversal of the chloride gradient and a change in GABAergic activity from excitatory to inhibitory (Liu et al., 2006). Calcium signaling through the α7-nAChR causes increased expression of the chloride transporter KCC2 which pumps chloride out of the cell (Campbell et al., 2011; Liu et al., 2006). Changes in GABAergic signaling are also responsible for morphologic changes within the neuronas well.

Despite the profound impact of the GABAergic excitation/inhibition transition on the nervous system, little is known about the mechanisms that determine the timing of the transition or about the developmental consequences of the inhibitory GABAergic input (Liu et al., 2006). Inactivation of the α7 nAChR gene extends the developmental period in which GABA depolarizes (Campbell et al., 2011; Liu et al., 2006).

Developmental deficits persist for extended periods in some studies. The adult born neurons in α7 nAChR KO mice display reduced dendritic arbors compared to age matched WT even six weeks after final mitosis (Campbell et al., 2011).

α7 nAChR and Neuropathology

Nicotine, α7 nAChR and Anxiety

Nicotine has been shown to have dose dependent effects on anxiety. At low doses nicotine provides an anxiolytic effect; however, at high doses it induces anxiety

(Pandya & Yakel, 2013; Picciotto & Kenny, 2013). The effect of nicotine on anxiety also appears to be sex influenced. Chronic administration of nicotine increased anxious behavior in female but not male mice (Picciotto & Kenny, 2013). The prevalence of tobacco use among those who suffer from an anxiety disorders is high.

Nearly half of people suffering from anxiety smoke compared to less than one third in the control population. Many smokers report that cigarettes are able to successfully reduce their sense of anxiety and improve their mood control. Abstinence from 46 nicotine use has been demonstrated to increase anxiety (Fowler et al., 2008). Some speculate that deficits may exist in the brains of smokers who suffer from co-morbid anxiety disorders. These deficits may enhance the reinforcing effects of nicotine resulting in higher rates of tobacco dependence. While nicotine in tobacco products has been shown to have subjective anxiolytic properties, other evidence suggests that nicotine abuse may contribute to the development of anxiety disorders (Fowler et al.,

2008).

The α7 nAChR have been implicated in the anxiety behavioral phenotype. α7- nAChR KO mice were shown to spend more time in the center of an open field apparatus indicating that they had reduced levels of anxiety, although this observation is not consistent in other studies (Fowler et al., 2008). Nicotine withdrawal elicits a similar anxiety profile in both α5 and α7 KO mice in contrast to WT mice (Fowler et al., 2008). However, the behavioral effects of anxiogenic doses of nicotine in wild type mice can be abrogated by co-administration of MLA (Fowler et al., 2008).

Alpha-7 nAChR specific agonists have been demonstrated to induce anxious behaviors in animal models that are not reversible with MLA (Pandya & Yakel, 2013).

This may indicate either a lack of specificity of these agents or the fact that the α7 nAChR is involved with the induction of anxiety but not its maintenance (Pandya &

Yakel, 2013). Although direct α7-nAChR agonists have been shown to have anxiogenic properties in mouse models, α7 nAChR PAMs do not seem to have this effect. The mechanism of anxiety in this case is believed to be due to α7-mediated serotonin release from the dorsal hippocampus (Pandya & Yakel, 2013). Nicotine administration increases the firing of serotonergic neurons in the dorsal raphe nucleus

47 leading to a release of serotonin. Presynaptic α7-nAChRs are thought to induce this effect (Pandya & Yakel, 2013). This mechanism was validated in animal studies by extinguishing anxiety like behaviors precipitated by PNU-282987, an α7-nAChR specific agonist, with a serotonergic antagonist WAY-100135 (Pandya & Yakel,

2013). Although α7 nAChR specific agonists are being designed for treatment of neurodegenerative disorders, schizophrenia, and pain the unwanted and likely α7- nAChR mediated side effect of anxiety may make α7 nAChR PAMs a better overall choice as a pharmacological agent (Pandya & Yakel, 2013).

Alzheimer’s Disease

Alzheimer’s disease (AD) is characterized by progressively worsening memory and cognitive deficits that can progress to the point of incapacitation (Sharma

& Vijayaraghavan, 2008). The incidence of AD among smokers is less than among nonsmokers. The cholinergic hypothesis of AD has been present for a long time and is based on findings that the loss of basal forebrain cholinergic neurons is one of the earliest signs of AD. This observation initially led to the use of cholinesterase inhibitors. These agents can alleviate some of the symptoms of AD but cannot halt progression (Sharma & Vijayaraghavan, 2008). The basis of the effectiveness of increasing synaptic acetylcholine concentrations depends on the presence of intact projections and signaling after the receptors had been activated. Neuronal apoptosis severs these connections rendering increased synaptic acetylcholine concentrations almost completely useless (Sharma & Vijayaraghavan, 2008).

The amount of α7 nAChRs are decreased in postmortem analyses of brains from patients with AD (Sharma & Vijayaraghavan, 2008). The α7 nAChR is believed to be a target for the treatment of Alzheimer’s disease because its metabotropic and 48 anti-inflammatory properties may serve to increase neuronal survival and maintain the functionality of intact circuits. The activation of α7 nAChRs has been demonstrated to yield a neuroprotection in AD models (Sharma & Vijayaraghavan, 2008). A feature of

α7 nAChRs is their ability to decrease the formation of the pathological β-amyloid (1-

42) peptide, the key component of plaques found in the brains of AD patients (Sharma

& Vijayaraghavan, 2008). β-amyloid can bind to the α7 nAChRs with picomolar affinity. In mouse models β-amyloid (1-42) also demonstrated the ability to activate

MAPK and leading to increased hyperphosphorylation of tau which promotes the formation of the neurofibrillary tangles characteristic of AD pathophysiology (Sharma

& Vijayaraghavan, 2008). Activation of α7-nAChRs also lead to decreased production of intracellular reactive oxygen species (Egleton et al., 2009).

Neuroprotection mediated by α7 nAChR may be due in part to nicotine induced proliferation of vasculature. During aging the neurovasculature is continually modified. It can be accelerated in pathological conditions such as strokes and vascular dementia. Changes in neurovasculature can include aberrant angiogenesis and hypo- and hyperperfusion (Egleton et al., 2009). In Alzheimer’s disease there is a good correlation between the reduced length of capillaries in the CA1 region and clinical dementia. This would indicate that a reduced capillary density perhaps by reduced angiogenesis might be a factor that worsens the pathophysiological picture of the disease (Egleton et al., 2009). Nicotine’s observed neuroprotective effects could be two fold, it could be due to reduction in the amount of pathological β-amyloid made by activation of α7 containing nAChRs or in inflammation and increased angiogenesis

(Egleton et al., 2009).

α7 and Schizophrenia

Approximately 1% of the population suffers from schizophrenia. This disorder has many variants and is characterized by positive symptoms that compose a spectrum of disordered thought and behavior and negative symptoms, characterized by social withdrawal and inability to participate in normal social interactions (Lieberman et al.,

2013). The positive symptoms are considered “positive” because they are visibly abnormal and often alarming to others while the “negative” symptoms may be subtle and go unnoticed by others. The negative symptoms of schizophrenia may precede the onset of positive symptoms for many years and persist despite the remission of psychosis. Positive symptoms are highly responsive to antipsychotics, but there are no adequate treatments for the negative symptoms and cognitive symptoms that are a barrier for reentry into normal social roles (Lieberman et al., 2013).

Similar to anxiety and depression the rates of tobacco smoking are dramatically higher in schizophrenic patients compared with the general population

(Fowler et al., 2008). Estimates indicate that the prevalence of tobacco smoking in schizophrenic patients is between 60-90%. Several explanations have been developed to account for why this observation is seen. First, schizophrenics attempt to induce some correction for neuroleptic therapies that induce extrapyramidal side effects.

Second, the nicotine from the tobacco smoke may ameliorate the generalized cognitive gating deficits or sensory gating deficits associated with the disease. Third, the rewarding properties of nicotine may be increased in schizophrenic patients leading to a greater abuse potential in this population. The nicotine in tobacco smoke may also ameliorate some of the negative symptoms associated with the disease (Fowler et al.,

2008). 50

Accumulating evidence from human patient populations indicate that a genetic linkage may exist between mutations in the α7 nAChR and the development of schizophrenia (Fowler et al., 2008). Activation of the α7 nAChR by nicotine may be one of the reasons why nicotine is used to self-medicate in this population. Decreased densities of the α7 nAChRs are found on postmortem analysis. Abnormal sensory gating has been noted with polymorphisms in 15q13-14, the region in which the α7 gene lies. Nicotine has been reported to correct sensory gating and attention deficits in individuals with schizophrenia (Fowler et al., 2008). Individuals with schizophrenia demonstrated impaired performance in attention demanding tasks once they quit smoking (Fowler et al., 2008; Picciotto & Kenny, 2013). Genetic and functional studies have implicated α7 nAChRs in pre-pulse inhibition, a physiological marker associated with schizophrenia in animal models (Picciotto & Kenny, 2013). The high rates of smoking may reflect high motivation to obtain the stimulatory effects of nicotine on the α7 nAChRs thereby improving compromised cognitive performance

(Fowler et al., 2008). If the link is solvent then the possibility of α7 nAChR targeted therapies would be beneficial to the control of the disease while causing the patient to avoid the myriad of negative consequences of tobacco abuse. As a result α7 nAChR specific agonists and PAMs are under development to help treat this disorder.

Proteomics

Proteomics is a field of study concerned with identify and analyzing the structure, functions and interactions of proteins in biological systems.

Neuroproteomics is the study of the proteome, collection of proteins, encoded by the genes of an organism (Ramos-Ortolaza et al., 2010). Proteomic analysis of brain tissue

51 is becoming an integral component of neuropsychiatric research (Abul-Husn & Devi,

2006). Proteomic approaches are being used extensively to study global changes in expression profiles in various tissues and to compare the profiles between physiological and perturbed states (Abul-Husn & Devi, 2006). One of the chief advantages of proteomics research is that it can be used to study the differences of perturbed states without the need for prior hypothesis (Abul-Husn & Devi, 2006).

Typically, scientific research involves designing a hypothesis around what is believed to be a single mechanistic detail and perturbing it to better understand its role in the function of a given process. This can be a difficult approach for trying to understand the pathological mechanisms for neuropsychiatric diseases. These diseases are often clinical diagnoses in which an individual satisfies subjective rather than objective criteria. Many of these behavioral phenotypes are likely a final common outcome of heterogeneous perturbations arising in one or more interdependent pathways.

Proteomic profiling represents an exciting method of study for these disorders because identification of proteins that are consistently seen in the perturbed state can be used to generate the basis of new hypotheses forming the opportunity for more targeted exploration of neuronal pathways. Diseases of the central nervous system, neuropsychiatric disorders, and degenerative conditions, as well as substance abuse involve multiple interacting proteins (Abul-Husn & Devi, 2006). Being able to observe global derangements in protein signaling caused by nicotine exposure could lead to the generation of any number of mechanistic hypotheses that can be plumbed by a variety of biochemical means. Receptor proteomics has emerged as a primary approach in the molecular analysis of signaling and regulation (Kabbani & Levenson,

2007). Multi-protein complexes known as signal complexes have been found to interact with receptors like the NMDA receptor (Kabbani & Levenson, 2007). The development of mass spectrometry based proteomics has allowed for the identifications of large numbers of proteins in complex mixtures like those present in serum samples.

Mass Spectrometry Based Proteomics

The development of biological mass spectrometry (MS) in the 1990s provided a protein analysis tool superior to the previous method, Edman degradation. MS proved more efficient, sensitive, and required less technical expertise in the way of protein purification (Steen & Mann, 2004). Mass spectrometry based proteomics was immediately recognized as an exciting tool it allowed for the identification of multiple proteins present in a mixture. In one experiment is became possible to identify of tens of thousands of proteins that would then form the substrate for further validation and exploration of relationships and interactions through secondary biochemical techniques. The basic methology of MS first requires degradation of a protein mixture by an enzyme that cuts at specific and reproducible sites; often Trypsin is used (Steen

& Mann, 2004). The complexity of the mixture will then be reduced by fractionation with liquid chromatography (LC) before analysis by mass spectrometry and database searching (LC-MS), Figure 1.6. In some instances a sample will require pre- fractionation by 1 or 2D gel chromatography to simplify the protein mixture prior to initial peptide degradation and LC-MS (Steen & Mann, 2004). LC typically employs reversed phase columns to simplify the mixture on the basis of peptide hydrophobicity prior to MS analysis; however, additional refinement is possible through the

53 implementation of two sets of LC columns. A cation exchange column coupled in series with a reverse phased column is the basis of multidimensional protein identifying technology (MudPIT). When LC columns are coupled in tandem, maximum peptide mixture fractionation can be achieved (Banks, Kong, & Washburn,

2012). A sample with tens of thousands of proteins will have a large number of peptides after enzymatic degradation. This can easily overwhelm the detection limits of MS instrument. Peptide fractionation before analysis can prevent missing identifications of less abundant species. The challenge of employing MS in proteomic research is volume. Samples generally have tens of thousands of peptides, and modern computational methods and database searching techniques are now capable of producing datasets with almost as many protein identifications (Cociorva, D, & Yates,

2007; Steen & Mann, 2004). Since identifications are made by statistical and computational methods and protein database searching, rather than by biochemical verification, a large component of proteomic based MS analysis requires careful consideration of both false positive and negative identifications (Steen & Mann,

2004).

Several general strategies for biological sample analysis exist that are designed to be coupled to LC-MS. Shotgun-style proteomics involves whole cell lysates that are typically digested in solution with trypsin, the resulting massive number of peptides the undergo LC-MS (Banks et al., 2012; Steen & Mann, 2004). Bottom-up proteomics involves fractionating the proteins by some method like SDS-PAGE and then performing tryptic digests in-gel before analysis by MS (Steen & Mann, 2004). Top- down proteomics involves ionizing an entire protein rather than the peptides

54 components (Whitelegge, 2013). The experiments performed in this dissertation are a combination of the bottom-up and top-down methods. Digestions were done in-gel and then the resulting peptides were further fractionated by reverse phase LC.

Figure 1.6: Flow chart of botton up proteomic experiment (Steen & Mann, 2004)

Peptide Ionization Techniques

A significant portion of biological mass spectrometry focuses on the ionization of peptides. Once peptides are ionized their mass can be measured in a variety of ways that are dependent on the mass analyzer and detector of the instrument being used . Ionization sources can be coupled to different mass analyzers, although some combinations are preferable to others, such as MALDI-TOF or ESI-LTQ. The importance of the mass is that it is a distinguishing feature of a peptide. The longer the peptide, the higher its mass should be. When proteins are degraded by proteases that cut in well conserved areas like trypsin, then the lengths of the degraded peptides should be predictable and by extension so should the peptide masses. The pattern of peptide masses can serve as a “protein fingerprint”. It is essential that the chemical structure of the peptide is intact throughout the ionization process; this is referred to as soft ionization. In contrast, hard ionization techniques are employed in small molecule

MS. A chemical is fragmented by collision to induce the formation of ions that are then detected. The most common soft ionization methods used in biological MS are matrix assisted laser desorption ionization (MALDI), or electrospray ionization (ESI).

The character of the peptide itself determines ease of ionization. Peptides with more basic residues will be protonated more easily; peptides with many hydrophobic residues are more difficult to protonate. This difference confers a detection bias for peptides with more basic residues. This is more critical in ESI than MALDI. The net positive or negative charge of an ion is important because it is the mass-to-charge ratio

(m/z) that is important in analysis (Paul, Kumar, Gajbhiye, Santra, & Srikanth, 2013;

Steen & Mann, 2004).

In MALDI the analyte is mixed with a large excess of ultraviolet absorbing matrix, normally a low-molecular-weight aromatic acid (Paul et al., 2013; Steen &

Mann, 2004). Upon irradiation with a laser beam of the appropriate wavelength, the excess matrix molecules sublime and transfer the embedded non-volatile analyte molecules into the gas phase. After numerous ion–molecule collisions in the plume of ions and molecules, singly charged analyte ions are formed. These ions are then accelerated by electric potentials into a mass analyzer of choice, typically a time of flight analyzer (TOF) (Steen & Mann, 2004). It is essential that the peptide samples are as fractionated as possible otherwise the spectra can easily become too complicated to analyze accurately. This method of ionization is generally more tolerant of peptides that are hydrophobic.

For ESI, the tapered end of the LC column or a metal needle is held at a high electrical potential (several kV) with respect to the entrance of the mass spectrometer.

The liquid effluent containing the peptides that are eluting from the chromatography column is thereby electrostatically dispersed (Paul et al., 2013; Steen & Mann, 2004).

This generates multiply charged droplets, which are ususally positively charged in proteomics experiments, due to an excess of protons. Once the droplets are airborne, the solvent evaporates, decreasing the size and increasing the charge density of the droplets (Paul et al., 2013; Steen & Mann, 2004). Desolvated ions are generated by the desorption of analyte ions from the droplet surface due to high electrical fields and/or the formation of very small droplets due to repetitive droplet fission until each droplet contains, on average, only one analyte. Typically, ESI methods are analyzed by an ion trap. Ion traps alter the electric field around an ion causing it to propel toward the mass

58 detector (Steen & Mann, 2004). ESI can constantly produce ions and couples to LC very easily. It pairs well with mass analyzers with rapid duty cycles because ions are continually sprayed into the detector. This method is less tolerant of hydrophobic peptides because they have fewer residues to protonate. The peptides also run the risk of being sprayed outside of the detector; the constantly generated peptides can deluge the detector.

Tandem Mass Spectrometry

It is difficult to confidently identify peptide ions that are present in complex mixtures because the instrument only measures m/z ratios. One single m/z ratio could correspond to thousands of different amino acid combinations with any number of charge states. More data per peptide ion can be acquired by selecting a single

“precursor” ion, fragmenting it by collision with inert gas molecules and detecting the resulting fragment ions, known as “product ions” that are designated as a, b, c, or, x, y, z depending on the side of the fragment that retains the charge. This process is known as collision induced dissociation (CID). It is one of many ways to create product ions

(Steen & Mann, 2004; Zubarev & Makarov, 2013). The process of selecting precursor ions, fragmenting them and measuring the masses of their product ions is known as tandem mass spectrometry (MS/MS); it is essential to analyzing mixtures of proteins.

The product ions fragmentation by CID is fairly predictable. Peptide ions breaks at their weakest point, the amide bond, to produce spectra almost entirely composed of y and b ions (Steen & Mann, 2004). The longer the peptide ions are the more y and b ions will be produced by CID. The data from the product ions is what is used to search protein databases to determine peptide identities. Typically, a precursor ion has to

59 have a certain abundance value that is set by the operator of the instrument to be selected for CID and MS/MS (Steen & Mann, 2004).

Mass Analyzers

Mass spectrometers are instruments that can have multiple ion sources coupled to a variety of mass analyzers. There are a wide variety of mass analyzers that vary considerably in mass accuracy, resolution, sensitivity, and dynamic range (Paul et al.,

2013). The sensitivity of advanced mass analyzers like the Orbitrap can be as low as femtogram levels (Paul et al., 2013). Mass accuracy is the ratio of the m/z measurement error to the true m/z and is often listed in ppm (Steen & Mann, 2004;

Zubarev & Makarov, 2013). The quantitative unit that m/z is usually expressed in is the Dalton. A mass accuracy of 1000 ppm corresponds to an accuracy of +/- 1 Dalton from the measured value (Steen & Mann, 2004). Mass resolution is the ability of an instrument to discern two closely spaced peaks. Instruments with poor resolution will detect two closely spaced masses as the same mass (Steen & Mann, 2004). The linear dynamic range is the range at which the signal of detection saturates. This is more of an issue in quantitative mass spectrometry where peak height measurements can be used as a basis to determine protein abundance in different samples. Instruments with low dynamic range would be limited in their abilities to estimate fold change. The

Time of Flight (TOF), quadrupole, ion trap are generally the most common analyzers

(Steen & Mann, 2004).

Figure 1.7: Types of peptide product ions that are formed by fragmentation (Steen & Mann, 2004)

A) Breakage points that form a,b,c and x,y,z ions. B) A series of y and b ions along a peptide, y and b ions are the dominat product ions seen in collision induced dissociation

TOF mass analyzers are often coupled to MALDI ion sources. It measures the time to travel through an electric field free flight tube that is under vacuum. In the ion source all of the ions are accelerated at the same kinetic energy; they travel through a field of known length to the detector. Since the kinetic energy is known and the distance of the tube is known, the calculation of the time of flight allows the mass of the ion to be calculated (KE=1/2mv 2). Smaller ions are lighter, so they travel faster giving them a smaller time of flight. Larger ions are slower and have a longer time of flight (Steen & Mann, 2004). Practical mass accuracy on a MALDI-TOF can be as high as 1000 ppm. They have no capacity for MS/MS unless additional mass analyzer components are added. Quadrupole mass analyzers are basically ion selection filters.

They restrict the passage of ions. By altering the sinusoidal potentials they step through different m/z values that they allow to pass to the detector (Steen & Mann,

2004). Quadrupole mass analyzers can exist in series and for MS/MS. Quadrupole ion traps, as their name implies, trap peptide ions in a dynamic electric field. The ions are then ejected sequentially according to their m/z value on to the mass detector with the assistance of a second electric field. Trapped ions can also be isolated and fragmented in the trap for MS/MS (Steen & Mann, 2004). The highest reported quadrupole ion trap mass accuracy was 12-15 ppm (Zubarev & Makarov, 2013).

Orbitrap

Orbitrap is a mass analyzer that is essentially an electron field ion trap where ions orbit around a central electrode (Zubarev & Makarov, 2013). Instead of being ejected into a mass detector, data gathered from oscillations of the ions within the electric field are used to compute their mass. “Ions remain on a nearly circular spiral

62 inside the trap, much like a planet in the solar system. At the same time axial electric field caused by special conical shape of electrodes, pushes ions towards the widest part of the trap initiating harmonic axial oscillations. Outer electrodes are then used as receiver plates for image current detection of these axial oscillations”(Zubarev &

Makarov, 2013). This allows masses to be calculated with accuracies of 1-2 ppm (+/-

0.001 Dalton) or less. This method also allows for extremely high resolution (Zubarev

& Makarov, 2013). The LTQ Orbitrap was introduced in 2005. Variations of this instrument were used to do much of the proteomic work featured in this dissertation.

The LTQ Orbitrap utilizes LC coupled electrospray, typically with nano-LC columns.

After ions are generated by electrospray, they enter an initial series of quadrupole mass analyzers and a possible collision cell (if CID is chosen) for MS/MS before entering the Orbitrap (Zubarev & Makarov, 2013).

SEQUEST

The mass spectrometry data that is generated by instrumentation are recorded as .RAW files. Several thousands of .RAW files are generated per experiment prohibiting manual database search to identify proteins impossible (Cociorva et al.,

2007; Steen & Mann, 2004). SEQUEST is one of many available algorithms designed to analyze .RAW files, assign peptide identities and correlate them to possible proteins of origin. DTASelect is a program that converts .RAW files into .dta files that

SEQUEST analyzes. DTASelect, or an equivalent program, acts as an initial filter to select quality spectra for SEQUEST to analyze (Cociorva et al., 2007). After

SEQUEST has finished the analysis the file is changed to an .out file (Cociorva et al.,

2007) that DTASelect (or an equivalent program) can organize. SEQUEST operates

63 by going through an entire database of proteins. It calculates peptide lengths and masses of an enzymatic digest of choice, typically trypsin. After these potential peptide generations are counted, SEQUEST then calculates the likely MS/MS spectrum resulting from them (Steen & Mann, 2004). These artificial spectra are correlated to experimentally acquired spectra and a score is assigned, termed the cross correlation score (X corr ) (Steen & Mann, 2004). The closer the artificial spectrum is to the actual spectrum the higher the score will be, Figure 1.8. There will be many X corr values per spectrum; these are ranked in a list. The closer the X corr values of the most likely and second most likely peptide identifications, the more ambiguous the assignment is (Cociorva et al., 2007). The difference between adjacent X corr values is termed ∆CN. The lower the score the more ambiguous the identification (Cociorva et al., 2007). Cut off values by peptide charge can be set so that ambiguous or poorly correlated identifications can be eliminated from the final list of identifications.

Figure 1.8: SEQUEST Algorithm (Steen & Mann, 2004) This figure is a flow chart of the cross correlation score (X corr )

Quantitative Mass Spectrometry

Mass Spectrometry can be used to determine quantitative data about peptide abundance as well as peptide identity (Steen & Mann, 2004). The basis of quantitative mass spectrometry involves evaluating changes in the amounts of proteins in two different samples (Ramos-Ortolaza et al., 2010; Steen & Mann, 2004). Changes in protein amount could correspond to changes in protein expression or changes in association to a specific affinity isolated protein. There are several types of experimental methodologies designed to facilitate quantitative analysis of mass spectrometry data. They can be divided into label free and label based methods. Label based methods involve the introduction of a weighted tag or stable heavy isotope to an experimental sample. This mass label allows for identical experimental and control peptides to be distinguished from one another. There are many types of label free methodologies as well.

Label free studies have significant advantages over label based methods.

Labeling strategies can be difficult to implement, have high associated costs, require significant sample quantity, and do not always uniformly label (Paul et al., 2013).

Label free strategies allow for the detection of greatest differences in protein abundance, offer relatively simple workflows, and the most direct comparison of physiological states (Zhu, Smith, & Huang, 2010). This is particularly important in understanding the physiological functions of biological systems as well as determining the functional implications of alterations in proteins in disturbed states such as those involved in neurodegenerative states and drugs of abuse (Ramos-Ortolaza et al.,

2010). The label free method chosen for the quantitative mass spectrometry experiments in this dissertation was the method of spectral counts.

Label Free Mass Spec with Spectral Counts

The advent of label free methods has circumvented some of the issues of labeling methods. Spectral counts have become a popular and successful method for relative protein quantification using mass spectrometry data (Leitch, Mitra, &

Sadygov, 2012), Figure 1.9. Spectral counts are routinely used to acquire quantitative proteomic information from complex biological samples for example, biomarkers in the human saliva proteome in type-2 diabetics, comparison of protein expression in yeast and mammalian cells under different culture conditions, and differentiating plasma membrane proteins from terminally differentiated mouse cell lines (Zhu et al.,

2010).

Spectral counting refers to counting all of MS/MS spectra assigned to a peptide

(Zhu et al., 2010). The more abundant a protein is in a sample the more of its peptides will be produced on enzymatic digest and the more often those peptides will be detected. The numbers of MS/MS spectra that are correlated to a single peptide are a direct marker of its abundance in a sample. The tally of MS/MS spectra for all unique peptides corresponding to a single protein is the spectral count for that protein (Dicker,

Lin, & Ivanov, 2010; Zhu et al., 2010). Spectral counts for identified proteins can be compared for the experimental and control samples to detect changes in abundance

(Zhu et al., 2010). Spectral counts have been demonstrated to be one of the best label free quantitative methodologies showing strong linear correlation with relative protein abundance ( r2 = 0.9997) with a dynamic range greater than 2 orders of magnitude

(Zhu et al., 2010). This technique is not without its limitations, which are important to consider when evaluating experimental data.

Limitations of Spectral Counting

One of the primary limitations to spectral counting is that large proteins will generate more peptides than smaller proteins after enzymatic digestion (Leitch et al.,

2012). Proteins within biological systems have varied structure based on their biological roles. The differences in structure can lead to differential abilities to be digested with trypsin. An assumption of proteomics is that enzymatic degradation is equivalent for all proteins in a sample; this assumption is clearly not always valid based on the level of hydrophobic domains within a given protein (Leitch et al., 2012).

These same hydrophobic domains offer the possibility of retaining a peptide on the LC column as well, preventing it from entering the mass spectrometer and being detected.

Not all peptides have the same ability to ionize (Leitch et al., 2012). These scenarios can either falsely elevate or lower spectral count data. The ability of peptide structural analysis programs (SEQUEST, MASCOT) to correctly assign peptide identity for all

MS/MS spectra in a peptide’s tally is a variable as well (Leitch et al., 2012; Zhu et al.,

2010). Another limitation of this technique is that spectral counts do not make full use of information from peaks in the LC-MS domain because they only quantify detected

MS/MS spectra that can lead to loss of efficiency (Dicker et al., 2010).

All of these possible scenarios represent challenges to reproducibility among replicate samples that is a necessity for quantitative mass spectrometry data. The number of replicates is itself a variable because samples can have tens of thousands of peptides

Figure 1.9: Flow Chart Illustrating Spectral Count Based Quantitative Mass Spectrometry (Zhu et al., 2010)

69 but due to cost considerations and sample preparation abilities, the number of replicates per experiment are typically small (Leitch et al., 2012). Because of these variables statistical analysis and validation is critical to the interpretation of results. Of several statistical tests compared in previous studies to evaluate the significance of comparative quantification by spectral counts the Fisher’s exact test and Student’s t- test were found to be the best when 3 or greater replicates were available. When replicates were limited to 2 or less the G-test was the best (Zhu et al., 2010). Currently because of issues like protein length, multiple hypothesis testing, and missing data from replicate samples Bayes factors have become a tool of choice for evaluating statistical confidence in normalized spectral counting data (Leitch et al., 2012).

QSPEC

Significance analysis of normalized spectral count data in the quantitative proteomic studies performed in this thesis was performed using QSPEC, a recently published algorithm for determining the statistical significance of differences in spectral counting data (Choi & Nesvizhskii, 2008). This algorithm used the Bayes

Factor in lieu of the p-value, as a measure of evidential strength (Goodman, 1999;

Leitch et al., 2012). By convention, a Bayes factor greater than 10 suggests strong evidence that a particular protein was differentially expressed between the two cohorts, thus a value of 10 was used as our significance threshold (Goodman, 1999;

Jeffreys, 1961). The advantage of using Bayes factors is that they take into account multiple hypothesis testing that corresponds to the many proteins in the dataset. They correct for protein length differences and account for missing data from replicate samples (Choi & Nesvizhskii, 2008; Leitch et al., 2012). This statistical algorithm has

70 the capacity to correct for many of the limitations of spectral counting to indicate quantitative results that are significant.

PANTHER

Protein analysis through evolutionary relationships (PANTHER) is a comprehensive software system for inferring the functions of genes based on their evolutionary relationships (Mi et al., 2010; Mi, Muruganujan, & Thomas, 2013). The

PANTHER website is freely available at http://www.pantherdb.org and also includes software for the analysis of genomic data relative to known and inferred gene functions. Phylogenetic trees of gene families form the basis of PANTHER. These families are subsequently annotated with ontology terms (Mi et al., 2010; Mi et al.,

2013). One of the main applications of PANTHER is the accurate prediction of the functions of uncharacterized genes based on their evolutionary relationships to genes with functionalities determined by experiments (Mi et al., 2010; Mi et al., 2013).

For analysis of the trees, the Gene tree Inference in the Genomic Age (GIGA) algorithm is used, this algorithm makes use of known species trees and presumably complete gene sets to infer accurate gene trees and locate gene duplication events relative to speciation events (Mi et al., 2010; Mi et al., 2013). The algorithm also performs rapid approximate reconstruction of ancestral protein sequences at each node in the tree using an iterative process starting at the leaves of the tree that considers the descendant sequences and the nearest outgroup (Mi et al., 2010). This approach has practical advantages for annotation of gene function but sacrifices some accuracy in some rare but important evolutionary relationships (Mi et al., 2013).

PANTHER is a useful tool in classifying the biochemical role of proteins identified in mass spectrometry data. It is an efficient program that can classify multiple biological roles for each identified protein and give indications for which physiological pathways and disease states that it participates in (Gollapalli et al., 2012;

Leitch et al., 2012).

PANTHER applies both software tools and manual curation to perform these inferences as accurately as possible and keep them up to date with the current results and opinions of the field (Mi et al., 2010; Mi et al., 2013). The function of gene products such as proteins is described using terms from gene ontology (GO) or from representative molecular pathways (Mi et al., 2010; Mi et al., 2013).

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CHAPTER 2: FURTHER ANALYSIS OF POTENTIAL INTERACTING PROTEINS OF THE α7 NACHR William Brucker, Joao Paulo, Edward Hawrot

Introduction

Neuronal nicotinic acetylcholine receptors (nAChRs) are a diverse class of pentameric ligand gated ion channels that are important mediators of fast synaptic transmission in the central and peripheral nervous systems. Nicotinic AChRs are important mediators of cognition, attention, emotion, memory, and addiction. 12

Various combinations of 12 different acetylcholine receptor subunits form the functional pentameric channels of neuronal nAChRs. These subunits ( α2- α10) and

(β2- β4), assemble as either homo ( α7, α8, and α9) or heteropentamers with a ( α)2(β)3 stoichiometry (Egleton, Brown, & Dasgupta, 2009; Fowler, Arends, & Kenny, 2008).

These differential subunit assemblies confer unique pharmacological and physiological properties to each nAChR subtype (Fowler et al., 2008). The α4β2 nAChRs are the most abundant in the CNS while the α7 nAChRs are the second most abundant. These receptors differ considerably in their pharmacological profiles with the α7 nAChR having a high affinity for bungarotoxin (Bgtx) and a low affinity for nicotine while the α4β2 has a high affinity for nicotine and a low affinity for Bgtx.

The α7 nAChR has a higher permeability to calcium, the Ca 2+ : Na + permeability ratio of the α7 nAChR is 10:1 compared to the 4:1 ratio of the α4β2 nAChRs (Gahring &

Rogers, 2005). The highly selective Bgtx affinity of the α7 nAChR has been a driving force of both its physiological study and its ability to be localized anatomically. 125 I-

Bgtx was used as a probe in many previous studies to demonstrate the presence of α7 nAChRs in many brain regions most notably the hippocampus, cortex, and VTA and its relative paucity in the thalamus (Gotti et al., 2007). As well as having different gross anatomical locations the α7 nAChR has different subcellular localizations as well.

In presynaptic locations the α7 subtype influences the release of glutamate,

GABA, norepinephrine, and dopamine (Picciotto & Kenny, 2013; Sharma &

Vijayaraghavan, 2008). Post-synaptic α7 nAChRs mediate excitatory impulses

(Campbell, Fernandes, John, Lozada, & Berg, 2011; Hu, Liu, Chang, & Berg, 2002;

Pandya & Yakel, 2013). The α7 nAChR is a membrane protein, previously thought to only be expressed in the cell membrane, but has subsequently been demonstrated on intracellular mitochondrial membranes as well (Gergalova et al., 2012). These mitochondrial α7 nAChRs are believed to play a role in cell survival and anti- apoptotic signaling (Gergalova et al., 2012). The α7 is present in a wide variety of non-neuronal tissues. In the CNS the α7 nAChR is expressed on astrocytes and microglia (Campbell et al., 2011; Pandya & Yakel, 2013). Studies suggest that the α7 nAChR plays a role in astrocyte-mediated modulation of synaptic activity and microglial mediated anti-inflammatory neuroprotection (Sharma & Vijayaraghavan,

2008). The α7 nAChR is present on a wide variety of other cell types like keratinocytes, epithelial cells, smooth muscle cells, lymphocytes, neutrophils, macrophages, and endothelial cells (Benhammou et al., 2000; De Simone, Ajmone-

Cat, Carnevale, & Minghetti, 2005; Egleton et al., 2009; Maus et al., 1998; Sharma &

Vijayaraghavan, 2001, 2008; Shytle et al., 2004; Wada, Naito, Kenmochi, Tsuneki, &

Sasaoka, 2007; H. Wang et al., 2003; Y. Wang et al., 2001; Zia, Ndoye, Lee, Webber,

& Grando, 2000).

Multiple studies demonstrate that the α7 nAChR has an active role in intracellular signaling in a variety of different tissues and that the majority of signaling is calcium dependent. Many studies indicate the high calcium permeability of the α7 nAChR allows it to participate in calcium signaling pathways (Egleton et al., 2009;

Gahring & Rogers, 2005). The activation of the α7 nAChR can induce calcium induced calcium release (CICR) and activation of calcium dependent second messengers. This confers a metabotropic signaling character to this more traditionally associated ionotropic receptor (Conroy, Liu, Nai, Margiotta, & Berg, 2003; Gahring &

Rogers, 2005). This amplification of intracellular calcium may also mean that relatively few receptors need to be activated to have noticeable biological effects.

Adding to this diversity of function are studies that suggest on macrophages α7 nAChRs do not conduct ions but rather their activation is coupled directly to phospholipase C and calcium mobilization from IP3-sensitive calcium channels

(Suzuki et al., 2006), suggesting that in some cells the α7 nAChR was a purely metabotropic receptor. In both macrophages and microglia α7 nAChR activation leads to decreased release of inflammatory mediators. The α7 nAChR interacts with G protein coupled signaling pathways as well. The activation of the α7 nAChRs in keratinocytes was able to activate small G-protein signaling through the

Ras/Raf/MEK1/ERK1/2 cascade leading to the up-regulation of α2-integrin

(Chernyavsky, Arredondo, Qian, Galitovskiy, & Grando, 2009).

Some of the signaling pathways associated with α7 nAChR activation are related to the physiology of long-term memory formation. Studies in PC12 cells link the activation of the α7 nAChR to ERK phosphorylation. Phosphorylation of ERK1/2 is an important α7 nAChR mediated pathway in neurons where it leads to the phosphorylation of the cyclic AMP–responsive element binding protein (CREB), a transcription factor that affects long-term synaptic changes and long-term memory

(Chernyavsky et al., 2009). Phosphorylated ERK1/2 is increased in hippocampal brain regions following long-term memory consolidation. Rodent studies have shown that the inhibition of ERK prevents long-term memory formation (El Kouhen, Hu,

Anderson, Li, & Gopalakrishnan, 2009). CREB is activated by phosphorylation and leads to gene regulation. Defects in CREB mediated transcription are associated with a congenital form of intellectual disability, Rubinstein-Taybi syndrome (Bito,

Deisseroth, & Tsien, 1996; El Kouhen et al., 2009).

Alpha-7 nAChR dependent signaling is crucial to the nervous system during development. In rodents this is the postnatal period but in humans this corresponds to the third trimester of pregnancy (Damborsky & Winzer-Serhan, 2012). During this period activation of the α7 nAChR leads to changes in gene regulation and leads to the transcription of the mature chloride transporter KCC2 which reverses the intracellular chloride gradient and changes GABAergic activity from excitatory to inhibitory, a critical event in neurodevelopment (Liu, Neff, & Berg, 2006). This α7 dependent change in chloride gradient also results in a cytoskeletal and morphological change in

81 the neuron leading to increased growth of dendritic spines (Liu et al., 2006). Another example of α7 dependent morphological changes is the development and strengthening of glutaminergic synapses during this same period of development

(Lozada et al., 2012). Αlpha-7 nAChR dependent signaling is also important in the processing of amyloid precursor protein (Nie et al., 2010). Activation of α7 with a specific agonist, GTS-21, reduced β-amyloid formation by decreasing gamma secretase activity while leaving α and β secretase unaffected which results in decreased pathological β-amyoid formation and secretion (Nie et al., 2010). These changes in protein expression and regulation linked to α7 nAChR activation indicate that the α7 nAChRs have an active role in intracellular signaling as well as ion conduction.

There may be a structural basis for the α7 nAChR’s activity to participate in intracellular signaling. Each subunit in the receptor pentamer has a long N-terminal extracellular domain and four transmembrane sequences, M1-M4, followed by a short

C-terminus (Picciotto et al., 2001). In nAChRs there is a long cytoplasmic loop of variant length that connects M3 to M4 and is highly divergent in both sequence and length among different nAChR subunits (Picciotto et al., 2001). It is typically 100-150 amino acids in length (Egleton et al., 2009). This loop is a point of intracellular communication; it has multiple phosphorylation sites that can affect the gating and kinetic properties of the channel (Picciotto et al., 2001). Phosphorylation of the muscle nAChR by protein kinase A, protein kinase C, and tyrosine kinases affect channel function and receptor expression (Picciotto et al., 2001). Phosphorylation of the cytoplasmic loop can occur in response to nicotine exposure and may be a key event in

82 nicotine mediated receptor up-regulation (Picciotto et al., 2001). The divergent sequences also specify trafficking and subcellular localization (Egleton et al., 2009;

Kabbani, Woll, Levenson, Lindstrom, & Changeux, 2007; Picciotto et al., 2001).

Although the α7 nAChR is normally localized perisynaptically, replacement of its cytoplasmic loop with the cytoplasmic loop of α3 will target the chimeric receptor to the synapse (Picciotto et al., 2001).

The cytoplasmic loop of the α7 nAChR is believed to be regulated by both phosphatases and kinases, inhibition of calmodulin kinase prevented the rapid rundown that occurs after activation. The rate of rundown is increased with the inhibition of calcineurin (Picciotto et al., 2001). Calcineurin substrates have two consensus sequences, PxIxIT and LxVP. Neither of these is present in the α7 nAChR’s cytoplasmic loop or the rest of the nAChR (H. Li, Rao, & Hogan, 2011; Roy & Cyert,

2009; Rusnak & Mertz, 2000). Analysis of the consensus sequence of PKACa

(rxRRl Slx) yielded limited homology in the cytoplasmic loop of the α7 nAChR

(KPRR CSL) . Structural analysis of the cytoplasmic loop (amino acids 318-469) of the

α7 nAChR was performed using the Eukaryotic Linear Motif Resource for Functional

Sites in Proteins ( http://elm.eu.org ), (Figure 2.1, Table 2.1). The analysis showed interaction with many proteases like N-Argdibasic convertase, furin, NEC1, NEC2, and subtilisin (Fry, O'Regan, Sabir, & Bayliss, 2012; Shen, Peng, Xu, & Zhao, 2013).

There are recognition sites for Cdh1 and cdc20, two proteins involved in interaction with the AP complex and cell cycle dependent protein degradation (Pe'er et al., 2013).

NEC1 and NEC2 are believed to have a role in the cell cycle as well (Fry et al., 2012).

There were sequence sites for multiple interactions with kinases. There were multiple

83 phosphorylation binding motifs and docking sites including a phosphoserine binding motif, a SRC 2 binding motif (SH2), and a motif for MAPK docking. There were multiple sites for phosphorylation by Casein Kinase 1, Casein Kinase 2, Glycogen synthase kinase 3, and two sites for Protein Kinase A.

The signaling pathways of the α7 nAChrR are of considerable therapeutic interest as α7 specific agonists and positive allosteric modulators are being investigated for treatment of schizophrenia, Alzheimer’s disease, neuropathic pain, visceral pain, and inflammation (Callahan, Hutchings, Kille, Chapman, & Terry, 2013;

Campbell et al., 2011; Costa, Motta, Manjavachi, Cola, & Calixto, 2012; Pandya &

Yakel, 2013). The disadvantage of α7 agonists over more targeted agonists is that the desensitization kinetics of the α7 nAChR are rapid which may lead to decreased effectiveness for drugs that are consistently present in the serum. This hypothesis is substantiated by the increased efficacy that PAMs, which decrease receptor desensitization rates, have over the α7 specific agonists in cognitive enhancement studies (Callahan et al., 2013; Costa et al., 2012; Pandya & Yakel, 2013).

Identification of proteins in the signaling pathway could lead to the development of highly targeted pharmacotherapy that is able to avoid the desensitization kinetics and perhaps some of the possible adverse side effects of specific and selective α7 nAChR agonists like increased anxiety.

Advances in biological mass spectrometry and protein identification software create the possibility of identifying numerous interacting partners of the α7 nAChR in an in vivo model. Examining the signaling pathway of the α7 nAChR in whole brain tissue could lend insight into the mechanistic framework of the numerous α7 nAChR

Figure 2.1: The cytoplasmic loop of the α7 nAChR as a proposed site for protein interactions A. The cytoplasmic loop connects M3 and M4 and is a proposed site of multiple protein interactions B. The highlighted areas of the sequence are regions of molecular interaction identified by the Linear Motif Resource for Functional Sites in Proteins. Details of those interactions are featured in Table 2.1.

HHHDPDGG KMPKWTRIILLNWCAWFLRMKRP GEDKVRPACQH KPRRCSL

ASVELSAGAGPPTSNGNLL YIGF RGLEGMH CAPTPDSGV VCGR LACSPTHD

EHLMHGTHPSDGDPDLAKILEEVR YIAN RFRCQDESEVICSEWKFAACVVDR

Table 2.1: Location of molecular interactions on the cytoplasmic loop of the α7 nAChR

Table 2.1 (cont)

87 related pathologies. The availability of α7 knockout mice allows for the acquisition of control tissues for these studies. One primary challenge of studying the in vivo intracellular interacting partners of the α7 nAChR is the isolation of the α7 nAChR from a tissue source. A detergent solubilized membrane preparation would produce a mixture of tens of thousands of proteins of which the α7 nAChR would be a minor component. In order to isolate α 7-nAChRs from the surrounding proteins a specific molecular probe would be necessary to separate these solubilized receptors from the solubilized milieu.

Fortunately, Bgtx is a selective antagonist of the α7 nAChR that is frequently used as a probe to detect surface expression of α7 nAChRs in the brain. Bgtx was the primary tool used to characterize the anatomical distributions of the α7 nAChR though

α-bungarotoxin, although it can also bind to the GABA β3 subunit (Pohanka, 2012)

(Gotti 2006). The α7 subunit’s Loop C (residues 186-197) and loop IV (residues159-

165) confer high affinity Bgtx binding (Marinou & Tzartos, 2003). Since the α7 nAChR is a homopentamer it has five Bgtx binding sites (Marinou & Tzartos, 2003).

The α1 nAChR subunit is present in muscle type nAChRs, a subunit that has high homology to the loop C and loop IV regions of the α7 nAChR. Toxin affinity has previously been used to purify muscle type nAChRs from Torpedo Californica membranes. Α-Cobratoxin was conjugated to solid phase beads and used to isolate muscle type nAChRs from solubilized membrane extracts prepared from the electric organs of Torpedo Californica (Meinen, Lin, Ruegg, & Punga, 2012; Patrick &

Lindstrom, 1973). Previously this solid phase affinity isolation technique was shown to work with Bgtx conjugated sepharose beads in the isolation of α7 nAChRs from

Triton X-100 solubilized membrane extracts prepared from murine WT whole brain tissue (Paulo, Brucker, & Hawrot, 2009). The Bgtx sensitive complexes were eluted from the affinity beads with 1 M Carbamylcholine (carbachol) (K i for α7 nAChR=18-

580 µM ), a nonspecific, low molecular weight cholinergic agonist and their presence in the eluent was confirmed by Western Blot for α7 nAChR (Anand, Peng, Ballesta, &

Lindstrom, 1993; Gopalakrishnan et al., 1995; Paulo et al., 2009; Quik, Choremis,

Komourian, Lukas, & Puchacz, 1996).

The focus of the experiments in this section of the dissertation serve as a follow up study to the initial α7 nAChR interactome characterization conducted by

Paulo et al. which was the first proteomic study of the interacting partners of the α7 nAChR. As mentioned previously, Paulo et al. employed Bgtx conjugated affinity beads to isolate α7 nAChR complexes from detergent (Triton X-100) solubilized membrane extracts prepared from the whole brain tissue of wild type and α7 nAChR knockout (KO) mice. The Bgtx sensitive complexes isolated from the WT and KO samples were released from the affinity beads by elution with 1M carbachol. The recovered proteins were fractionated by SDS-PAGE and digested in-gel with trypsin before the peptides were analyzed by mass spectrometry and database search. The result of that study was the identification of 55 proteins that were seen in one or more of 3 WT datasets, but never in the three KO datasets. These proteins were organized into several different classes: basic metabolism, cellular structure, protein chaperoning, proteolytic pathway and signal transduction.

The 55 potential α7 nAChR interacting proteins exist as a list of potential protein interacting partners for the α7 nAChR that require further analysis to

89 determine the validity and the nature of their association. These proteins were identified by the SEQUEST algorithm and through protein database searching. The

SEQUEST algorithm makes peptide identifications based on degree of correlation to theoretical predicted specta created from proteins in a database, these proteins were never identified or confirmed through direct biochemical study and thus cannot labeled as definite interacting partners. The studies in this chapter focus on repeating the isolation of Bgtx sensitive complexes from DSME prepared from the whole brain tissues of WT and α7 nAChR KO mice to produce an additional WT and KO dataset to test the reproducibility of the data base driven identifications featured in Paulo et al.

Combining this new dataset with the three previously produced allowed for demonstration of the limited reproducibility of these proteomic identifications with 11 of the 55 proteins being identified as being present in WT datasets but absent from KO datasets. Eleven of these reproducibly identified, potential α7 nAChR interacting partners appeared in 3 of the 4 WT datasets but never in a KO dataset. There were 26 proteins identified in this WT dataset that had previously only been seen in one WT dataset but also never in a KO dataset. These proteins were not able to be published in the list of potential α7 nAChR interacting partners previously but now that they identification has been reproduced they satisfy the criteria to be included on the list of

“potential interacting α7 nAChR partners.” Of the original 55 proteins, 6 were eliminated as potential α7 nAChR interacting partners because they were detected in the new KO dataset. Several proteins identified in WT datasets but not in the KO datasets were selected for further biochemical analysis by Western Blot. These included: GAP-43, NMDA zeta , Guanine nucleotide-binding protein G(I)/G(S)/G(T)

90 subunit β-3 (GNB3), and GLUR2 (an AMPA receptor subunit). None of these proteins were identified as being present in Bgtx sensitive isolates from WT samples and were absent in KO mice. Interestingly, although the result of the Western blot demonstrated that GNB3 was not observed in the Bgtx isolates from either WT or KO mice, it did indicate a link between GNB3 and α7 nAChR. The abundance of GNB3 was diminished in DSME from KO mice compared to WT mice. This suggests an undefined but present relationship between α7 nAChRs and GNB3.

Materials

DSME preparation

Triton X-100 (807426) was purchased from MP Biomedicals (Solon, OH).

Complete, Mini protease inhibitor cocktail was purchased from Roche (Mannheim,

Germany). Cyanogen bromide (CNBr) activated sepharose beads (C9142), carbachol

(C4382), α-bungarotoxin (T3019) and general chemicals for buffers were purchased from Sigma-Aldrich (St. Louis, MO). The water used in making all solutions and used in all experimentation Millipore ultrafiltered, protein free water.

Western Blotting

Rabbit anti-α7 nAChR antibody (ab10096) was purchased from Abcam

(Cambridge, MA). Horseradish peroxidase-conjugated rabbit anti goat (sc-2774), goat anti-NMDA zeta (sc-31557), goat anti-GAP43 (sc-7458), rabbit anti-GNB3 (sc-381), and goat anti-GLUR2 (sc-7611) were purchased from Santa Cruz Biotechnology

(Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-rabbit antibody

(A9169) was purchased from, Sigma-Aldrich (St. Louis, MO). CL-XPosure Film

(34090) and Supersignal West Pico Chemiluminescence substrate (34080) were

91 purchased from Pierce (Rockford, IL). MagicMark XP (LC5602) and positive control protein standards were purchased from Invitrogen (Carlsbad, CA).

Proteomics

Trypsin Gold, mass spectrometry grade (V5280) was purchased from the

Promega Corporation (Madison, WI). BenchMark Pre-Stained ladder (10748-010) and

Brilliant Blue G-Colloidal Coomassie stain (B2025) was purchased from Sigma-

Aldrich (St. Louis, MO). Precast gels (NuPAGE Novex, 4-12% Bis-Tris Gels, 10 wells, 1 mm thickness) (NPO321BOX) were purchased from Invitrogen (Carlsbad,

CA).

Production of α-Bungarotoxin Conjugated Affinity Beads

The production of ligand conjugated affinity beads was used previously to isolate nAChR subunits from homogenized tissues (Meinen et al., 2012; Patrick &

Lindstrom, 1973; Paulo et al., 2009). The production of the affinity beads began with hydrating 1.5 grams of CNBr-activated sepharose 4B beads in 5 mL of 1 mM HCl for

30 minutes in a fume hood. The beads were washed on a coarse, glass filter with 500 mL of 1 mM HCL with vacuum assistance. After the washing was complete the dried beads were scraped into a 50 mL Falcon tube, then were resuspended in 7.5 mL of coupling buffer (0.25 M NaHCO3, 0.5 M NaCl, pH 8.3) and centrifuged for 5 minutes at 1,500 × g. The beads were pelleted and resuspended in 15 mL of coupling buffer that received the addition of 3 mg of Bgtx. The Bgtx and the beads underwent gentle agitation for 12-16 hours at 4 o C.

After this incubation period, the beads were pelleted by centrifugation for 5 minutes at 1,500 × g and resuspended in 15 mL of a glycine blocking solution (0.2 M glycine in 80% coupling buffer) for the purpose of conjugating glycine to sites on the

92 beads not occupied by Bgtx. The glycine blocking solution was incubated with the beads for 12-16 hours at 4 o C with gentle agitation. After the incubation with the blocking solution, the beads were centrifuged at 1,500 x g and underwent several washing steps on a coarse glass filter with vacuum assistance. The beads were washed with 100 mL of Buffer A (0.1 M NaHCO3, 0.5 M NaCl, pH 8.0), followed by another wash with 100 mL of Buffer B (0.1 M NaCH3CO2, 0.5 M NaCl, pH 4.0), then again with 100 mL of Buffer A, followed by 100 mL of coupling buffer. The beads received two more 100 mL washes with Tris-buffered saline (TBS, 50 mM Tris, 150 mM NaCl, pH 7.4-7.6). After the washing steps were completed the beads were stored in TBS with 0.1% TritonX-100.

Preparation of Detergent Solubilized Membrane Extracts (DSME)

Mice were sacrificed. Their whole brain tissues were extracted and stored in

1.5 mL Eppendorf tubes before being frozen on dry ice and stored at -80 o C until used for membrane preparation. The average mouse brain weight was 400 mg. Four mouse brains were used per biological replicate.

The frozen whole brain tissue was thawed at room temperature and homogenized in ice-cold Homogenization buffer (TBS supplemented with protease inhibitors (TBSp)) at a ratio of 2 mL homogenization buffer per 400 mg of tissue. The homogenization was performed with 20-25 strokes of a Potter-Elvehjem glass homogenizer. After the homogenization was complete, the soluble portion was separated from the membrane-containing portion by centrifugation at 100,000 × g for

60 minutes using an ultracentrifuge at 4° C. After centrifugation the soluble proteins in the supernatant were decanted and the membrane-containing pellet was resuspended in ice-cold solubilization buffer (TBSp with addition of Triton X-100 to make a 1% 93

Triton X-100 solution). The ratio used is 1mL solubilization buffer per 400 mg of tissue. The membrane-containing pellet was once again homogenized with 20-25 strokes of a Potter-Elvehjem glass homogenizer and incubated on ice with gentle agitation for 2 hours. During this time membranes were solubilized by the detergent.

The suspension was centrifuged again at 100,000 × g for 60 minutes using an ultracentrifuge at 4° C. After centrifugation, the supernatant (detergent solubilized membrane extract (DSME)) was retained; the pellet (detergent insoluble portion of the membrane) was discarded. Isolation of Bgtx sensitive complexes was performed by incubating the DSME with Bgtx conjugated affinity beads.

Isolation of Bgtx Sensitive Complexes with Bgtx Conjugated Affinity Beads

The volume of DSME used per biological replicate was 3 mL. One hundred microliters of the Bgtx conjugated bead slurry was added to the DSME. The suspension incubated 14-16 hours at 4 o C with gentle agitation to keep the beads circulating through the solution. After the incubation period ended the beads were centrifuged at 2,000 × g for 5 minutes and then washed 3 times with 1.5 mL of ice cold solubilization buffer to reduce the amount of proteins binding to the beads in nonspecific fashion. After the washing steps were complete, the beads were incubated with 50 µL of a 1 M carbachol dissolved in solubilization buffer. The proteins were then precipitated from solution with acetone before being fractionated by SDS-PAGE.

Four times the sample volume of chilled (-20° C) acetone was added to the samples.

After the addition of the acetone, the samples were mixed by vortexing and incubated for 1 hour at -20° C before the precipitate was pelleted by centrifugation at 13,000 x g for 10 minutes. The supernatant was aspirated and the pellet was allowed to air dry.

Fractionation with SDS-PAGE

The protein pellet was resuspended in 10µL of SDS-loading buffer (100 mM

Tris-HCl, pH 8, 2% SDS, 100 mM DTT, 20% glycerol, 0.002% bromophenol blue) and heated at 55° C for 30 minutes. Samples were loaded into 15% polyacrylamide gels and SDS-PAGE was run at 125 Volts for approximately 30 minutes.

Protein Visualization and In-Gel Tryptic Digestion

After gel electrophoresis was complete the gels were fixed in a solution of 50% methanol and 10% glacial acetic acid for 1 hour before overnight staining with colloidal coomassie. The gels were destained by two washes with destaining solution

(40% methanol and 10% glacial acetic acid) for 30 minutes with agitation. After the proteins were visualized with stain, they were excised with a razor blade in pieces no larger than 1 mm before being washed several times to remove detergent and other impurities, then digested with trypsin “in-gel”. Solution was added after each washing.

The slices were incubated at 37 o C for 15 minutes. The solution was aspirated. The slices were washed twice with a washing solution (50 mM ammonium bicarbonate in

50% water/ 50% acetonitrile) before being completely dehydrated with 100% acetonitrile, then \completely rehydrated in a digestion buffer (50 mM ammonium bicarbonate). This cycle of washing, dehydration, rehydration was repeated twice before the slices were completely dehydrated in 100% acetonitrile. The acetonitrile was aspirated; the gel slices were dried by speed vacuum before 100 µg of trypsin in digestion buffer was added per slice for in-gel digestion to occur at 37 o C for 24 hours.

After the gel slices were digested, peptides were extracted by two sequential elution steps. The first elution was performed with a 1% formic acid solution; the second was performed with a 1% formic acid solution in 50% Millipore filtered water

95 and 50% acetonitrile. The peptide-containing liquid was reduced completely by drying with a vacuum concentrator and then resuspended in 100 µL of a 0.1 % acetic acid solution before being analyzed by mass spectrometry.

Mass Spectrometry

Mass spectrometry was performed with nano scale HPLC coupled to ESI-

LTQ-Orbitrap instrumentation at the Taplin Biological Mass Spectrometry Facility at

Harvard Medical School (Boston, MA). The samples were reconstituted in 10 µL of

HPLC solvent A (2.5% acetonitrile, 0.1% formic acid). A nanoscale reversed phase

HPLC capillary was prepared with 5 µm C18 spherical silica beads packed into a fused silica capillary (100 µm inner diameter and approximately 12 cm in length) with a flamedrawn tip. After equilibrating the column, the sample was loaded via a Famos autosampler (LC Packings, San Francisco, CA). A gradient was formed and peptides were eluted with increasing concentrations of solvent B (97.5% acetonitrile, 0.1% formic acid). The eluted peptides were ionized by ESI before being analyzed by the mass spectrometry instrument. The scanning done by the mass spectrometry was data- dependent with 1 MS scan followed by 5 MS/MS scans. MS/MS spectra were generated and automatically searched against the mouse NCBI nonredundant protein database using the SEQUEST algorithm provided with BioWorks 3.2 SR

(ThermoFinnigan, San Jose, CA). The list of predicted proteins was imported into a custom-made FileMaker Pro (FileMaker, Santa Clara, CA) relational database for data analysis. The proteins in the dataset represent Bgtx isolated proteins from one WT and one α7 knockout sample.

Data Processing

Peak list files were created by the program “extract_msn.exe” that was installed with BioWorks version 3.2 SR using the following parameters: the mass must fall within the range of 600 to 4500 Daltons and the minimum total ion current for the scan must exceed 1000. The total ion current is a chromatographic measurement that corresponds to the abundance of peptides being scanned. A value below the threshold of 1000 would indicate too few ions to analyze by MS/MS. The precursor tolerance for grouping was +/- 1.5 Da with no differing intermediate scans allowed and only a single scan required to create a peak file. The minimum signal-to- noise for a peak to be written to the peak file was 3; 25 such peaks must be found for a peak file to be created. The program calculated charge states of the peptide; however, in cases of ambiguity, peak files for both the +2 and +3 charge states were created. No smoothing or deisotoping was performed. The precursor-ion tolerance was 2.0 Da and the fragment-ion tolerance was 0.8 Da. The possible modifications allowed were iodoacetamide alkylation of cysteine and oxidation of methionine. Enzymatic digestion was specified as trypsin with up to 2 missed cleavages allowed. Searches were performed using the NCBI nonredundant mouse database. A list of reversed- sequences of proteins in this database was created from these entries and appended to the original database for searching so that false positive rates could be estimated. Final false positive rates for peptide identity assignment were estimated at approximately

1%. Each peptide identified had a list of the ten best peptide matches that were ranked according to the correlation score X corr and the difference between X corr scores, the

∆CN. Xcorr scores were used as a basis for screening spectra for validity. Mass tolerance parameters were based on the charge of the peptide. Peptides with charge of 97

+1, +2, or +3 required respective X corr values greater than 1.5 and, 2.0, and 2.5 to be considered valid. Peptides with charge of +1, +2, or +3 required respective ∆CN values greater than 0.28, 0.30, and 0.37

Western Blotting

Western Blotting was performed to identify the following protein targets: α7- nAChR (ab23832), GAP-43 (neuromodulin) (sc-7458), NMDA zeta (sc-31557),

GNB3 (sc-381), and GLUR2 (sc-7611). For every respective western blot, after proteins were fractionated by SDS-PAGE they were transferred from the polyacrylamide gels onto a nitrocellulose membrane at 100 V for 100 minutes at 4° C in electroblotting buffer (25 mM Tris-HCl, 0.2 M glycine, 20% methanol). After the transfer, the proteins were blocked for 1 hour at room temperature in a solution of

TBS-T (TBS supplemented with Tween-20 to a 0.1% solution) for 1 hour at room temperature with agitation. After the blocking step the primary antibody was diluted in

TBS (1:500 dilution) and incubated at 4 o C with the blocked membrane overnight (14-

16 hours) with agitation. After the primary incubation was complete the membrane received two washes in TBS-T for 5 minutes before being probed with the appropriate secondary antibody at a 1:20,000 dilution in TBS. All secondary antibodies were conjugated with horseradish peroxidase (goat anti-rabbit antibody (A9169), rabbit anti goat (sc-2774)). The secondary antibodies incubated with the membrane at room temperature with agitation for 1 hour before receiving 2 more 5 minute washes; one in

TBS followed by another one in TBS-T. After the washing steps were complete the membrane was incubated with a chemiluminescence substrate for 5 minutes. The membrane was picked up with tweezers to allow the substrate to run off of it before being placed in between a Staples brand, plastic sheet protector (Item: 40713 Model: 98

105240). The membrane was exposed to X-ray film (CL-XPosure Film (34090)) for

30 seconds before the film was developed.

Results

Mass Spectrometry Studies

In our previous study of the α7 nAChR interactome, (Paulo et al., 2009), there were three datasets for Bgtx sensitive proteins isolated from the DSME of WT mice and 3 datasets for Bgtx sensitive proteins identified in α7 nAChR knockout mice. Two of the 3 datasets were analyzed by LTQ instrumentation while 1 of the 3 was analyzed by LTQ-Orbitrap. The Orbitrap assisted LTQ has the advantage of higher mass accuracy and resolution than LTQ alone and can produce more numerous and confident peptide identifications. The identifications are weighted equally. One peptide identification in the LTQ analyzed data is considered as valid as the identifications made through analysis of LTQ-Orbitrap spectra. Paulo and colleagues identified 55 proteins in 2 of 3 WT datasets and never in a KO dataset (Table 2.2).

This current experiment added an additional dataset to each category to determine the reproducibility of protein identifications, add validity to those that appear consistently, and eliminate previously identified potential interacting partners that appear in the new

KO dataset. There are no standardized criteria for the number of peptide identifications a protein must have to be a “confirmed” member of a dataset. Single peptide identifications are sufficient for membership. Although statistically, regardless of instrumentation used, there is a 99% probability that the peptide was correctly identified given the estimated 1% false positive rate. Being considered a member of a dataset implies a less rigorous association with the α7 nAChR than a status of

“interacting partner” which would require biochemical validation. The data generated 99 from this study identifies a list of potential interacting partners that can be further investigated with more focused studies with the proteins to satisfy the most rigorous inclusion criteria as the most likely candidates.

The goal of these proteomic studies was to establish consistency of identifications over the 4 datasets. None of the previously published “potential α7 nAChR interacting proteins” satisfied the maximum consistency requirements of appearing in every WT data set and none of the KO datasets. There were 11 proteins that appeared in 3 out of 4 wild type data sets that were never seen in a knockout data set (Table 2.3). There were 26 proteins that were seen only once in a previous wildtype data set that was identified in the new wildtype data set. These proteins never appeared in any KO data set (Table 2.4). There were 6 proteins that satisfied inclusion criteria in Paulo et al. that appeared in the new KO dataset that eliminates them from the list of potential α7-nAChR interacting partners (Table 2.5).

The 11 most consistent potential α7 nAChR interacting proteins were: Protein disulfide isomerase A2 , Endophilin-A2 , Gamma-soluble NSF attachment protein ,

Isoform Long of 14-3-3 protein β/α, Contactin 1 , Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit β-3, Transgelin-3, Isoform 1 of Gelsolin , Isoform

1 of Tropomodulin-2, Isoform 1 of Protein SET , and Isoform 2 of

Serine/threonine-protein phosphatase 2B catalytic subunit β isoform. The data for this study were gathered in 2010; the data from the previous studies were gathered in

2007. Changes to the database likely occurred in that timeframe. As a result there were

5 proteins that were identified as isoforms that did not have that designation in our previous study: Isoform Long of 14-3-3 protein β/α, Isoform 1 of Gelsolin, Isoform

100

1 of Tropomodulin-2 Isoform 2 of Serine/threonine-protein phosphatase 2B catalytic subunit β isoform. The sequence of the peptide identifications corresponding to these isoforms was searched against the sequence of the original entity. The identified peptides were found in both sequences so the two different proteins were ruled to be equivocal. They were included in the list as a result. This isoform investigation was done for every category.

The 26 proteins identified in this follow-up study represent a list of “newly considered potential nAChR” interacting partners as they satisfy the dual dataset identification requirement established in Paulo et al. and have never been included in a

KO dataset. One of the interesting aspects of this list of proteins is the previous instrumentation that acquired their spectra. All of the proteins identified in the most recent series of datasets were analyzed by LTQ-Orbitrap instrumentation. Of the 26 total proteins in this list 18 had were previously identified in the LTQ-Orbitrap data set from Paulo et al. while 8 proteins were previously identified in either of the two previously acquired LTQ datasets which raises the possibility of instrumentation bias in the proteomic analysis of peptides. The proteins previously identified through LTQ-

Oribitrap instrumentation were: Isoform 1 of Dihydrolipoyllysine-residue succinyltransferase component of 2-oxoglutarate dehydrogenase complex, mitochondrial, Isoform Mitochondrial of Fumarate hydratase, mitochondrial

Pyruvate dehydrogenase E1 component subunit α, somatic form, mitochondrial

Isoform Glt-1A of Excitatory amino acid transporter 2, Elongation factor 1-α 1,

Isoform 1 of Paralemmin, Thy-1 membrane glycoprotein, Isoform HSP105-α of

Heat shock protein 105 kDa, Α-internexin, Casein kinase II subunit α, Excitatory

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Table 2.2: Proteins from Wild-Type Mouse Brain Tissue (Paulo et al., 2009) Identified by Nano-ESI of Peptides Originating from In-Gel Digested Proteins Isolated by Selective Elution from Bgtx Conjugated Affinity Beads .

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Table 2.3: Proteins seen in 3 of 4 WT datasets and never in a KO dataset

Frequency Uniport Protein Name of Peptide Accession Idenification Number

Contactin 1 31 P12960

Isoform 1 of Tropomodulin-2 30 Q9JKK7-1

Transgelin-3 6 Q9R1Q8 Guanine nucleotide-binding protein G(I)/G(S)/G(T) 4 Q61011 subunit beta-3

Endophilin-A2 3 Q62419

Isoform Long of 14-3-3 protein beta/alpha 3 Q9CQV8-1

Protein disulfide isomerase A2 2 P27773

Isoform 1 of Gelsolin 2 P13020-1 Isoform 2 of serine/threonine-protein phosphatase 2 P48453-1 2B catalytic subunit beta isoform

Gamma-soluble NSF attachment protein 1 Q9CWZ7

Isoform 1 of Protein SET 1 Q9EQU5-1

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Table 2.4: Proteins previously identified in at least one WT dataset that were also identified in the current WT dataset but not in a KO dataset.

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Table 2.5: Proteins Ruled out of Interactome by Appearing in New KO Dataset

105 amino acid transporter 1, Isoform 1 of Putative tyrosine-protein phosphatase auxilin, Synaptic vesicle glycoprotein 2A, Synaptophysin, F-box only protein 2,

Actin-related protein 2/3 complex subunit 5-like protein, Neuronal growth regulator 1, and Isoform Long of Clathrin coat assembly protein AP180. While the 8 proteins previously identified through LTQ instrumentation were Isoform 1 of

Glutamate receptor 2, Isoform 2 of Gamma-adducin, Isoform Long of Clathrin coat assembly protein AP180, Rho GDP-dissociation inhibitor 1, Voltage- dependent anion channel 3, Mitogen-activated protein kinase 1, Nucleosome assembly protein 1-like 1, and Isoform A of Drebrin .

The 6 proteins that previously satisfied inclusion criteria in Paulo et al. but were identified in this more recent are 14-3-3 eta, 14-3-3 sigma, L-lactate dehydrogenase A chain, BASP, Syntaxin 1A, Syntaxin 1B . This represents an elimination of more than 10% of the previous list of potential α7 nAChR interacting proteins in Paulo et al., reflective of the labile nature of proteomic data. Most of the identifications for “ruled out” proteins on this list were previously by 1 peptide identifications which could mean that they were misidentified in the original study.

The “ruled out” terminology is excessively pejorative because a misidentification could result in a false negative as easily as a false positive. Identifying members of an interactome in this fashion is stochastic. While these proteins are “ruled out” in this artificial construct generated for determining interactome components, they should not be completely eliminated from consideration. The knockout model is missing the α7 nAChR, but the rest of the proteome is intact and the possibility exists that they interact specifically with the α7 but can also be isolated in nonspecific fashion with the

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Bgtx conjugated beads. There could also be a change in the quantity of isolation in both of these situations where more of a protein is seen in specific isolation than nonspecific isolation. This cannot be directly answered by qualitative proteomic studies. A quantitative proteomic study might give more information on these types of interactions. The presence of these proteins in KO datasets should not prevent their study if other literature has cited possible physiological links that are determined through biochemical means.

Western Blot Studies

Following the previous proteomic investigations several protein targets were selected for investigation by secondary biochemical means. The α7 nAChR was the first protein to be probed for by Western blot. This protein was detected in Paulo et al. as being isolated from DSME prepared from whole brain tissue by Bgtx conjugated beads and was eluted from them by elution with 1M carbachol, Figure 2.2. The ability to isolate the α7 nAChR in this fashion validates the core mechanistic principles of this study.

Peptides from GNB3 were identified in 3 out of the 4 WT datasets but never in a KO dataset, which makes GNB3 one of the most frequently identified potential α7 nAChR interacting proteins and a high priority target for secondary biochemical investigation. The Western blot, Figure 2.3, did not demonstrate GNB3 in Bgtx isolated proteins from the DSME of WT or KO mice. It did demonstrate reduced abundance in the pure DSME prepared from the whole brain tissue of KO mice compared to DSME from WT mice with 20 µg of protein being loaded per lane.

Although the protein was not able to be isolated with Bgtx affinity the reduced basal levels in the KO mouse indicate that there is a functional relationship between the α7 107 nAChR and GNB3. The relationship between these two proteins has never been documented in literature previously.

GAP- 43 (neuromodulin) was identified in 2 of the 4 WT datasets and never in any KO dataset. The Western blot, Figure 2.4, demonstrated that GAP-43 was not able to be isolated through Bgtx affinity. However, like GNB3, with equal loading amounts of 20 µg of protein per lane the quantity of GAP-43 seen in DSME from KO brain tissue is less than that in DSME prepared from WT mice. The difference in abundance is visible but not to the same extent as in the case of GN B3.

NMDA zeta was detected only in the most recent LTQ-Orbitrap acquired WT dataset and has never appeared in a KO dataset. Compared to more frequently identified proteins, NMDA zeta was a low probability target, but numerous functional associations between the NMDA receptor and the α7 nAChR have been previously reported. Chronic nicotine exposure promoted complex formation between the α7 nAChR and the NMDA (S. Li, Li, Pei, Le, & Liu, 2012). The formation of this complex was increased by the activation of the α7 nAChR. Its activity was able to increase activity of ERK and cue induced reinstatement of nicotine exposure in rat models (S. Li et al., 2012). During development activation of the α7 nAChR increases the formation of glutaminergic synapses (Lozada et al., 2012). Western blot, Figure

2.5, demonstrated that NMDA zeta were able to be detected in the Bgtx isolated proteins from the DSME of both WT and KO mice. This would indicate that it might bind to the beads in nonspecific fashion.

SME was prepared from α7 KO and WT brains and then incubated with Bgtx affinity beads and then probed with N-19 at a dilution of 1:500, and with the

108 appropriate secondary diluted to 1: 20,000. Signal was seen in Bgtx isolates from both the WT and KO mice indicating that GLUR2 could be isolated with Bgtx in a non α7 nAChr dependent manner and was unlikely a direct member of the α7 nAChR interactome.

GLUR2 was identified in 2 of the 4 WT datasets and never in a KO dataset.

Western blot demonstrated that GLUR2 was detectable in Bgtx isolated proteins from

DSME prepared from the whole brain tissue of both WT and KO mice. GLUR2 was not seen in samples of WT whole brain DSME that were incubated with glycine conjugated beads indicating that it does have some specificity to binding Bgtx. Most of the binding studies with Bgtx have been done on receptors expressed on cell surfaces. Once the membranes have been solubilized with detergent, there is a possibility that secondary, high affinity Bgtx epitopes are exposed. This result does not give conclusive evidence about the status of GLUR2 as α7 nAChR interacting protein. Much like the NMDA receptor, there is significant evidence that GLUR2 is functionally related to the α7 nAChR (Tanaka, Ohashi, Moue, & Kobayashi, 2012).

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Figure 2.2: Western Blot Probing for the α7 nAChR with antibody ab23832 A. In this Western blot the α7-nAChR is successfully identified at its target molecular weight of 60 kDa. B. Coomassie stained SDS-PAGE of Bgtx isolates from WT and KO DSME, the arrow signifies the gel band believed to correspond to the α7-nAChR, it is absent in the KO lane.

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Figure 2.3: Western Blot Probing for GNB3 GNB3 is not present in Bgtx isolates and is less abundant in α7 knockout DSME.

kDa

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Figure 2.4: Western Blot Probing for GAP- 43 (neuromodulin) and NMDA zeta from the DSME of WT or KO mice A. Probing for neuromodulin. B. Probing for NMDA zeta

kDa

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Figure 2.5: Western Blot Probing for GLUR2 Signal for GLUR2 is present in Bgtx isolates from both KO and WT mice indicating that it is isolated with Bgtx affinity as it is not present in unconjugated bead isolates incubated with wild type DSME. The Bgtx based isolation is α7 nAChR independent.

kDa

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Discussion

Although the α7 nAChR is the focus of the affinity isolation and Western blot confirmed isolation, it has not been detected in any of the 4 WT datasets. It is unusual not to detect peptides resulting from the target of protein enrichment. The possibility exists for numerous detection biases in this case. One possibility behind the absence of

α7 nAChR peptides is that because the α7 nAChR is a membrane protein with 4 transmembrane domains its tryptic digestion would produce numerous hydrophobic peptides that would have a harder time ionizing than more basic peptides and thus being detected by the mass spectrometry instrument. Those same hydrophobic domains may make the α7 nAChR difficult to degrade with trypsin and result in the generation of fewer peptides for analysis. Also reverse phase columns are used to fractionate the peptides before their ionization by electrospray, so there is a possibility that these peptides are getting retained on the column. Because the α7 nAChR subunit has so many hydrophobic domains it may be difficult to degrade with trypsin and thus result in the generation of fewer peptides. As seen in Paulo et al., the only time α7 nAChR peptide was confidently identified was by post source decay done with

MALDI-TOF on in-gel chymotryptic digests. MALDI ionization is independent of the hydrophobicity of the peptide and chymotrypsin is more tolerant of hydrophobic domains in a protein than trypsin is which would serve to validate these hypotheses to some degree.

These studies serve to emphasize the role of proteomic data as a guide and corroborating evidence rather than definitive conclusions. GNB3 had never previously been thought to have a relationship with the α7 nACHR, but it appeared in 3 out of 4

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WT datasets and none of the KO datasets. The proteomic data indicated that there might be a functional relationship between GNB3 and the α7 nAChR also demonstrated by Western blot. Although GNB3 was not present in the Bgtx isolated proteins from whole brain WT DSME, it was markedly reduced in the KO whole brain

DSME. A connection between the α7 nAChR and GNB3 might be related to vascular physiology. As well as being expressed in the nervous system the α7 nAChR is also expressed in the vascular system (Egleton et al., 2009). KO mice demonstrate a decreased baroreceptor reflex that manifests in maintenance of a normal heart rate in response to vasodilation (Picciotto et al., 2001). The typical response evoked by a decrease in blood pressure is a compensatory increase in heart rate. This could represent a role for the α7 nAChR in carrying out the signaling of the sympathetic nervous system. A gain of function mutation in GNB3 (GNB3825T) seen in humans is associated with primary hypertension (hypertension without known etiology) and is the subject of numerous epidemiological studies. This mutation is also associated with tachycardia (Bae et al., 2007; Kabadou et al., 2013; Siffert, 2003). These physiological relationships combined with the data from the Western blot may indicate a solid physiological connection between the α7 nAChR and GNB3 and certainly warrants future studies to uncover the degree of their connection.

Neuromodulin was identified in two WT datasets and never in a KO dataset but western blot failed to confirm. The sensitivity of LTQ-Orbitrap mass spectrometry can be subfemtomolar; chemiluminescence mediated detection by western blot is typically picograms and can be as low as mid femtograms. Mass spectrometry has a slight edge in sensitivity. There is some possibility that Neuromodulin would be

115 detected by mass spectrometry but not visualized in a western blot. The proteomic data cannot be used to make a definitive conclusion about neuromodulin’s status as α7 nAChR interacting protein, but it cannot be entirely excluded either.

The Western blots for both the NMDA zeta and GLUR2 were both inconclusive because the proteins were detected in Bgtx isolates from the WT and the

KO. There was not a noticeable difference in quantity in both samples. The NMDA zeta was only detected in 1 WT dataset and the GLUR2 was detected in 2, There are many corroborating studies that indicate a relationship between both of these proteins and the α7 nAChR. The studies by Li et al. suggest a formation of a physical complex between the α7 and NMDA receptors (S. Li et al., 2012). Another study by Tanaka et al. indicated that α7 nAChRs can affect the transcription of Glutamate receptor 2

(GluR2) at the mRNA level (Tanaka et al., 2012). Nicotine treatments in cell culture produced transient increases in GluR2 mRNA that could be inhibited by co administration of Bgtx with nicotine (Tanaka et al., 2012). Further investigation determined that activation of the α7 nAChR in turn activates the calcium dependent

PI3K/Akt pathway that releases a DNA/RNA binding protein from GluR2 mRNA, allowing the protein to be translated (Tanaka et al., 2012). The α7 nAChR activation is heavily associated with the AKT pathway and calcium dependent signaling (Gahring

& Rogers, 2005; Martin, de Fiebre, & de Fiebre, 2004). The previous studies indicate a high likelihood that the α7 nAChR and these proteins are involved in the same interaction network even though the Western blot may indicate that the interaction is nonspecific.

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Many of the proteins in this study have previously suggested relationships with the α7 nAChR and structural motifs on the cytoplasmic loop. Casein kinase 2 subunit-

α is the catalytic domain of the kinase (Jahn, Nastainczyk, Rohrkasten, Schneider, &

Hofmann, 1988). Structural analysis of the cytoplasmic loop of the α7 nAChR demonstrated 2 phosohorylation sites, one for casein kinase 1 and another for casein kinase 2. Synaptophysin is a glycoprotein associated with vesicles and is frequently implicated and used as a marker in studies of Alzheimer’s disease models, a pathology strongly linked to the α7-nAChR (Unger, Svedberg, Schutte, Bednar, & Nordberg,

2005).

Mitogen-activated protein kinase 1, also known as ERK2 has been linked to the α7 nAChR through both inflammatory pathways and calcium signaling pathways due to the mutual interaction between both proteins and protein kinase C. In the prefrontal cortex the α7 nAChR has been shown to promote the phosphorylation of

ERK2 (Dickinson, Kew, & Wonnacott, 2008). In addition a MAPK docking motif appears on the cytoplasmic loop of the α7 nAChR.

Isoform A of Drebrin is involved in cytoskeletal interaction in the neuron and is essential in synaptic plasticity and axonal growth. The α7 nAChR is involved with multiple structural proteins as well as neurite outgrowth. An interaction between these two proteins is possibile (Mizui et al., 2009). Protein disulfide isomerase A2 is involved, as its name implies, with the formation of disulfide bonds. It may play a role in the assembly of receptor subunits or the formation of disulfide bonds to link another protein to the α7-nAChR. Endophilin-A2 has never been directly linked to either nicotine or the α7 nAChR, but it is strongly associated with dynamin 2, shown to

117 associate with the α7 nAChR. Endophilin associates with Dynamin 2 and is an essential protein in the process of endocytosis (Ross et al., 2011).

Contactin 1 is a membrane protein found in neurons that is related to cell adhesion (Dunckley & Lukas, 2006). Contactin has previously associated links with the α7 nAChR. In nicotine studies involving SHY5Y cells, the expression of contactin was altered by chronic nicotine exposure (Dunckley & Lukas, 2006). Since the

SHY5Y cells express the α7 nAChR this strengthens the likelihood that there is a real connection between the two proteins. Transgelin-3 is a cytoskeletetal protein strongly linked to Alzheimer’s disease and the hyper phosphorylation of Tau (Feuillette et al.,

2010). Alzheimer’s disease is strongly linked to the α7 nAChR. The protective role the

α7 nAChR is believed to play in reducing β-amyloid production. β-amyloid has been shown to activate the α7 nAChR and lead to the hyper phosphorylation of tau via

MAPK activation (Sharma & Vijayaraghavan, 2008).

Isoform 1 of Gelsolin has never been directly linked to the α7 nAChR but the two do share a link through pathology. The gelsolin gene is believed to be a candidate gene for schizophrenia a disease to which the α7 nAChR is strongly associated and is believed to play a role in the auditory gating deficit (Sharma & Vijayaraghavan, 2008;

Xi et al., 2004). Schizophenic mouse models have lower levels of α7 expressions in their brains (Callahan et al., 2013; Picciotto et al., 2001).

Isoform 2 of Serine/threonine-protein phosphatase 2B catalytic subunit β isoform (PP2B) has not been directly linked to the α7 nAchR, but there is some evidence that the two proteins may be negative regulators of each other in regards to the establishment of long-term potention, learning and memory. The α7 nAChR is

118 thought to help promote learning and memory by leading to CREB phosphorylation through the activity of ERK1/2. Isoform 2 of Serine/threonine-protein phosphatase 2B interferes with CREB phosphorylation and long-term depression. It is possible that the two are modulators of one another (Mauna, Miyamae, Pulli, & Thiels, 2011). There are no consensus sequences for PP2B in the cytoplasmic loop of the α7 nAChR.

Of the proteins that were “ruled out” for being in the knockout dataset no firm conclusion can be made without secondary biochemical evidence. The Western blot data indicates that both the NMDA zeta and GLUR2 could be ruled out as potential α7 nAChR interacting partners, but there are corroborating studies that indicate that they do interact with the α7 nAChR. A firm “rule out” is not possible with proteomic data on its own and based on numbers of peptides identified and frequency of protein identifications proteomic data should classify α7 nAChR interacting partners as more or less likely rather than ruled in or ruled out. A future direction could be a quantitative proteomic study involving the wild type and knockout mouse to determine if proteins held in common between the two datasets are up regulated or down regulated. This would be increasingly informative and would generate a lot of new information from a set of proteins that are effectively marginalized in a qualitative study like this unless they were also evaluated by Western blot.

Another aspect of the α7 nAChR interacting partners is the possibility that the

α7 nAChR does not have a uniform interactome and that it may differ based on the tissue it is being expressed in or its subcellular localization. With the α7 nAChR being expressed on intracellular membranes like the mitochondria it raises the possibility of different interacting partners based on localization (Gergalova et al., 2012). This may

119 explain why many mitochondrial proteins like succinyltransferase component of 2- oxoglutarate dehydrogenase complex, mitochondrial, Isoform Mitochondrial of

Fumarate hydratase, mitochondrial Pyruvate dehydrogenase E1 component subunit α, somatic form, mitochondrial Isoform Glt-1A of Excitatory amino acid transporter 2, and Excitatory amino acid transporter 1 are detected in this study. However, these proteins are often abundant and frequently detected in proteomic studies.

The α7 nAChR is present in multiple tissues. The whole brain homogenate contains a mix of neurons, astrocytes, microglia, neutrophils, lymphocytes, endothelial cells, and platelets all of which express the α7 nAChR albeit in different amounts

(Campbell et al., 2011; Egleton et al., 2009; Sharma & Vijayaraghavan, 2008). Once the DSME is prepared, the possibility exists of incorporating interacting proteins from all of these α7 nAChR expressing tissues into proteomic data. This increases the utility of the proteomic data. The proteins could have different significances, especially when protein moonlighting is considered.

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Picciotto, M. R., Caldarone, B. J., Brunzell, D. H., Zachariou, V., Stevens, T. R., & King, S. L. (2001). Neuronal nicotinic acetylcholine receptor subunit knockout mice: physiological and behavioral phenotypes and possible clinical implications. Pharmacol Ther, 92 (2-3), 89-108. Picciotto, M. R., & Kenny, P. J. (2013). Molecular mechanisms underlying behaviors related to nicotine addiction. Cold Spring Harb Perspect Med, 3 (1), a012112. Pohanka, M. (2012). Alpha7 nicotinic acetylcholine receptor is a target in pharmacology and toxicology. Int J Mol Sci, 13 (2), 2219-2238. Quik, M., Choremis, J., Komourian, J., Lukas, R. J., & Puchacz, E. (1996). Similarity between rat brain nicotinic alpha-bungarotoxin receptors and stably expressed alpha-bungarotoxin binding sites. J Neurochem, 67 (1), 145-154. Ross, J. A., Chen, Y., Muller, J., Barylko, B., Wang, L., Banks, H. B., et al. (2011). Dimeric endophilin A2 stimulates assembly and GTPase activity of dynamin 2. Biophys J, 100 (3), 729-737. Roy, J., & Cyert, M. S. (2009). Cracking the phosphatase code: docking interactions determine substrate specificity. Sci Signal, 2 (100), re9. Rusnak, F., & Mertz, P. (2000). Calcineurin: form and function. Physiol Rev, 80 (4), 1483-1521. Sharma, G., & Vijayaraghavan, S. (2001). Nicotinic cholinergic signaling in hippocampal astrocytes involves calcium-induced calcium release from intracellular stores. Proc Natl Acad Sci U S A, 98 (7), 4148-4153. Sharma, G., & Vijayaraghavan, S. (2008). Nicotinic Receptors: Role in Addiction and Other Disorders of the Brain. Subst Abuse, 2008 (1), 81. Shen, L., Peng, H., Xu, D., & Zhao, S. (2013). The next generation of novel low- density lipoprotein cholesterol-lowering agents: Proprotein convertase subtilisin/kexin 9 inhibitors. Pharmacol Res . Shytle, R. D., Mori, T., Townsend, K., Vendrame, M., Sun, N., Zeng, J., et al. (2004). Cholinergic modulation of microglial activation by alpha 7 nicotinic receptors. J Neurochem, 89 (2), 337-343. Siffert, W. (2003). G-protein beta3 subunit 825T allele and hypertension. Curr Hypertens Rep, 5 (1), 47-53. Suzuki, T., Hide, I., Matsubara, A., Hama, C., Harada, K., Miyano, K., et al. (2006). Microglial alpha7 nicotinic acetylcholine receptors drive a phospholipase C/IP3 pathway and modulate the cell activation toward a neuroprotective role. J Neurosci Res, 83 (8), 1461-1470. Tanaka, T., Ohashi, S., Moue, M., & Kobayashi, S. (2012). Mechanism of YB-1- mediated translational induction of GluR2 mRNA in response to neural activity through nAChR. Biochim Biophys Acta, 1820 (7), 1035-1042. Unger, C., Svedberg, M. M., Schutte, M., Bednar, I., & Nordberg, A. (2005). Effect of memantine on the alpha 7 neuronal nicotinic receptors, synaptophysin- and low molecular weight MAP-2 levels in the brain of transgenic mice over- expressing human acetylcholinesterase. J Neural Transm, 112 (2), 255-268. Wada, T., Naito, M., Kenmochi, H., Tsuneki, H., & Sasaoka, T. (2007). Chronic nicotine exposure enhances insulin-induced mitogenic signaling via up- regulation of alpha7 nicotinic receptors in isolated rat aortic smooth muscle cells. Endocrinology, 148 (2), 790-799.

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Wang, H., Yu, M., Ochani, M., Amella, C. A., Tanovic, M., Susarla, S., et al. (2003). Nicotinic acetylcholine receptor alpha7 subunit is an essential regulator of inflammation. Nature, 421 (6921), 384-388. Wang, Y., Pereira, E. F., Maus, A. D., Ostlie, N. S., Navaneetham, D., Lei, S., et al. (2001). Human bronchial epithelial and endothelial cells express alpha7 nicotinic acetylcholine receptors. Mol Pharmacol, 60 (6), 1201-1209. Xi, Z. R., Qin, W., Yang, Y. F., He, G., Gao, S. H., Ren, M. S., et al. (2004). Transmission disequilibrium analysis of the GSN gene in a cohort of family trios with schizophrenia. Neurosci Lett, 372 (3), 200-203. Zia, S., Ndoye, A., Lee, T. X., Webber, R. J., & Grando, S. A. (2000). Receptor- mediated inhibition of keratinocyte migration by nicotine involves modulations of calcium influx and intracellular concentration. J Pharmacol Exp Ther, 293 (3), 973-981.

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CHAPTER 3: 125 I-α-BUNGAROTOXIN BINDING AS A PROBE FOR METHOD OPTIMIZATION AND QUANTIFICATION William Brucker and Edward Hawrot

Introduction

The ability to identify the α7 nAChR by Western Blot was a valuable assay and was a vital tool in optimizing aspects of the Bgtx affinity isolation. A problem that emerged during the proteomic studies in Chapter 2 was an eventual loss of specificity of the anti-α7 antibody (ab23832) to the extent that it was detecting “ α7 nAChRs” in

Bgtx isolates and in pure DSME prepared from KO mice. The mice were genotyped to assure that there had been no breeding errors. Other commercially available anti-α7 antibodies shared similar lack specificity in this regard. Fortunately, alternatives were available in the form of commercially available 125 I-Bgtx.

Bgtx is a well-known specific antagonist of α7 nAChRs. It also has some affinity for GABA β3 subunits (Pohanka, 2012). Numerous studies have employed

125 I-Bgtx autoradiography to reliably probe for the presence of the α7 nAChR in anatomical regions or levels of α7 nAChR expression in disease states (Ferreira et al.,

2001; Picciotto et al., 2001). Because the α7 nAChR is a homopentamer it has 5 high affinity Bgtx binding sites. Assuming only one site is occupied by Bgtx conjugated to the affinity bead, several others are free to bind 125 I-Bgtx. The commercial availability of 125 I-Bgtx provides an excellent tool to both detect and quantify the amount of α7 nAChR in proteins isolated by Bgtx-conjugated beads. In the following chapter the

126 bead 125 I-Bgtx binding assay is used to optimize the isolation of the α7 nAChR and quantify it in both non-neuronal and neuronal tissues of other species. The focus of the experiments in this section was to identify and validate negative control models that could be used in α7 nAChR containing tissues.

Materials

Cyanogen bromide (CNBr) activated sepharose beads (C9142), carbachol

(C4382), α-bungarotoxin (T3019) and general chemicals for buffers were purchased from Sigma-Aldrich (St. Louis, MO). Triton X-100 (807426) was purchased from MP

Biomedicals (Solon, OH). Complete, Mini protease inhibitor cocktail was purchased from Roche (Mannheim, Germany). The water used in making all solutions and used in all experimentation Millipore ultra filtered, protein free water. 125 I-Bgtx (NEX-126) was purchased from Perkin Elmer (Shelton, CT). The gamma emission from the 125 I-

Bgtx was determined using a Wallac 1275 gamma counter (Perkin Elmer, Shelton,

CT). Paper Spin Cup Filters (product # P169700) were purchased from Pierce

(Rockford, IL).

Methods

After the preparation of detergent solubilized membrane extracts (DSME) from whole brain tissue and receptor isolation with Bgtx affinity beads detailed in Chapter

2, the slurry is centrifuged at 1,500 x g to separate the affinity beads from the DSME.

The DSME is then aspirated and the complex bound Bgtx beads are then resuspended in 2 mL of solubilization buffer and triturated. The 2 mL suspension is then divided into 1 mL aliquots. One of the 1 mL aliquots (total aliquot) will be used to determine total 125 I-Bgtx binding while the other will be used to determine nonspecific 125 I-Bgtx

127 binding (nonspecific aliquot). The two equally portioned aliquots are centrifuged at

1,500 x g before aspiration of the respective supernatants. The total aliquot receives an addition of 5 nM 125 I-Bgtx in a 1 mL solution of solubilization buffer while the nonspecific aliquot receives an addition of 5 nM 125 I-Bgtx + 1 µM Bgtx. The respective samples incubate for 1 hour at room temperature. After the incubation period both aliquots are centrifuged at 1,500 x g for 1 minute and the respective radioactive supernatants are aspirated. The beads from each aliquot are resuspended in

400 µL of solubilization buffer and transferred to respective paper spin cup filters. The spin cup filters are centrifuged at 1,500 x g for 30 seconds and the flow through is discarded. The beads are washed three times with 300 µL of solubilization buffer, after each centrifugation the flow through is discarded. After the third wash is complete the spin cup is removed from the filter. The top of the spin cup filter is trimmed with a razor blade so that it can fit inside of a glass test tube. A gamma counter quantifies the radioactivity. The counts per minute from the nonspecific binding aliquot are subtracted from the counts per minute of the total binding aliquot to determine the specific binding of the sample, Figure 3.1.

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125 Figure 3.1: On-Bead I-Bgtx Binding Assay Protocol

1. Aspirate DSME 2. Re-suspend beads with 5 nM 125 I-Bgtx+1 μM Bgtx 5 nM 125 I-Bgtx 2 mL Sol Buffer/50 μL Beads

α7 α7 α7 1 mL 1 mL α7

α7

α7 α7 α7 α7 1. Aspirate Radioactive Solution 2. Wash beads 3 times with Solubilization Buffer 3. Approximate decays/min with Gamma Counter 4. Calculate Specific Binding

Total Binding Specific Binding Nonspecific binding

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Validation of Negative Control Models with on Bead 125 I-Bgtx Binding Assay

Depending on the nature of an experiment, it may be useful to have a negative control model that is not a KO mouse. KO mice have been without the α7 for the entirety of their development, resulting physiological compensation may interfere with other systems (Fowler, Arends, & Kenny, 2008). In some cases a more appropriate model could be one where the α7 nAChR is excluded from binding to preserve the types of background proteins that would occur during normal development. The creation of a negative control model would also be very useful in studying tissues where α7 knockout models are not available like human brain tissue or cell lines that naturally express the α7 nAChR. The purpose of this experiment was to demonstrate that negative control models other than the KO mouse could be produced, Figure 3.2.

The Bgtx-conjugated affinity isolation of wild type whole brain DSME served as a positive control while the Bgtx-conjugated affinity isolation of KO whole brain DSME served as the negative control. The experimental negative controls were the MLA inhibited model and the glycine conjugated bead model. Each model had 3 biological replicates.

Bgtx Conjugated Affinity Bead Positive Control

Fifty microliters of Bgtx-conjugated affinity beads were added to DSME prepared from WT whole brain tissue and incubated overnight. 125 I-Bgtx binding was evaluated in the manner described above.

KO DSME Negative Control

Fifty microliters of Bgtx-conjugated affinity beads were added to DSME prepared from KO whole brain tissue and incubated overnight. 125 I-Bgtx binding was evaluated in the manner described above.

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Methyllycaconitine (MLA) Negative Control

A possible negative control model would involve the addition of MLA. MLA is α7 specific antagonist (Ki 0.69-10.3 nM) (Barrantes, Rogers, Lindstrom, &

Wonnacott, 1995; Davies et al., 1999; Gopalakrishnan et al., 1995; Macallan et al.,

1988; Quik, Choremis, Komourian, Lukas, & Puchacz, 1996). It is possible that the addition of an excess of MLA would prevent the Bgtx conjugated beads from binding the α7 nAChR complexes in DSME during the overnight incubation period. For this study MLA was added to the wildtype whole brain DSME to a concentration of 1 mM to place it excess. This MLA containing DSME was incubated with Bgtx conjugated sepharose beads overnight. 125 I-Bgtx Binding was evaluated in the manner described above.

Glycine Conjugated Sepharose Beads Negative Control

Bgtx conjugated affinity beads are blocked with glycine. A positive, negative control would be sepharose beads that were conjugated only with glycine and no Bgtx.

Without Bgtx to bind the α7 nAChRs the beads should have no capacity to isolate α7 nAChRs unless it binds to them in nonspecific fashion. Fifty microliters of “Glycine only” beads were added to DSME prepared from wildtype whole brain tissue and incubated overnight. 125 I-Bgtx binding was evaluated in the manner described above.

The MLA inhibited and glycine-conjugated negative control models had specific binding that was as low as the KO DSME indicating that both methods are effective at producing a negative control in an α7 nAChR containing sample. The glycine-conjugated negative control model had the lowest specific 125 I-Bgtx binding and could be used as a pre-clearing strategy to remove proteins before the addition of

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Bgtx –conjugated sepharose beads. This may reduce the inclusion of nonspecific binding proteins in the proteomic data.

Use of on Bead 125 I-Bgtx Binding Assays to Validate and Quantify Elution Conditions from Bgtx Affinity Beads

The negative control studies demonstrated the ability of on-bead 125 I-Bgtx binding assays to detect and quantify α7 nAChRs isolated from murine whole brain

DSME. The purpose of these elution studies was to determine if the 1 M carbachol elution was the most efficient way to release the α7 nAChR interacting complexes from the Bgtx conjugated beads. If an on bead 125 I-Bgtx binding assay was performed after an elution that removed all α7-nAChRs from the beads then there should be no specific 125 I-Bgtx binding detected. The purpose of Bgtx affinity beads is their pharmacological means of action. They bind the α7-nAChR and release it when outcompeted by excess carbachol. Carbachol is a nonspecific cholinergic agonist with a low molecular weight (Ki for α7 nAChR= 18-580 µm) (Anand, Peng, Ballesta, &

Lindstrom, 1993; Gopalakrishnan et al., 1995; Quik et al., 1996). The previous elution protocol used a large excess of carbachol (1 M) to ensure that all α7 nAChRs were removed from the beads. Several elution studies were performed to maximize the release of the α7 nAChRs from the beads.

In this experiment α7 nAChRs were isolated in standard fashion from equivalent amounts of WT whole brain DSME with Bgtx conjugated affinity beads.

After the isolations were complete, several different elution methods were performed.

Each elution solution was incubated with the Bgtx beads for 5 minutes before being

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Figure 3.2: Specific Bgtx Binding in Negative Control Models for On-Bead Binding Assay

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aspirated and on-bead 125 I-Bgtx binding assays were performed. Each experiment used only one biological replicate but the results were reproducible in future elution studies.

Elution with Solubilization Buffer

This was the negative control. Solubilization buffer is not designed to inhibit or interfere with Bgtx binding. Its use as an eluent should not remove α7 nAChRs from the beads and therefore specific 125 I-Bgtx binding should not be altered.

Elution with Carbachol in Solubilization Buffer

Elutions with both 1 M and 2 M carbachol in solubilization buffer were performed. 1 M carbachol is in excess and 2 M carbachol would be even greater.

These low molecular weight cholinergic agonists should outcompete the binding of

Bgtx to the α7 nAChrs and release them from the affinity beads. Elution with 1 M carbachol in solubilization buffer was the standard protocol and its ability to remove

α7 nAChRs from the affinity beads was validated by Western blot for the α7 nAChR.

High Salt Elutions

Carbamylcholine chloride is the carbachol salt dissolved in solubiliztion buffer before elution. This raises the hypothesis that the α7 nAChR could be released from the beads by the high salt conditions just as easily as the pharmacological competition provided by the carbachol. High salt elutions were performed with 1 M and 2 M NaCl dissolved in solubilization buffer.

Boiling Beads

Post incubation with DSME, Bgtx conjugated sepharose beads were boiled at

100 o C in SDS-PAGE loading buffer for 5 minutes. After boiling the beads were

134 washed once with solubilization buffer to remove denaturing compounds in the SDS loading buffer before on-bead 125 I-Bgtx binding assays were performed.

The specific 125 I-Bgtx binding was unaffected by either of the high salt elutions as both the 1 M and 2 M NaCl elutions were equivalent to elution with solubilization buffer. The carbachol elutions had a paradoxical effect where the specific binding was greatly increased (1 M Carbachol=37.06 fmol Bgtx binding/mg protein, 2 M

Carbachol=39.96 fmol Bgtx binding/mg protein) compared to the solubilization buffer eluted control (11.41 fmol Bgtx binding/mg protein). Boiling the beads eliminated nearly all of the specific binding (0.3565 fmol Bgtx binding/mg protein), Figure 3.4.

This study indicated that boiling the beads would result in a less specific eluent but would ensure that all α7 nAChRs were removed. To reduce the inclusions of proteins that bind in nonspecific fashion in the proteomic data, a 2 M NaCl elution could be performed prior to boiling to reduce the protein abundance of the sample while ensuring that no α7 nAChR complexes were retained on the beads.

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Figure 3.3 : Use of On Bead 125 I-Bgtx Binding Assays to Validate and Quantify Elution Cond itions from Bgtx Affinity Beads

45 39.96 40 37.06 35

15 12.93 12.91 11.41 10

5 0.36

fmol Specific Bgtx Binding/mg solubilized protein solubilizedBinding/mgBgtx Specific fmol 0

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Discussion

The on-bead 125 I-Bgtx binding assays proved to be an effective assay for α7 nAChR detection and method optimization. In the future these assays could be used gain quantitative information about the ability of the Bgtx conjugated Sepharose bead to isolate α7 nAChRs from other neural α7 containing tissues like rat or human whole brain tissues as well as the host of non-neuronal tissues that express α7 nAChRs.

Because they yield quantitative information, the on-bead 125 I-Bgtx binding assays could be used to estimate tissue needs for a proteomic study. MLA inhibited or glycine-conjugated beads could be used for negative control models in tissues that do not have a KO model. This could pave the way for an Alzheimer’s disease verses healthy aged-matched control study. This technique could be used to probe non- neuronal tissues like rat and mouse spleen that are packed with α7-nAChR expressing leukocytes.

The paradoxical effect of carbachol elution increasing specific binding is an interesting finding. The most important aspect is that it represents α7 nAChRs being retained on the beads. The fact that the signal amplification did not occur in the high salt elutions indicates that it is a pharmacological effect of carbachol rather than high salt conditions opening up new 125 I-Bgtx binding epitopes. Carbachol being used in such great excesses as 1-2 M should easily remove all α7 nAChRs. It is possible that the carbachol is releasing the α7 nAChRs from the Bgtx, but they are being retained on the beads by other protein interactions. The α7 nAChR has five Bgtx binding sites, a possible cause of the signal amplification is that carbachol is opening up more Bgtx binding sites for the 125 I-Bgtx to interact with resulting in a signal amplification.

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Elution with 1 M carbachol is sufficient to remove α7 nAChRs as seen by the Western blot, Figure 2.2, but these binding assays indicate that even in excess concentrations carbachol cannot remove them all. Boiling the beads is reliable and ensures that all proteins are removed. The disadvantage of boiling beads is that the specificity of the elution is lost. However, some of this may be compensated for by preclearing the

DSME with glycine conjugated beads before Bgtx-conjugated affinity beads are added and using a 2 M NaCl elution before boiling the beads to remove more proteins that bind in nonspecific fashion. These changes were adopted for future proteomic studies, detailed in Chapter 4.

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References

Anand, R., Peng, X., Ballesta, J. J., & Lindstrom, J. (1993). Pharmacological characterization of alpha-bungarotoxin-sensitive acetylcholine receptors immunoisolated from chick retina: contrasting properties of alpha 7 and alpha 8 subunit-containing subtypes. Mol Pharmacol, 44 (5), 1046-1050. Barrantes, G. E., Rogers, A. T., Lindstrom, J., & Wonnacott, S. (1995). alpha- Bungarotoxin binding sites in rat hippocampal and cortical cultures: initial characterisation, colocalisation with alpha 7 subunits and up-regulation by chronic nicotine treatment. Brain Res, 672 (1-2), 228-236. Davies, A. R., Hardick, D. J., Blagbrough, I. S., Potter, B. V., Wolstenholme, A. J., & Wonnacott, S. (1999). Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling alpha 7-type neuronal nicotinic acetylcholine receptors. Neuropharmacology, 38 (5), 679-690. Ferreira, M., Ebert, S. N., Perry, D. C., Yasuda, R. P., Baker, C. M., Davila-Garcia, M. I., et al. (2001). Evidence of a functional alpha7-neuronal nicotinic receptor subtype located on motoneurons of the dorsal motor nucleus of the vagus. J Pharmacol Exp Ther, 296 (2), 260-269. Fowler, C. D., Arends, M. A., & Kenny, P. J. (2008). Subtypes of nicotinic acetylcholine receptors in nicotine reward, dependence, and withdrawal: evidence from genetically modified mice. Behav Pharmacol, 19 (5-6), 461-484. Gopalakrishnan, M., Buisson, B., Touma, E., Giordano, T., Campbell, J. E., Hu, I. C., et al. (1995). Stable expression and pharmacological properties of the human alpha 7 nicotinic acetylcholine receptor. Eur J Pharmacol, 290 (3), 237-246. Macallan, D. R., Lunt, G. G., Wonnacott, S., Swanson, K. L., Rapoport, H., & Albuquerque, E. X. (1988). Methyllycaconitine and (+)-anatoxin-a differentiate between nicotinic receptors in vertebrate and invertebrate nervous systems. FEBS Lett, 226 (2), 357-363. Picciotto, M. R., Caldarone, B. J., Brunzell, D. H., Zachariou, V., Stevens, T. R., & King, S. L. (2001). Neuronal nicotinic acetylcholine receptor subunit knockout mice: physiological and behavioral phenotypes and possible clinical implications. Pharmacol Ther, 92 (2-3), 89-108. Pohanka, M. (2012). Alpha7 nicotinic acetylcholine receptor is a target in pharmacology and toxicology. Int J Mol Sci, 13 (2), 2219-2238. Quik, M., Choremis, J., Komourian, J., Lukas, R. J., & Puchacz, E. (1996). Similarity between rat brain nicotinic alpha-bungarotoxin receptors and stably expressed alpha-bungarotoxin binding sites. J Neurochem, 67 (1), 145-154.

139

CHAPTER 4: QUANTITATIVE AND QUALITATIVE APPROACHES TO UNCOVER THE ROLE OF α7 NACHR IN THE NEUROBIOLOGY OF NICOTINE ADDICTION William Brucker, Joao Paulo, Edward Hawrot

Smoking is the number one preventable cause of mortality and morbidity in the world. Despite the established health costs of smoking, many tobacco users find it difficult to quit smoking (Wang, Yuan, & Li, 2011). Smoking is a behavior reinforced by nicotine, a psychoactive component found within tobacco (Wang et al., 2011). Abuse of tobacco products is responsible for more than 5 million deaths per year (Yeom, Shim,

Lee, & Hahm, 2005). Addiction is defined as abnormal behavior characterized by compulsive use of the substance in question despite serious negative consequences. The behaviors that comprise addiction are not immediately present at the onset of substance use but evolve gradually and progressively. Once addictive patterns of behavior are reinforced they can be difficult to extinguish even after use of the drug has been discontinued (Wang et al., 2011). Nicotine can stimulate the mesocorticolimbic dopamine system of the cell bodies in the VTA and their projections into the nucleus accumbens, striatum, and prefrontal cortex (Hwang & Li, 2006). It is believed that nicotine and other drugs of abuse cause changes in synaptic plasticity in this “reward circuitry” causing the act of administering the drug to be over-learned (Niehaus, Murali, & Kauer, 2010). The long-term potentiation of excitatory synapses is a mechanism that underlies the ability of neuronal circuits to form memories in addiction or other brain functions (Niehaus et al.,

140

2010). An understanding of the mechanisms by which nicotine activated nAChRs cause

this long-term potentiation and changes in synaptic strength in the mesocorticolimbic

system could lead to the development of targeted pharmacotherapies to prevent or abolish

the reinforcing properties of nicotine.

Nicotine causes changes in this circuitry through the activation of nAChRs

(Fowler, Arends, & Kenny, 2008). The first step towards understanding how synaptic plasticity is altered by nicotine is to monitor global changes in the protein interacting partners of nAChRs after nicotine exposure. The ability to use Bgtx as a probe to isolate the α7 nAChR from in vivo tissue sources creates the potential for identifying nicotine induced changes in the interacting partners of the α7 nAChR (Paulo, Brucker, & Hawrot,

2009). Although α7 nAChRs are present on neurons in the VTA, their role in nicotine reinforcement is unclear. Current consensus supports a modulator role in contrast to the mechanisms described for β2 nAChRs (Levin et al., 2009; Picciotto & Kenny, 2013).

Some groups have hypothesized, based on animal studies, that while β2 nAChRs are

essential to initiating reinforcement, α7 nAChRs play a greater role in maintaining it

during chronic nicotine exposure (Levin et al., 2009). This potential role in chronic

reinforcement is more clinically relevant because while most animal studies last for

several weeks human smokers will smoke for several years (Levin et al., 2009).

Varenicline, an effective smoking cessation pharmacotherapy, is a partial agonist at α4β2 nAChRs and a full agonist at α7 nAChRs (Sharma & Vijayaraghavan, 2008). The basis for establishing the role of the α7 nAChR in nicotine reinforcement originated from behavioral studies. Since the function of α7 nAChR seems to be more nuanced, a

141

proteomic analysis of the effects of nicotine exposure on its interacting partners may

elucidate its true physiological role more effectively than a behavioral study.

Role of Proteomics in the Study of Addiction

Proteomic analyses of in vivo CNS tissues are becoming an increasingly important part of neuroscience research (Abul-Husn & Devi, 2006). Diseased states of the CNS like neurodegenerative disorders, neuropsychiatric disorders and nicotine abuse involve multiple interacting proteins (Abul-Husn & Devi, 2006). Exposure to drugs of abuse cause changes in gene regulation and protein expression in both neurons and supporting glial cells that mirrors increases in addictive behavior patterns (Wang et al.,

2011). Such changes suggest alterations in a large array of protein interacting partners

(Wang et al., 2011). Proteomic study allows for qualitative and quantitative assessment of global alterations in protein interacting partners when comparing physiologic verses pathologic states (Abul-Husn & Devi, 2006). Recognition of the patterns of altered pathway regulation may inform addiction pathophysiology and targets for directed therapy to abrogate the reinforcement of drugs of abuse. Proteomic investigations have been used in the past to profile how expression patterns were altered by exposure to drugs of abuse such as amphetamines, alcohol, nicotine, cocaine, and morphine (Wang et al.,

2011). Despite the fact that nicotine is the most widespread drug of abuse only two neuroproteomic studies have been performed (Hwang & Li, 2006; Wang et al., 2011;

Yeom et al., 2005).

Previous Neuroproteomic Studies of Nicotine

The studies performed by Yeom et al. 2005 and Hwang et al. 2006 were similar in that they both examined global proteomic changes in anatomic regions of the rat brain

142 following nicotine exposure for 1 week. Yeom and collegues examined proteomic changes in the striatum after administration of nicotine at 0.4 mg/kg twice daily by subcutaneous injection for 7 days. The study by Hwang and collegues involved dissection of multiple regions of the rat brain (amygdala, nucleus accumbens, prefrontal cortex, striatum, and ventral tegemental area (VTA)) by coronal section followed by punch biopsy after administration of nicotine bitartrate at a dose of 3.15 mg/kg/day through osmotic minipump for 7 days. Both studies employed 2D SDS-PAGE as methods of protein fractionation followed by in-gel tryptic digestions of selected gel regions,

MALDI-TOF mass spectrometry and protein database searching. Protein quantification in both cases was accomplished using densitrometry and comparison of 2D gels of nicotine exposed and control models. Yeom et al. also correlated mRNA levels to the changes in densitrometry. Both studies identified many proteins whose expression may have been effected by nicotine (Table 4.1). Yeom et al. identified 7 proteins in the striatum that demonstrated significant down or up regulation in response to nicotine exposure. Many densitrometry studies correlated well with the mRNA levels of respective proteins (Wang et al., 2011; Yeom et al., 2005). Zi nc-finger binding protein-89, cyclic nucleotide phosphodiesterase, and deoxyribonuclease 1-like 3 demonstrated up regulation in response to nicotine exposure while tandem pore domain halothane-inhibited potassium channel 2, brain-specific hyaluronan-binding protein, death effecter domain containing protein, and brain-derived neurotrophic factor were downregulated (Wang et al., 2011;

Yeom et al., 2005). Hwang et al. demonstrated differential changes in protein abundance by brain region in response to nicotine exposure. For example, Dynamin 1 was up- regulated in the prefrontal cortex and nucleus accumbens but was down-regulated in the

143 amygdala and ventral tegmental area. Dynamin1, laminin receptors, aldolase A, SNAP-β, and N-ethylmaleimide-sensitive fusion protein were identified in multiple brain regions and demonstrated both down and up-regulation indicating that nicotine induced expression changes may not be uniform throughout the CNS (Hwang & Li, 2006; J.

Wang, Yuan, & Li, 2011). For example, proteomic data is often subject to great variability depending on sample preparation, which can lead to inconsistent results from multiple replicates within the same study. The accuracy of peptide identification by

MALDI-TOF is dependent on the purity of the sample. 2D SDS-PAGE will fractionate proteins more thoroughly than 1D SDS-PAGE; however, the gel bands are still not entirely composed of uniform protein populations. It is not surprising, therefore, that there was no correlation between the identifications made in the striatum region of proteins identified by Yeom et al. and Hwang et al. It is difficult to conclude from this data alone if nicotine had variable effects on protein expression in different regions, but it is an attractive target for future quantitative proteomic studies with more sophisticated instrumentation capable of MS/MS, multiple biological replicates, and statistical analysis.

The neuroproteomic study presented here employs Bgtx affinity isolation and quantitative mass spectrometry with LTQ-Orbitrap instrumentation to determine nicotine induced changes in the interacting partners of the α7 nAChR and is the most instrumentally

sophisticated to date. It is also the only study of its kind to focus on the α7 nAChR.

144

Table 4.1: Effect of nicotine exposure on protein expression in different regions of rat brain. Upregulated Protein Expression Downregulated Protein Expression Whole brain a zinc-finger binding protein-89 brain-derived neurotr ophic factor death effecter domain containing prote in cyclic nucleotide phosphodiesterase brain-specific h yaluronan-binding protein tandem pore domain halotha ne-inhibited deoxyribonuclease 1-like 3 Prefrontal Pyruvate kinase, muscle Pyruvate dehydrogenase, (lip oamide) beta Similar to N-ethylmaleimide-sensitive f actor Cortex b Malate dehydrogenase Isocitrate dehydrogenase 3 (NAD 1) alpha Synapsin Iib Mitochondrial, N -Ethylmaleimide sensitive NADH dehydrogenase 1, alpha subcomplex 10-like N-Ethylmaleimide sensitive fusion protein fusion protein protein attachment protein α Dynamin 1 Ubiquinol-cytochrome c reductase core prot ein I Protein phosphatase 2a catalytic subunit α Voltage-dependent, anion channel 1 Valosin-containin g attachment protein, beta Peroxiredoxin 2 Similar to CCT (chaperonin containing TCP-1) GTP-binding protein alpha o Guanine nucleotide binding protein beta-1 subunit zeta subunit Chaperonin 60 Amygdala b N-Ethylmaleimide sensitive fusion protein OTU domain isoform 1 Enolase 1, α Ubiquitin aldehyde binding 1 Dynamin 1 Dipeptidylpeptidase 3 Laminin receptor 1 Dihydrolipoa mide dehydrogenase Hsc70-ps1 Glutamine synthetase Syntaxin binding prote in 1 Heat shock protein 8 Peptidylprolyl isomerase A Heat shock protein 1 Prohibitin Hsc70-ps1, Similar to ATPase Laminin receptor 1 H1 transporting Ubiquitin C-terminal hydrolase PGP9. 5 V1 subunit A Protein phosphatase 3, catalytic subunit, α Nucleus b alpha, Dipeptidylpeptidase 3 Protein phosphatase 3 c atalytic subunit α isoform Lactate dehydrogenase B accumbens Hsc70-ps1 Ubiquitin C-terminal hydrolase Triosephosph ate isomerase 1 Dynamin 1 PGP9.5 Aldolase A Enolase 1 α Laminin receptor 1 Peroxiredoxin 1 b Striatum Annexin A5 Glucose regulated protein, 58 kDa Glutathi one S-transferase omega 1 Crystallin, mu flavoprotein (Fp) Septin 5 Similar to N-ethylmaleimide-sensitive factor Succinate dehydrogenase complex subunit A Aldo-keto reductase family 1, member B4 attachment protein beta Triosephosphate isomerase 1 Glutathione S-transferas e pi 1 NADH dehydrogenase (ubiquinone) Fe-S protein 2 Sirtuin 2 Glutamate dehydrogenase 1 VTA b Dihydrolipoamide acetyltransferase M2 pyruvate kinase Dynamin 1 Aconitase 2, mitochondrial Aldolase A Glutamate oxaloacetate transaminase 1 Transketolase Phosphoglycerate mutase 1 H1 transporti ng unc-18 protein homolog ATP synthase Similar to succinate semialdehyde dehydrogenase Gars_predicted protein mitochondrial F1 complex, α subunit, isoform 1 (NAD1-dependent) a Rats received 0.4mg/kg/dose of nicotine injected tw ice daily subcutaneously for 7 days, Yeom et al. 20 05. b Rats received 3.15 mg/kg/day through osmotic minipu mp for 7 days, Hwang et al. 2006.

145

146

Materials

Nicotine Administration: Nicotine hydrogen tartrate salt (N5260) was purchased from Sigma Aldrich (St. Louis, MO). Water used for production of nicotine solution was standard drinking water of quality set by the Brown University Animal Control

Facility. Mice, aged 6 weeks, were purchased from Charles River (Wilmington, MA) and housed in the Brown University Animal Control Facility. All experimental protocols were carried out in accordance with the Brown University Institutional

Animal Care and Use Committee (IACUC).

DSME preparation: Triton X-100 (807426) was purchased from MP Biomedicals

(Solon, OH). Complete, Mini protease inhibitor cocktail was purchased from Roche

(Mannheim, Germany). Cyanogen bromide (CNBr) activated sepharose beads

(C9142), carbachol (C4382), α-bungarotoxin (T3019) and general chemicals for buffers were purchased from Sigma-Aldrich (St. Louis, MO). The water used in making all solutions and used in all experimentation was Millipore ultra filtered, protein free water.

Proteomics: Trypsin Gold, mass spectrometry grade (V5280) was purchased from the

Promega Corporation (Madison, WI). BenchMark Pre-Stained ladder (10748-010) and

Brilliant Blue G-Colloidal Coomassie stain (B2025) was purchased from Sigma-

Aldrich (St. Louis, MO). Precast gels (NuPAGE Novex, 4-12% Bis-Tris Gels, 10 wells, 1 mm thickness) (NPO321BOX) were purchased from Invitrogen (Carlsbad,

CA). Paper Spin Cup Filters (product # P169700) were purchased from Pierce

(Rockford, IL).

Nicotine Exposure

147

Rationale for Oral Nicotine Exposure in a Mouse Model

The most common method of nicotine administration in humans is smoking.

Voluntary inhalation of nicotine is not a method that can easily be modeled in animals.

The closest approximation to human smoking behavior in a murine model is oral nicotine delivery through drinking water. This is a commonly employed method

(Grabus et al., 2005; Matta et al., 2007) and can be used on large population animals making it an appealing choice for proteomic studies that require large tissue volumes.

Oral route provides continuous nicotine exposure without introducing potentially confounding variables such as the stress evoked from procedures (mini-pump insertion) or injections for drug administration (Matta et al., 2007). Oral administration has the additional benefit of producing numerous small peaks in plasma nicotine levels that more closely approximate the peaks seen in human smokers (Matta et al., 2007). The oral route is pharmacologically relevant with serum nicotine concentrations reaching levels as high as 15.85 µM (200 µg nicotine/mL drinking water), vastly higher than what would be seen in human smokers (500 nm-1 µM)

(Matta et al., 2007). The strain, sex and age of the mice used affect how much nicotine that they consume. C3H mice can consume 4mg/kg per day while C57/Bl/6 mice can consume as much as 12 mg/kg/day (Matta et al., 2007). Adolescent female mice consume greater volumes and higher concentrations of nicotine containing solutions than their male counterparts (Matta et al., 2007). Chronic oral nicotine intake can produce tolerance, up-regulation of α4β2 nAChRs and precipitate signs of withdrawal

(somatic signs and hyperalgesia) if administration is stopped (Grabus et al., 2005;

Matta et al., 2007). These effects can be seen with administration of nicotine containing solutions of 50–200 µg/mL for more than 42 days (Matta et al., 2007). 148

Low Nicotine Affinity of the α7 nAChR

The hypothesis of this study is dependent on nicotine activating the α7 nAChRs. While the α7 nAChRs have a low affinity for nicotine there is considerable evidence to suggest that they are activated at serum concentrations seen in typical smokers. The EC50 of the α7 nAChR as evidenced by channel conductance is estimated to be 27-55 µM (Delbono et al., 1997; Papke & Porter Papke, 2002; Sharma

& Vijayaraghavan, 2008). While serum nicotine levels in human smokers (500 nm-1

µM) are far below this level, nicotine use is shown to improve auditory gating deficits in schizophrenics, an effect dependent on α7 nAChR activation (Sharma &

Vijayaraghavan, 2008). Also in vitro studies demonstrate α7 nAChR dependent intracellular signaling changes can be accomplished with administration of 1 µM nicotine solutions (Hu, Liu, Chang, & Berg, 2002). One possible reason for low levels of receptor activation having physiological responses is the metabotropic nature of α7 nAChR signaling through calcium dependent pathways. A low level of α7 nAChR activation might not cause a large current, but the calcium that passes through the channel could induce CICR, leading to the activation of a much larger number of proteins (Gahring & Rogers, 2005). The α7 nAChR has rapid desensitization kinetics, so it is possible that a low agonist concentration could activate small numbers of them continuously rather than large numbers of them briefly before they desensitize. Other evidence suggests that due to the lipophilic nature of nicotine, its serum concentration may be far less than the concentration at the synapse which some have estimated to be

5 times higher (Sharma & Vijayaraghavan, 2008). Mice are more tolerant of high serum nicotine levels than humans and with chronic nicotine exposure have a greater likelihood of achieving plasma levels closer to the EC50 of the α7 nAChR. 149

Nicotine exposure

Sixty female C57BL/6 genetic mice were used for this study at age 12 weeks and evenly divided into nicotine exposed and control groups. Female mice were chosen because they consume more nicotine than male mice. C57BL/6 mice also consume more nicotine than other strains. Mice in the nicotine exposed group received drinking water supplemented with nicotine at a concentration of 70 µg/mL and 2% saccharin for 8 weeks while mice in the control group received drinking water supplemented with 2% saccharin only for the duration of the exposure period. The purpose of the saccharin additive was to give the water a sweet taste that would encourage the consumption of nicotine. The nicotine concentration of 70 µg/mL is below the concentration where nicotine would cause avoidance (Matta et al., 2007). At the end of 8 weeks, a period of time consistent with the literature that is long enough to cause dependence and model chronic exposure, all mice in the study were sacrificed by asphyxiation with CO 2. Their whole brain tissues were extracted and immediately frozen with dry ice then stored at -80 o C until used for experimentation.

Tissue Preparation

The whole brain tissues of nicotine exposed and control mice were treated in parallel. Detergent solubilized membrane extracts (DSME) were prepared by first homogenizing the whole brain tissue in homogenization buffer (50 mM Tris, 150 mM

NaCl, pH 7.4, protease inhibitors) at a ratio of 2 mL homogenization buffer/400mg tissue. Ten nicotine exposed (4 g) and 10 control brains (4 g) were used in this experiment. Once the homogenate was generated the membrane-containing portion was separated from the soluble fraction by centrifugation at 100,000xg for 1 hour. The soluble fraction was then decanted and DSME was prepared by homogenizing the 150 membrane pellet in detergent containing solubilization buffer (50m M Tris, 150m M

NaCl, pH 7.4, protease inhibitors, 1% Triton X-100) at a ratio of 1 mL/400 mg of starting tissue. After the homogenates were prepared they were agitated at 4 o C for 2 hours to promote the formation of micelles. After the incubation period, the homogenate was centrifuged at 100,000g for 1 hour to separate the DSME from insoluble particles. This procedure resulted in the generation of 8 mL of DSME for both the nicotine exposed and control tissues. Proteins binding to the sepharose beads in non-specific fashion were depleted by a pre-clearing step. Glycine conjugated sepharose beads, 100 µL, were added per mL of DSME and allowed to incubate with agitation at 4 o C for 2 hours. After the incubation period was over the suspension was separated by centrifugation at 1,500xg for 2 minutes and the DSME was collected. 25

µL of Bgtx conjugated sepharose beads (50 µL of slurry) were added per every 1 mL of DSME and incubated at 4 o C overnight with agitation.

Isolation of Bgtx Sensitive Proteins

On day 2 the DSME was separated from the beads by centrifugation at 1,500xg for 2 minutes after which the DSME was aspirated and the beads were resuspended in

3 mL of solubilization buffer. 1 mL of the slurry was then added to three Pierce paper spin cup filters (product number 69700). The suspension was separated by centrifugation at 3,500xg. The beads for each individual experiment were washed 3 times in this fashion with 300 µL of solubilization buffer before a final wash with 2 M

NaCl in solubilization buffer. After the high salt elution, the beads were resuspended in 100 µL of solubilization buffer and transferred to a 1.5 mL Eppendorf tube. The samples were centrifuged at 3,500xg for 30 seconds and the solubilization buffer was aspirated before 100 µL of gel loading buffer (10 % SDS, 10 mM DTT, 20% glycerol, 151

0.2 M tris HCl, 0.05% bromophenol blue, pH 6.8) was added to the beads. The beads were boiled at 100 o C for 10 minutes. After being boiled the beads were pelleted by centrifugation at 3,500xg. The supernatant was loaded into precast polyacrylamide gels (NuPAGE Novex 4-12% Bis-Tris Gels) with 50 µL of loading buffer added per lane. This resulted in 6 groups: 3 nicotine exposed (N1, N2, N3) and 3 control groups

(C1, C2, C3). SDS-PAGE was performed at 120 V for 30 minutes. After the proteins were separated, bands were visualized by staining with colloidal coomassie blue

(Pierce G-250) according to the protocol of the manufacturer.

Each gel lane was excised and cut into 16 slices of equal size. Each of the slices was cut in half to reduce its size for in gel tryptic digestion producing 32 gel slices per biological replicate. The slices were washed multiple times to remove detergents and other impurities before the proteins were reduced with 50 mM TCEP and alkylated with 100 mM iodoacetamide. The reduction step is to reduce disulfide bonds and unfold proteins. The alkyation step is performed to alkylate the free thiol groups and prevent disulfide bonds from reforming. The elongated proteins are more susceptible to degradation with trypsin (Sechl 1998). After the reduction and alkylation steps the gel slices are washed again before being digested in-gel with trypsin. After the in-gel tryptic digestion, peptides were extracted by two elution steps.

The elution fluids from each half gel slice were collected into a common tube. Each lane was divided into 32 slices and peptides from each half slice were recombined creating 16 peptide collections per lane and then analyzed by mass spectrometry.

152

Gel Washing Steps, Reduction, Alkylation, In-Gel Tryptic Digestion, and Peptide Extraction

The in-gel tryptic digestion of all slices occurred on the same day using one preparation of every buffer and solution. 500 µL of washing solution (50 mM ammonium bicarbonate in 50% water and 50% acetonitrile) was added to all gel slices. They were incubated at 37 o C for 15 minutes after which the supernatant was aspirated. The slices were washed twice in this fashion before being dehydrated with an addition of 500 µL of 100% acetonitrile per slice. The slices were rehydrated with

500 µL digestion buffer (50 mM ammonium bicarbonate) at 37 o C for 15 minutes.

Each slice received 200 µL of reducing solution (50 mM TCEP in digestion buffer) and was heated at 60 o C for 10 minutes. After the samples cooled the reducing solution was aspirated and all slices received an addition of 200 µL of alkylating solution (100 mM iodoacetamide in digestion buffer) and incubated at room temperature for one hour in the dark. The alkylating solution was then aspirated and the slices received 500

µL of washing solution and were incubated at 37 o C for 15 minutes. The washing solution was aspirated and this washing step was repeated twice more with the same volume of washing solution. The slices were completely dehydrated with the addition of 500 µL of acetonitrile. The acetonitrile was aspirated and the gel slices were dried by a vacuum concentrator before each slice received an addition of 10 µL of tryptic digest solution (10 µg/µL trypsin in digestion buffer) and incubated at 37 o C for 24 hours.

The peptide rich tryptic digest solutions from each divided slice were recombined into a single 1.5 mL Eppendorf tube. Each slice received an addition of

200 µL of 1% Formic acid and incubated at 37 o C for 15 minutes after which time the

153 solution was collected and recombined into each slice-pair’s respective common tube.

The second elution step was done with 200 µL of 1% formic acid solution in 50% water/acetonitrile and the slices were incubated at 37 o C for 15 minutes. The peptides from each slice-pair were once again recombined. The 16 peptide-containing solutions were reduced to a volume of less than 10 µL by vacuum concentrator. These concentrated peptide-containing solutions were then resuspended in 100 µL of 0.1 % acetic acid before being analyzed by mass spectrometry.

Qualitative Mass Spectrometry

Mass spectrometry was performed at the Brown Proteomics Core Facility

(Providence, RI). The resulting .RAW files were analyzed at the Taplin Biological

Mass Spectrometry Institute at Harvard University (Boston, MA). Nano-scale reversed phase HPLC columns packed with 5 µm of C 18 silica beads were used to separate peptides on the basis of hydrophobicity before being introduced into the LTQ-Orbitrap mass spectrometer by nano scale ESI. Nano-ESI is advantageous over standard ESI because the flow of peptides into the mass spectrometer is reduced allowing for greater sensitivity, increased ability to create ions, and increased tolerance of solvent impurities (Schmidt, Karas, & Dulcks, 2003).

The column was allowed to come to equilibrium and then the samples were injected with an auto sampler onto the column. The peptides were eluted from the column with solvents of increasing hydrophobicity. After being eluted from the column the peptides were subject to electrospray ionization and were detected by an

LTQ Orbitrap in MS/MS was acquired in data dependent fashion. The .RAW Files generated from the MS/MS were collected and analyzed at the Taplin Facility. Data

154 were searched against the UniProt mouse database (downloaded May 1, 2012) using the MaxQuant (v. 1.2.2.5) Andromeda search engine (Cox 2011). Search parameters are detailed in Table 4.2. Two missed cleavages were allowed per peptide and mass tolerances of +/- 10 ppm for precursor and of +/- 0.8 Da for fragment ions were used.

Amino acid modification parameters were a fixed carbamidomethyl (Cys) and variable deamidation (Asn/Gln), oxidation (Met), and Acetylation (N-term). Our false discovery rate (FDR) of 1% at the protein level was determined by searching the same dataset against the target database and a decoy database. The latter featured the reversed amino acid sequences of all the entries in the database above (Elias, Gibbons,

King, Roth, & Gygi, 2004; Moore, Young, & Lee, 2000).

Table 4.2: Parameters for MaxQuant Andromeda Parameter Setting Database Mouse SwisProt (Downloaded May 1, 2012) Missed cleavages 2 Enzyme specificity Typsin Precursor mass tolerance 10 ppm Fragment mass tolerance 0.8 Da Dynamic modifications Deamidated (NQ), Oxidation (M), Acetylation (N-term) Static modifications Carbamidomethyl (C) Protein False Discovery Rate 1%

Venn diagrams

The VENNY on-line Venn diagram plotter was used to obtain lists of unique and common proteins (Oliveros, 2007).

Protein Networks and Functional Analysis of Protein

Proteins identified in both the qualitative and quantitative proteomic studies were subjected to functional pathway analysis using protein analysis through evolutionary relationships (PANTHER) (www.pantherdb.org) system version 8 and

155 database for annotation, visualization and integrated discovery (DAVID)

(http://david.abcc.ncifcrf.gov/) version 6.7.

QSPEC spectral counting analysis

Relative protein quantification was accomplished using a label-free technique, spectral counting. Spectral counting compares the number of identified tandem mass spectra for the same protein across multiple data sets. To search for differences in the protein profile among datasets, spectral counts were normalized based on the total spectral counts, as outlined previously (Dong et al., 2007). Specifically, spectral counts of each protein were first divided by the total spectral counts of all proteins from the same sample, and then scaled by multiplying the total spectral counts of the sample by the maximum total number of spectral counts. Significance analysis of our normalized spectral count data was performed using QSPEC, a recently published algorithm for determining the statistical significance of differences in spectral counting data from two samples (Choi & Nesvizhskii, 2008). This algorithm used the

Bayes Factor in lieu of the p-value, as a measure of evidential strength (Goodman,

1999). By convention, a Bayes factor greater than 10 suggests strong evidence that a particular protein was differentially expressed between the two cohorts; thus a value of

10 was used as our significance threshold (Jeffreys , 1961 ). Proteins with a Bayes factor of 10 or greater demonstrated fold changes of +/- 1.5.

Results

Overview of Datasets

The 2337 unique proteins identified in this study were evaluated both quantitatively and qualitatively. There were 1902 total proteins identified in the control datasets (C1, C2, C3) and 1869 proteins identified in the nicotine-exposed data 156 sets (N1, N2, N3), Figure 4.1. the nicotine-exposed datasets contained 435 unique proteins (Appendix A.1); The control datasets contained 468 unique proteins

(Appendix A.2). The proteins unique to the nicotine exposed and control groups were evaluated qualitatively while the 1434 proteins (Appendix A.3) that were common between the two groups were evaluated quantitatively.

Analysis of Consistency between Data sets

Quantitative proteomic studies involve comparing spectral counts from proteins identified in both the nicotine-exposed and control datasets. Consistent identification between the replicates in each dataset is a vital part of the fidelity of this comparison. If a protein produces a massive amount of spectral counts in one dataset replicate but none in the other two then that protein is less reliable for quantitative comparison than a protein with similar spectral counts in all three replicate datasets.

Many of the proteins identified in both control and nicotine-exposed datasets were consistently identified in all three respective replicate datasets. 901 (76%) proteins identified in the nicotine-exposed dataset appeared in all 3 data sets while 851 (73%) proteins were identified in all 3 control datasets. Both the nicotine-exposed and control datasets had 8% of identified proteins appear only once in a respective dataset (Figure

4.2 and 4.3). A comparison was also done to determine the spectral count consistency within the nicotine exposed and control data sets (Figure 4.4 and 4.5). The spectral counts for all proteins from replicate datasets were compared to one another. If the datasets were identical then all of the spectral counts should be identical for every protein. In the case of two identical datasets if spectral counts per protein from one dataset was plotted against the spectral counts per protein from its identical complementary dataset a linear relationship should exist and the R 2 value would be 1. 157

The spectral counts were highly consistent between the control replicates (C1 vs. C2

(R 2=0.9265), C1 vs. C3 (R 2=0.9473), C3 vs. C2 (R 2=0.9078)) and more divergent in the nicotine exposed samples reflecting the spectral counts in N1 as being less consistent than N2 and N3 (N1 vs. N2 (R 2=0.8384), N1 vs. N3 (R 2=0.7584), N3 vs.

N2 (R 2=0.949)). These differences in spectral counts within the nicotine-exposed group may be due to the multiple handling steps involved in peptide processing, variations in individual in-gel tryptic digestions, and the stochastic nature of mass spectrometry based peptide identification.

Quantitative Results

Fifty-three proteins were identified that met the criteria for statistically significant change, fold change of +/- 1.5 and a Bayes factor value of 10 or greater. There were

16 proteins that seemed to be downregulated by nicotine exposure, and 37 that were indicated to be up-regulated by nicotine exposure.

Qualitative Analysis of Proteins Unique to Nicotine and Control Datasets

Qualitative analysis focused on proteins that were uniquely identified in either the nicotine-exposed or control datasets. The DAVID ontology database was used to determine potential intracellular roles of identified proteins, Figures 4.7 and 4.8. The

PANTHER ontology database was used to determine potential pathways that the proteins may participate in for each of the three groups: proteins only identified in nicotine-exposed datasets (Table 4.9), only in control datasets (Table 4.10), and in both datasets (Table 4.11). These analyses also characterized the nature of the identified proteins to determine if clear differences in global pathway participation could be attributed to chronic nicotine exposure. No obvious global differences in

158 pathway participation was obvious from the qualitative data; however, the incorporation of proteins that demonstrated significant fold change due to nicotine- exposure demonstrated multiple up-regulated proteins in the ATP synthesis (3 proteins up-regulated) and synaptic vesicle trafficking (3 proteins). Although a number of unique proteins were identified in both the nicotine and control datasets, several of the unique proteins belonged to pathways shared by proteins identified in the common dataset. Several pathways were only present in the datasets of uniquely identified proteins. Some identified proteins participated in multiple pathways, like

Synaptosomal-associated protein 25 which is involved in 23 different pathways. This may reflect that activation of the α7 nAChR could lead to a wide variety of intracellular effects.

159

Figure 4.1: Venn diagram of proteins exclusive to and in common with nicotine exposed and control groups. A total of 468 unique proteins were specific to the control groups (C1, C2, C3) while 435 proteins were specific to the nicotine exposed groups (N1, N2, N3). 1434 proteins were found in both data sets. The proteins unique to each data set were evaluated qualitatively while those in common were analyzed in quantitative fashion with spectral counts.

160

Figure 4.2: Venn diagrams of the distribution of unique identified proteins in nicotine exposed and control data sets. An analysis of protein distributions in: A. the nicotine exposed data sets (N1, N2, N3) and B. the control data sets (C1, C2, C3).

A) B)

161

Figure 4.3: Reproducibility of protein identifications in the same dataset. A) The percentage of proteins identified in 1, 2, or all 3 of the nicotine-exposed datasets; 76% of proteins were observed in all three sets. B) The percentage of proteins identified in 1, 2, or all 3 of the control datasets; 73% of the proteins identified in all three samples.

162

Figure 4.4: Comparison of Spectral Count consistency in control datasets (C1, C2, C3) Graphs comparing the spectral counts of proteins common in all 3 control data sets: A. C1 vs. C2 (R 2=0.9265). B. C1 vs. C3 (R 2=0.9473). C. C3 vs. C2 (R 2=0.9078). A. 800 R² = 0.926 600

400 C2 200

0 0 200 400 600 800 1000 C1

1200 1000 R² = 0.947 800

C3 600 400 200 0 0 200 400 600 800 1000 C1

1200 1000 R² = 0.907 800 600 C2 400 200 0 0 200 400 600 800 C3

163

Figure 4.5: Comparison of Spectral Count consistency in nicotine-exposed datasets (N1, N2, N3) Graphs comparing the spectral counts of proteins common in all 3 nicotine exposed data sets: A. N1 vs. N2 (R 2=0.8384). B. N1 vs. N3 (R 2=0.7584). C. N3 vs. N2 (R 2=0.949).

800 R² = 0.838 600 400

N2 200 0 -200 0 100 200 300 400 500 N1

800 R² = 0.758 600 400 N3 200 0 0 100 200 300 400 500 N1

800 R² = 0.949 600 400 N2 200 0 0 200 400 600 800 N3

164

Figure 4.6: The statistical basis for assigning significance to fold change of +/- 1.5 . A graph of log2 (fold change) verses frequency of protein identification, 95% of the data is between +0.5 and -0.5. Proteins with fold changes greater than +0.5 or less than -0.5 have undergone significant quantitative change in response to nicotine exposure.

250 95% of the data falls between the red lines 200

150

100 Frequency 50

0 1 0 1 - 1.6 1.4 1.2 0.8 0.6 0.4 0.2 0.2 0.4 0.6 0.8 1.2 1.4 ------>1.6 < log2(fold change)

165

Table 4.3: Proteins with less than 1.5 fold decrease in response to nicotine exposure

166

Table 4.4: Proteins with greater than 1.5 fold increase in response to nicotine exposure

167

Figure 4.7: Analysis of functionalities (A) and intracellular roles (B) of proteins identified in nicotine-exposed and control datasets. A.

40% Nicotine-exposed Control 35% 30% 25% 20% 15% 10% 5% 0%

25% Nicotine-exposed Control

20%

15%

10%

0% transport transport cell cycle cycle cell apoptosis localization reproduction reproduction cell adhesion adhesion cell system process system cellular process process cellular metabolic process process metabolic cell communication communication cell homeostatic process process homeostatic response to stimulusresponse cellular organization organization cellular developmental process process developmental immune system system process immune generation metabolites of generation

168

Figure 4.8: Further analysis of intracellular roles and functionalities of proteins identified in nicotine exposed and control datasets. A. Distribution of subcellular localizations for proteins in nicotine exposed and control datasets. B. Analysis of functionalities and intracellular roles of proteins in the nicotine exposed and control data sets.

A. 80% Nicotine -exposed Control 70% 60% 50% 40% 30% 20% 10% 0% intracellular complex complex protein complex complex protein ribonucleoprotein plasma membrane membrane plasma extracellular region region extracellular

14% Nicotine-exposed Control 12% 10% 8% 6% 4% 2% 0% lyase lyase ligase kinase kinase binding - receptor protease hydrolase isomerase chaperone transferase transporter phosphatase storage protein storage oxidoreductase calcium structural protein structural enzyme modulator enzyme signaling molecule molecule signaling transcription factor transcription cytoskeletal protein cytoskeletal cell junction protein junctioncell nucleic acid binding binding acid nucleic transfer/carrier protein transfer/carrier cell adhesion molecule molecule adhesion cell transmembrane receptor transmembrane membrane traffic protein traffic membrane defense/immunity protein defense/immunity extracellular matrix protein matrix extracellular

169

Table 4.5: Summary of pathways participated in by proteins unique to the nicotine-exposed datasets. The pathways in bold are unique to nicotine-exposed datasets.

Uniprot Pathway Protein name ID

5-Hydroxytryptamine degredation (P04372) P24549 Retinal dehydrogenase 1

Q8K009 Mitochondrial 10-formyltetrahydrofolate dehydrogenase

5HT1 type receptor mediated signaling pathway (P04373) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

5HT2 type receptor mediated signaling pathway (P04374) Q8R570 Synaptosomal-associated protein 47

1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase Q8R3B1 delta-1 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

5HT3 type receptor mediated signaling pathway (P04375) Q8R570 Synaptosomal-associated protein 47

5HT4 type receptor mediated signaling pathway (P04376) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Adrenaline and noradrenaline biosynthesis (P00001) Q8R570 Synaptosomal-associated protein 47

Alpha adrenergic receptor signaling pathway (P00002) Q8R570 Synaptosomal-associated protein 47

Alzheimer disease-presenilin pathway (P00004) P34960 Macrophage metalloelastase

P62737 Actin, aortic smooth muscle

Angiotensin II-stimulated signaling through G proteins and beta-arrestin (P05911) Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2

Apoptosis signaling pathway (P00006) E9QNL4 Inhibitor of nuclear factor kappa-B kinase subunit alpha

Axon guidance mediated by Slit/Robo (P00008) Q80TR4 Slit homolog 1 protein

170

Uniprot Pathway Protein name ID

Axon guidance mediated by semaphorins (P00007) P70206 Plexin-A1

B cell activation (P00010) F6UND7 Tyrosine-protein kinase HCK

E9QNL4 Inhibitor of nuclear factor kappa-B kinase subunit alpha

P18052 Receptor-type tyrosine-protein phosphatase alpha

Beta1 adrenergic receptor signaling pathway (P04377) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12 E9PZQ0 Ryanodine receptor 1

Beta2 adrenergic receptor signaling pathway (P04378) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12 E9PZQ0 Ryanodine receptor 1

Beta3 adrenergic receptor signaling pathway (P04379) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Blood coagulation (P00011) P33587 Activation peptide

Cadherin signaling pathway (P00012) Q9WTR5 Cadherin-13

P62737 Actin, aortic smooth muscle

P67871 Casein kinase II subunit beta

F8VPK8 Protein Pcdh9

Q9Z0M3 Cadherin-20

Cholesterol biosynthesis (P00014) P54869 Hydroxymethylglutaryl-CoA synthase, mitochondrial

Cortocotropin releasing factor receptor signaling pathway (P04380) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Cytoskeletal regulation by Rho GTPase (P00016) P54227 Stathmin

P62737 Actin, aortic smooth muscle

171

Uniprot Pathway Protein name ID

DNA replication (P00017) P20664 DNA primase small subunit

Dopamine receptor mediated signaling pathway (P05912) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2

EGF receptor signaling pathway (P00018) Q3UHC7 Disabled homolog 2-interacting protein

P62071 Ras-related protein R-Ras2

Endogenous_cannabinoid_signaling (P05730) Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2

Enkephalin release (P05913) Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

FAS signaling pathway (P00020) Q921K2 Poly (ADP-ribose) polymerase family, member 1

P14733 Lamin-B1

FGF signaling pathway (P00021) Serine/threonine-protein phosphatase 2A 55 kDa regulatory Q925E7 subunit B delta isoform

Fructose galactose metabolism (P02744) Q9JIA6 Galactokinase

GABA-B_receptor_II_signaling (P05731) Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2

General transcription regulation (P00023) E9QAP7 Protein Taf4a

Glycolysis (P00024) P06745 Glucose-6-phosphate isomerase

E9PYX4 Glyceraldehyde-3-phosphate dehydrogenase

Gonadotropin releasing hormone receptor pathway (P06664) Q62048 Astrocytic phosphoprotein PEA-15

P62835 Ras-related protein Rap-1A

Q68ED7 CREB-regulated transcription coactivator 1

P00405 Cytochrome c oxidase subunit 2

Q9JJZ2 Tubulin alpha-8 chain

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 O54950 5'-AMP-activated protein kinase subunit gamma-1

172

Uniprot Pathway Protein name ID Heterotrimeric G-protein signaling pathway-Gi alpha and Gs alpha mediated pathway

(P00026) P62835 Ras-related protein Rap-1A

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12 Heterotrimeric G-protein signaling pathway-Gq alpha and Go alpha mediated

pathway (P00027) P62835 Ras-related protein Rap-1A

(P00028) Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Histamine H1 receptor mediated signaling pathway (P04385) 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase Q8R3B1 delta-1 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Histamine H2 receptor mediated signaling pathway (P04386) Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Huntington disease (P00029) Q8VEH3 ADP-ribosylation factor-like protein 8A

E9Q8T7 Protein Dnahc1

E9QAP7 Protein Taf4a

E9PYX4 Glyceraldehyde-3-phosphate dehydrogenase

O35350 Calpain-1 catalytic subunit

P62737 Actin, aortic smooth muscle

Q80TB8 Synaptic vesicle membrane protein VAT-1 homolog-like

Hypoxia response via HIF activation (P00030) P51450 Nuclear receptor ROR-gamma

Inflammation mediated by chemokine and cytokine signaling pathway (P00031) 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase Q8R3B1 delta-1 P62071 Ras-related protein R-Ras2

P62737 Actin, aortic smooth muscle

173

Uniprot Pathway Protein name ID Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Integrin signalling pathway (P00034) P62835 Ras-related protein Rap-1A

E9QPX1 Endostatin

Q61140 Breast cancer anti-estrogen resistance protein 1

Interleukin signaling pathway (P00036) E9QNL4 Inhibitor of nuclear factor kappa-B kinase subunit alpha

Q60837 Interleukin-12 receptor subunit beta-1

Ionotropic glutamate receptor pathway (P00037) Q8R570 Synaptosomal-associated protein 47

P56564 Excitatory amino acid transporter 1

JAK/STAT signaling pathway (P00038) P18052 Receptor-type tyrosine-protein phosphatase alpha

Mannose metabolism (P02752) Q8K0C9 GDP-mannose 4,6 dehydratase

Metabotropic glutamate receptor group II pathway (P00040) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 O35526 Syntaxin-1A

Metabotropic glutamate receptor group III pathway (P00039) Q8R570 Synaptosomal-associated protein 47

P56564 Excitatory amino acid transporter 1

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 O35526 Syntaxin-1A

Muscarinic acetylcholine receptor 1 and 3 signaling pathway (P00042) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 O35526 Syntaxin-1A

Muscarinic acetylcholine receptor 2 and 4 signaling pathway (P00043) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 O35526 Syntaxin-1A

Nicotine degradation (P05914) Q8K2I3 Dimethylaniline monooxygenase [N-oxide-forming] 2

G3X982 Aldehyde oxidase 3, isoform CRA_a

Nicotinic acetylcholine receptor signaling pathway (P00044) Q8R570 Synaptosomal-associated protein 47

P62737 Actin, aortic smooth muscle

174

Uniprot Pathway Protein name ID O35526 Syntaxin-1A

Opioid prodynorphin pathway (P05916) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Opioid proenkephalin pathway (P05915) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Opioid proopiomelanocortin pathway (P05917) Q8R570 Synaptosomal-associated protein 47

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12

Oxytocin receptor mediated signaling pathway (P04391) Q8R570 Synaptosomal-associated protein 47

PDGF signaling pathway (P00047) Q6PE55 Platelet-derived growth factor receptor-like protein

Q6ZQ12 Ninein-like protein

Parkinson disease (P00049) Q9CWH6 Proteasome subunit alpha type-7-like

Q9WTX6 Cullin-1

O55042 Alpha-synuclein

Q91ZZ3 Beta-synuclein

F6UND7 Tyrosine-protein kinase HCK

Q9WUD1 STIP1 homology and U box-containing protein 1

P67871 Casein kinase II subunit beta

Pentose phosphate pathway (P02762) P06745 Glucose-6-phosphate isomerase

Phenylethylamine degradation (P02766) P24549 Retinal dehydrogenase 1

Q8K009 Mitochondrial 10-formyltetrahydrofolate dehydrogenase

Pyruvate metabolism (P02772) Q8BMF3 NADP-dependent malic enzyme, mitochondrial

175

Uniprot Pathway Protein name ID

Synaptic_vesicle_trafficking (P05734) Q8R570 Synaptosomal-associated protein 47

P46097 Synaptotagmin-2

O35526 Syntaxin-1A

T cell activation (P00053) E9QNL4 Inhibitor of nuclear factor kappa-B kinase subunit alpha

P18052 Receptor-type tyrosine-protein phosphatase alpha

TGF-beta signaling pathway (P00052) P62071 Ras-related protein R-Ras2

Thyrotropin-releasing hormone receptor signaling pathway (P04394) Q8R570 Synaptosomal-associated protein 47

Toll receptor signaling pathway (P00054) E9QNL4 Inhibitor of nuclear factor kappa-B kinase subunit alpha

Transcription regulation by bZIP transcription factor (P00055) E9QAP7 Protein Taf4a

Ubiquitin proteasome pathway (P00060) P62334 26S protease regulatory subunit 10B

Vasopressin synthesis (P04395) Q00493 Carboxypeptidase E

Wnt signaling pathway (P00057) Q9WTR5 Cadherin-13

P62737 Actin, aortic smooth muscle

E9Q4N7 Protein Arid1b

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit P63213 gamma-2 P67871 Casein kinase II subunit beta

F8VPK8 Protein Pcdh9

Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit Q9DAS9 gamma-12 Q9Z0M3 Cadherin-20

p53 pathway by glucose deprivation (P04397) O54950 5'-AMP-activated protein kinase subunit gamma-1

176

Table 4.6: Pathways participated in by proteins identified only in the control datasets. The pathways in bold are unique to the control datasets.

Pathway Uniprot ID Protein name

5-Hydroxytryptamine degredation (P04372) Delta-1-pyrroline-5-carboxylate dehydrogenase, Q8CHT0 mitochondrial

5HT1 type receptor mediated signaling pathway (P04373) cAMP-dependent protein kinase catalytic subunit P05132 alpha

5HT2 type receptor mediated signaling pathway (P04374) Q91UZ1 Phospholipase C beta 4

Alpha adrenergic receptor signaling pathway (P00002) Q91UZ1 Phospholipase C beta 4

Alzheimer disease-amyloid secretase pathway (P00003) P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 P29121 Proprotein convertase subtilisin/kexin type 4

Alzheimer disease-presenilin pathway (P00004) Q65CL1 Catenin alpha-3 P70701 Protein Wnt-10a P29121 Proprotein convertase subtilisin/kexin type 4

Angiogenesis (P00005) P70701 Protein Wnt-10a Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf Neuronal proto-oncogene tyrosine-protein kinase P05480 Src D3YTR0 Adenomatous polyposis coli protein 2 Angiotensin II-stimulated signaling through G proteins and beta-arrestin

(P05911) Q63844 Mitogen-activated protein kinase 3 P70227 Inositol 1,4,5-trisphosphate receptor type 3

Apoptosis signaling pathway (P00006) Q63844 Mitogen-activated protein kinase 3

177

Pathway Uniprot ID Protein name

Arginine biosynthesis (P02728) Carbamoyl-phosphate synthase [ammonia], Q8C196 mitochondrial

Axon guidance mediated by semaphorins (P00007) Q60875 Rho guanine nucleotide exchange factor 2 Q6P9R4 Rho guanine nucleotide exchange factor 18

B cell activation (P00010) Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf Q9QUN3 B-cell linker protein P62204 Calmodulin P70227 Inositol 1,4,5-trisphosphate receptor type 3

Beta1 adrenergic receptor signaling pathway (P04377) cAMP-dependent protein kinase catalytic subunit P05132 alpha

Beta2 adrenergic receptor signaling pathway (P04378) cAMP-dependent protein kinase catalytic subunit P05132 alpha

Blood coagulation (P00011) P12388 Plasminogen activator inhibitor 2, macrophage

Cadherin signaling pathway (P00012) Q60737 Casein kinase II subunit alpha Q65CL1 Catenin alpha-3 P70701 Protein Wnt-10a Neuronal proto-oncogene tyrosine-protein kinase P05480 Src

Cytoskeletal regulation by Rho GTPase (P00016) Q5SX40 Myosin-1 Q60875 Rho guanine nucleotide exchange factor 2 B1B1A8 Myosin light chain kinase, smooth muscle Q6P9R4 Rho guanine nucleotide exchange factor 18 G3UW82 MCG140437, isoform CRA_d P13542 Myosin-8 A2AQP0 Myosin-7B

De novo pyrmidine ribonucleotides biosythesis (P02740) Q8C196 Carbamoyl-phosphate synthase [ammonia], mitochondrial

178

Pathway Uniprot ID Protein name

Dopamine receptor mediated signaling pathway (P05912) Q60829 Protein phosphatase 1 regulatory subunit 1B cAMP-dependent protein kinase catalytic subunit P05132 alpha

EGF receptor signaling pathway (P00018) Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf Dual specificity mitogen-activated protein kinase P70236 kinase 6

Endothelin signaling pathway (P00019) Q91UZ1 Phospholipase C beta 4 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf cAMP-dependent protein kinase catalytic subunit P05132 alpha P70227 Inositol 1,4,5-trisphosphate receptor type 3

Enkephalin release (P05913) cAMP-dependent protein kinase catalytic subunit P05132 alpha

FGF signaling pathway (P00021) Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf

GABA-B_receptor_II_signaling (P05731) cAMP-dependent protein kinase catalytic subunit P05132 alpha

Gamma-aminobutyric acid synthesis (P04384) P48320 Glutamate decarboxylase 2

Glycolysis (P00024) E9PX99 Glyceraldehyde-3-phosphate dehydrogenase

Gonadotropin releasing hormone receptor pathway (P06664) P47811 Mitogen-activated protein kinase 14 Q64727 Vinculin Q63844 Mitogen-activated protein kinase 3

179

Pathway Uniprot ID Protein name Dual specificity mitogen-activated protein kinase P70236 kinase 6 Neuronal proto-oncogene tyrosine-protein kinase P05480 Src P70227 Inositol 1,4,5-trisphosphate receptor type 3 Heterotrimeric G-protein signaling pathway-Gi alpha and Gs alpha mediated

pathway (P00026) Q7TSH2 Phosphorylase b kinase regulatory subunit beta cAMP-dependent protein kinase catalytic subunit P05132 alpha P62204 Calmodulin Heterotrimeric G-protein signaling pathway-Gq alpha and Go alpha mediated

pathway (P00027) Q60875 Rho guanine nucleotide exchange factor 2 Q6P9R4 Rho guanine nucleotide exchange factor 18 Q91UZ1 Phospholipase C beta 4 Voltage-dependent R-type calcium channel E9QK20 subunit alpha-1E P70227 Inositol 1,4,5-trisphosphate receptor type 3 Heterotrimeric G-protein signaling pathway-rod outer segment

phototransduction (P00028) cAMP-dependent protein kinase catalytic subunit P05132 alpha P62204 Calmodulin

Histamine H1 receptor mediated signaling pathway (P04385) Q91UZ1 Phospholipase C beta 4 P70227 Inositol 1,4,5-trisphosphate receptor type 3

Histamine H2 receptor mediated signaling pathway (P04386) P05132 cAMP-dependent protein kinase catalytic subunit alpha

Huntington disease (P00029) O08529 Calpain-2 catalytic subunit Q62108 Disks large homolog 4 E9PX99 Glyceraldehyde-3-phosphate dehydrogenase

Inflammation mediated by chemokine and cytokine signaling pathway (P00031) Q5SX40 Myosin-1 G5E852 Tyrosine-protein kinase B1B1A8 Myosin light chain kinase, smooth muscle

180

Pathway Uniprot ID Protein name Q91UZ1 Phospholipase C beta 4 Calcium/calmodulin-dependent protein kinase Q923T9 type II subunit gamma G3UW82 MCG140437, isoform CRA_d Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf cAMP-dependent protein kinase catalytic subunit P05132 alpha P13542 Myosin-8 P70227 Inositol 1,4,5-trisphosphate receptor type 3 A2AQP0 Myosin-7B Insulin/IGF pathway-mitogen activated protein kinase kinase/MAP kinase

cascade (P00032) A2AR50 Ras-specific guanine nucleotide-releasing factor RalGPS1 Q63844 Mitogen-activated protein kinase 3

Integrin signalling pathway (P00034) O88990 Alpha-actinin-3 P11087 Collagen alpha-1(I) chain O70585 Dystrobrevin beta G5E874 Laminin subunit gamma-2 Q01149 Collagen alpha-2(I) chain Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 Q61001 Laminin subunit alpha-5 Q64727 Vinculin Q63844 Mitogen-activated protein kinase 3 G3UW84 Actinin alpha 2 P04627 Serine/threonine-protein kinase A-Raf P05480 Neuronal proto-oncogene tyrosine-protein kinase Src Q80X90 Filamin-B

Interferon-gamma signaling pathway (P00035) G5E852 Tyrosine-protein kinase P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3

181

Pathway Uniprot ID Protein name

Interleukin signaling pathway (P00036) G5E852 Tyrosine-protein kinase Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf

Ionotropic glutamate receptor pathway (P00037) Q923T9 Calcium/calmodulin-dependent protein kinase type II subunit gamma Voltage-dependent R-type calcium channel E9QK20 subunit alpha-1E

JAK/STAT signaling pathway (P00038) G5E852 Tyrosine-protein kinase P47811 Mitogen-activated protein kinase 14

Metabotropic glutamate receptor group I pathway (P00041) Q91UZ1 Phospholipase C beta 4 P05132 cAMP-dependent protein kinase catalytic subunit alpha

Metabotropic glutamate receptor group II pathway (P00040) E9QK20 Voltage-dependent R-type calcium channel subunit alpha-1E cAMP-dependent protein kinase catalytic subunit P05132 alpha

Metabotropic glutamate receptor group III pathway (P00039) E9QK20 Voltage-dependent R-type calcium channel subunit alpha-1E cAMP-dependent protein kinase catalytic subunit P05132 alpha

Muscarinic acetylcholine receptor 1 and 3 signaling pathway (P00042) Q91UZ1 Phospholipase C beta 4 P70227 Inositol 1,4,5-trisphosphate receptor type 3

Muscarinic acetylcholine receptor 2 and 4 signaling pathway (P00043) P05132 cAMP-dependent protein kinase catalytic subunit alpha

Nicotine pharmacodynamics pathway (P06587) Q60829 Protein phosphatase 1 regulatory subunit 1B P05132 cAMP-dependent protein kinase catalytic subunit alpha

182

Pathway Uniprot ID Protein name

Nicotinic acetylcholine receptor signaling pathway (P00044) Q5SX40 Myosin-1 G3UW82 MCG140437, isoform CRA_d Q8R5C5 Beta-centractin Disheveled-associated activator of Q80U19 morphogenesis 2 P13542 Myosin-8 A2AQP0 Myosin-7B

Notch signaling pathway (P00045) Q9QZS3 Protein numb homolog O08919 Numb-like protein Q9WTS2 Alpha-(1,6)-fucosyltransferase

Oxidative stress response (P00046) P47811 Mitogen-activated protein kinase 14

Oxytocin receptor mediated signaling pathway (P04391) Q91UZ1 Phospholipase C beta 4

PDGF signaling pathway (P00047) G5E852 Tyrosine-protein kinase Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf F8VPQ4 SLIT-ROBO Rho GTPase-activating protein 3 P70227 Inositol 1,4,5-trisphosphate receptor type 3

PI3 kinase pathway (P00048) G5E852 Tyrosine-protein kinase Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1

Parkinson disease (P00049) Q60737 Casein kinase II subunit alpha P47811 Mitogen-activated protein kinase 14 O70435 Proteasome subunit alpha type-3 Q63844 Mitogen-activated protein kinase 3 Neuronal proto-oncogene tyrosine-protein kinase P05480 Src

Plasminogen activating cascade (P00050) P12388 Plasminogen activator inhibitor 2, macrophage

183

Pathway Uniprot ID Protein name

Ras Pathway (P04393) Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf Dual specificity mitogen-activated protein kinase P70236 kinase 6

T cell activation (P00053) Ras-specific guanine nucleotide-releasing factor A2AR50 RalGPS1 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf P62204 Calmodulin

TGF-beta signaling pathway (P00052) P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 Q9CS84 Neurexin-1-alpha

Thyrotropin-releasing hormone receptor signaling pathway (P04394) Q91UZ1 Phospholipase C beta 4 Voltage-dependent R-type calcium channel E9QK20 subunit alpha-1E

Toll receptor signaling pathway (P00054) P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 D3Z1I3 MCG49525

Ubiquitin proteasome pathway (P00060) Q9R1T2 SUMO-activating enzyme subunit 1 P62192 26S protease regulatory subunit 4 26S proteasome non-ATPase regulatory subunit Q99JI4 6 Q9D906 Ubiquitin-like modifier-activating enzyme ATG7 Q8BVQ9 26S protease regulatory subunit 7 D3Z1I3 MCG49525

VEGF signaling pathway (P00056) P47811 Mitogen-activated protein kinase 14 Q63844 Mitogen-activated protein kinase 3 P04627 Serine/threonine-protein kinase A-Raf

Wnt signaling pathway (P00057) Q60737 Casein kinase II subunit alpha

184

Pathway Uniprot ID Protein name Q5SX40 Myosin-1 Q65CL1 Catenin alpha-3 Q91UZ1 Phospholipase C beta 4 P70701 Protein Wnt-10a G3UW82 MCG140437, isoform CRA_d P13542 Myosin-8 P70227 Inositol 1,4,5-trisphosphate receptor type 3 A2AQP0 Myosin-7B D3YTR0 Adenomatous polyposis coli protein 2

mRNA splicing (P00058) Q62189 U1 small nuclear ribonucleoprotein A

p38 MAPK pathway (P05918) P47811 Mitogen-activated protein kinase 14 Q63943 Myocyte-specific enhancer factor 2D Dual specificity mitogen-activated protein kinase P70236 kinase 6

p53 pathway feedback loops 2 (P04398) P51943 Cyclin-A2 P97377 Cyclin-dependent kinase 2 P47811 Mitogen-activated protein kinase 14

p53 pathway (P00059) D3Z351 MCG1219 Werner syndrome ATP-dependent helicase O09053 homolog P97377 Cyclin-dependent kinase 2 P70124 Serpin B5 Q9R190 Metastasis-associated protein MTA2

185

Table 4.7: Pathways participated in by proteins identified in both nicotine- exposed and control datasets. Proteins up-regulated by nicotine exposure are highlighted in bold. Proteins downregulated by nicotine exposure are noted in italics. Proteins with previously reported links to α7 nAChR are underlined. Affected pathways have same notations.

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

5-Hydroxytryptamine degredation (P04372)

Cytosolic 10-formyltetrahydrofolate Q8R0Y6 99 79 dehydrogenase P47738 Aldehyde dehydrogenase, mitochondrial 61 55

Q3U367 4-trimethylaminobutyraldehyde dehydrogenase 17 10

Alpha-aminoadipic semialdehyde Q9DBF1 7 8 dehydrogenase

5HT1 type receptor mediated signaling pathway (P04373)

P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2 Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 cAMP-dependent protein kinase catalytic P68181 14 13 subunit beta Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha

5HT2 type receptor mediated signaling pathway (P04374)

P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P68404 Protein kinase C beta type 24 13

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Q8K394 Inactive phospholipase C-like protein 2 4 3

186

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

5HT3 type receptor mediated signaling pathway (P04375)

P60879 Synaptosomal-associated protein 25 68 112

5HT4 type receptor mediated signaling pathway (P04376)

P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas

ATP synthesis (P02721)

G3UWG1 MCG115977 8 65

Q03265 ATP synthase subunit alpha, mitochondrial 130 305

P56480 ATP synthase subunit beta, mitochondrial 114 378

Adrenaline and noradrenaline biosynthesis (P00001)

P60879 Synaptosomal-associated protein 25 68 112

Alpha adrenergic receptor signaling pathway (P00002)

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 P60879 Synaptosomal-associated protein 25 68 112

P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

Alzheimer disease-amyloid secretase pathway (P00003)

P63085 Mitogen-activated protein kinase 1 57 64

P68404 Protein kinase C beta type 24 13

Q8CD76 Uncharacterized protein 19 16

Q91YS4 Kinesin light chain 2 4 2

Amyloid beta A4 precursor protein-binding P98084 1 2 family A member 2

Alzheimer disease-presenilin pathway (P00004)

P68033 Actin, alpha cardiac muscle 1 886 238

P63260 Actin, cytoplasmic 2 638 328

Q8BFZ3 Beta-actin-like protein 2 301 316

P61161 Actin-related protein 2 104 101

Q02248 Catenin beta-1 94 78

Low-density lipoprotein receptor-related Q91ZX7 82 51 protein 1 515 kDa subunit Q9R1R2 Tripartite motif-containing protein 3 81 78

P26231 Catenin alpha-1 31 15

Q2NL51 Glycogen synthase kinase-3 alpha 2 2

187

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Aminobutyrate degradation (P02726)

4-aminobutyrate aminotransferase, P61922 24 52 mitochondrial

Androgen/estrogene/progesterone biosynthesis (P02727)

O70503 Estradiol 17-beta-dehydrogenase 12 3 3

Angiogenesis (P00005)

Q02248 Catenin beta-1 94 78

P63085 Mitogen-activated protein kinase 1 57 64

P68404 Protein kinase C beta type 24 13

E9QMQ8 Focal adhesion kinase 1 13 20

Tyrosine-protein phosphatase non-receptor P35235 12 8 type 11 P62746 Rho-related GTP-binding protein RhoB 5 14

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 E9Q753 GTPase NRas 3 2

E9QAN8 Protein Pik3c2b 2 2

Q2NL51 Glycogen synthase kinase-3 alpha 2 2

Angiotensin II-stimulated signaling through G proteins and beta-arrestin (P05911) P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

P63085 Mitogen-activated protein kinase 1 57 64

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 Guanine nucleotide-binding protein subunit P62881 14 13 beta-5

Apoptosis signaling pathway (P00006)

P63017 Heat shock cognate 71 kDa protein 776 783

P17156 Heat shock-related 70 kDa protein 2 384 390

Q7TSZ0 Heat shock protein 9 243 271

P17879 Heat shock 70 kDa protein 1B 127 207

P63085 Mitogen-activated protein kinase 1 57 64

P68404 Protein kinase C beta type 24 13

G3UWG1 MCG115977 8 65

D3Z5F7 Protein Gm20521 4 1

Eukaryotic translation initiation factor 2 Q6ZWX6 3 1 subunit 1

188

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed O08600 Endonuclease G, mitochondrial 1 2

Arginine biosynthesis (P02728)

Q91YI0 Argininosuccinate lyase 8 5

Q9D1A2 Cytosolic non-specific dipeptidase 3 10

Carbamoyl-phosphate synthetase 2, aspartate B2RQC6 27 8 transcarbamylase, and dihydroorotase P16460 Argininosuccinate synthase 9 7

Asparagine and aspartate biosynthesis (P02730)

P05202 Aspartate aminotransferase, mitochondrial 163 221

P05201 Aspartate aminotransferase, cytoplasmic 64 131

Axon guidance mediated by Slit/Robo (P00008)

B0V2N1 Receptor-type tyrosine-protein phosphatase S 9 6

E9QLZ9 Protein enabled homolog 8 1

P62746 Rho-related GTP-binding protein RhoB 5 14

Axon guidance mediated by netrin (P00009)

E9QLZ9 Protein enabled homolog 8 1

E9QAN8 Protein Pik3c2b 2 2

Axon guidance mediated by semaphorins (P00007)

O08553 Dihydropyrimidinase-related protein 2 484 520

O35098 Dihydropyrimidinase-related protein 4 47 25

Q9EQF6 Dihydropyrimidinase-related protein 5 42 39

P70207 Plexin-A2 15 2

Q80UG2 Plexin-A4 10 3

P49615 Cyclin-dependent kinase 5 8 19

B cell activation (P00010)

Serine/threonine-protein phosphatase 2B P48453 93 100 catalytic subunit beta isoform P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

P63085 Mitogen-activated protein kinase 1 57 64

P68404 Protein kinase C beta type 24 13

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 E9Q753 GTPase NRas 3 2

β1 adrenergic receptor signaling pathway (P04377)

P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 E9Q401 Ryanodine receptor 2 18 13

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5

189

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha

Beta2 adrenergic receptor signaling pathway (P04378)

P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 E9Q401 Ryanodine receptor 2 18 13

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha

Beta3 adrenergic receptor signaling pathway (P04379)

P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1

Blood coagulation (P00011)

E9PV24 Protein Fga 125 152

Cadherin signaling pathway (P00012)

P26231 Catenin alpha-1 31 15

P61161 Actin-related protein 2 104 101

Q02248 Catenin beta-1 94 78

P55288 Cadherin-11 8 6

Q2NL51 Glycogen synthase kinase-3 alpha 2 2

Q8BFZ3 Beta-actin-like protein 2 301 316

190

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

O54833 Casein kinase II subunit alpha' 3 4

P68033 Actin, alpha cardiac muscle 1 886 238

P63260 Actin, cytoplasmic 2 638 328

Cell cycle (P00013)

Eukaryotic translation initiation factor 3 Q9DCH4 14 13 subunit F

Cortocotropin releasing factor receptor signaling pathway (P04380)

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 P60879 Synaptosomal-associated protein 25 68 112

Cytoskeletal regulation by Rho GTPase (P00016)

P68033 Actin, alpha cardiac muscle 1 886 238

P63260 Actin, cytoplasmic 2 638 328

P68372 Tubulin beta-4B chain 477 736

Q7TMM9 Tubulin beta-2A chain 469 714

P99024 Tubulin beta-5 chain 447 713

Q9D6F9 Tubulin beta-4A chain 394 580

Q9ERD7 Tubulin beta-3 chain 373 543

Q8BFZ3 Beta-actin-like protein 2 301 316

Q9JM76 Actin-related protein 2/3 complex subunit 3 52 42

P59999 Actin-related protein 2/3 complex subunit 4 44 47

Q9CVB6 Actin-related protein 2/3 complex subunit 2 41 31

Q9JJV2 Profilin-2 35 41

Q9R0Q6 Actin-related protein 2/3 complex subunit 1A 33 29

E9PYM9 Rho-associated protein kinase 2 24 16

F8WGL3 Cofilin-1 23 43

P70429 Ena/VASP-like protein 10 9

E9QLZ9 Protein enabled homolog 8 1

P45591 Cofilin-2 6 12

P62746 Rho-related GTP-binding protein RhoB 5 14

Q9CPW4 Actin-related protein 2/3 complex subunit 5 5 7

Q8VDD5 Myosin-9 3 3

191

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Q69ZX3 MKIAA0866 protein 3 7

DNA replication (P00017)

Q99LF4 tRNA-splicing ligase RtcB homolog 275 257

De novo purine biosynthesis (P02738)

P54822 Adenylosuccinate lyase 30 28

E9PZF0 Nucleoside diphosphate kinase 2 3

Q9WTP7 GTP:AMP phosphotransferase, mitochondrial 7 10

P24547 Inosine-5'-monophosphate dehydrogenase 2 27 29

De novo pyrimidine deoxyribonucleotide biosynthesis (P02739)

E9PZF0 Nucleoside diphosphate kinase 2 3

De novo pyrmidine ribonucleotides biosythesis (P02740)

E9PZF0 Nucleoside diphosphate kinase 2 3

Carbamoyl-phosphate synthetase 2, aspartate B2RQC6 27 8 transcarbamylase, and dihydroorotase

Dopamine receptor mediated signaling pathway (P05912)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 P60879 Synaptosomal-associated protein 25 68 112

cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Serine/threonine-protein phosphatase PP1- P62137 4 4 alpha catalytic subunit Serine/threonine-protein phosphatase PP1-beta P62141 13 11 catalytic subunit Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit P49615 Cyclin-dependent kinase 5 8 19

Q8BTM8 Filamin-A 74 58

192

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

EGF receptor signaling pathway (P00018)

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 P84096 Rho-related GTP-binding protein RhoG 5 8

P68510 14-3-3 protein eta 90 139

E9QAN8 Protein Pik3c2b 2 2

Serine/threonine-protein phosphatase 2A P62715 9 5 catalytic subunit beta isoform Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 O70456 14-3-3 protein sigma 41 24

Q9Z268 RasGAP-activating-like protein 1 60 84

P63085 Mitogen-activated protein kinase 1 57 64

E9Q753 GTPase NRas 3 2

P62259 14-3-3 protein epsilon 106 183

P68404 Protein kinase C beta type 24 13

P63101 14-3-3 protein zeta/delta 161 296

Endogenous_cannabinoid_signaling (P05730)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1

Endothelin signaling pathway (P00019)

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta E9QAN8 Protein Pik3c2b 2 2

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha

193

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P63085 Mitogen-activated protein kinase 1 57 64

cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit P68404 Protein kinase C beta type 24 13

Enkephalin release (P05913)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit

FGF signaling pathway (P00021)

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 Q571J7 MCG141011, isoform CRA_e 26 27

Tyrosine-protein phosphatase non-receptor P35235 12 8 type 11 P68510 14-3-3 protein eta 90 139

E9QAN8 Protein Pik3c2b 2 2

Serine/threonine-protein phosphatase 2A P62715 9 5 catalytic subunit beta isoform Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 O70456 14-3-3 protein sigma 41 24

Q9Z268 RasGAP-activating-like protein 1 60 84

P63085 Mitogen-activated protein kinase 1 57 64

194

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed Serine/threonine-protein phosphatase 2A 65 Q76MZ3 35 49 kDa regulatory subunit A alpha isoform E9Q753 GTPase NRas 3 2

P62259 14-3-3 protein epsilon 106 183

P68404 Protein kinase C beta type 24 13

P63101 14-3-3 protein zeta/delta 161 296

Formyltetrahydroformate biosynthesis (P02743)

Monofunctional C1-tetrahydrofolate synthase, Q3V3R1 9 9 mitochondrial

Fructose galactose metabolism (P02744)

P17710 Hexokinase-1 80 137

GABA-B_receptor_II_signaling (P05731)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit

Gamma-aminobutyric acid synthesis (P04384)

4-aminobutyrate aminotransferase, P61922 24 52 mitochondrial P48318 Glutamate decarboxylase 1 5 2

General transcription regulation (P00023)

DNA-directed RNA polymerases I, II, and III Q923G2 5 4 subunit RPABC3 DNA-directed RNA polymerase II subunit E9Q898 12 9 RPB3

Glutamine glutamate conversion (P02745)

P26443 Glutamate dehydrogenase 1, mitochondrial 179 218

P15105 Glutamine synthetase 218 210

Glycolysis (P00024)

P09411 Phosphoglycerate kinase 1 32 24

P17183 Gamma-enolase 80 87

P21550 Beta-enolase 3 39

195

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

F7D6E8 Glyceraldehyde-3-phosphate dehydrogenase 27 21

P17710 Hexokinase-1 80 137

P47857 6-phosphofructokinase, muscle type 56 56

P12382 6-phosphofructokinase, liver type 28 31

P17182 Alpha-enolase 138 179

P52480 Pyruvate kinase isozymes M1/M2 112 189

Gonadotropin releasing hormone receptor pathway (P06664)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 Q62432 Mothers against decapentaplegic homolog 2 2 2

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 P17879 Heat shock 70 kDa protein 1B 127 207

P48036 Annexin A5 3 5

E9QMQ8 Focal adhesion kinase 1 13 20

Q7TSZ0 Heat shock protein 9 243 271

Q02248 Catenin beta-1 94 78

P39053 Dynamin-1 315 352

Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Q2NL51 Glycogen synthase kinase-3 alpha 2 2

PTK2 protein tyrosine kinase 2 beta, isoform G3X8V1 16 15 CRA_a Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P70662 LIM domain-binding protein 1 50 39

P17156 Heat shock-related 70 kDa protein 2 384 390

P63085 Mitogen-activated protein kinase 1 57 64

E9Q753 GTPase NRas 3 2

196

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit P63017 Heat shock cognate 71 kDa protein 776 783

P68368 Tubulin alpha-4A chain 408 373

5'-AMP-activated protein kinase subunit Q91WG5 1 5 gamma-2 P68404 Protein kinase C beta type 24 13

Small nuclear ribonucleoprotein-associated P63163 6 12 protein N

Hedgehog signaling pathway (P00025)

Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Q2NL51 Glycogen synthase kinase-3 alpha 2 2

cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit

Heme biosynthesis (P02746)

Q8CGC7 Bifunctional glutamate/proline--tRNA ligase 39 29

Q544X6 Ferrochelatase 5 7

Heterotrimeric G-protein signaling pathway-Gi alpha and Gs alpha mediated pathway

(P00026) Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta B1AWD9 Clathrin light chain A 33 44

Q6IRU5 Clathrin light chain B 36 80

Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Q2NL51 Glycogen synthase kinase-3 alpha 2 2

Phosphorylase b kinase gamma catalytic chain, P07934 4 2 skeletal muscle isoform Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2

197

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit Q9Z1E4 Glycogen [starch] synthase, muscle 121 121

Q9WUB3 Glycogen phosphorylase, muscle form 228 194

Heterotrimeric G-protein signaling pathway-Gq alpha and Go alpha mediated pathway

(P00027) B1AWD9 Clathrin light chain A 33 44

Q6IRU5 Clathrin light chain B 36 80

A2ALS4 Protein Rap1gap 3 7

Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P68404 Protein kinase C beta type 24 13

Heterotrimeric G-protein signaling pathway-rod outer segment phototransduction (P00028) cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1

Histamine H1 receptor mediated signaling pathway (P04385)

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 Q8K394 Inactive phospholipase C-like protein 2 4 3

P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P68404 Protein kinase C beta type 24 13

Histamine H2 receptor mediated signaling pathway (P04386)

cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Guanine nucleotide-binding protein subunit P62881 14 13 beta-5

198

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed Guanine nucleotide-binding protein G(s) Q6R0H7 11 32 subunit alpha isoforms XLas Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit

Huntington disease (P00029)

P68033 Actin, alpha cardiac muscle 1 886 238

P63260 Actin, cytoplasmic 2 638 328

P68372 Tubulin beta-4B chain 477 736

Q7TMM9 Tubulin beta-2A chain 469 714

P99024 Tubulin beta-5 chain 447 713

Q9D6F9 Tubulin beta-4A chain 394 580

Q9ERD7 Tubulin beta-3 chain 373 543

Q9JHU4 Cytoplasmic dynein 1 heavy chain 1 315 219

Q8BFZ3 Beta-actin-like protein 2 301 316

Q5SQX6 Cytoplasmic FMR1-interacting protein 2 148 150

Q7TMB8 Cytoplasmic FMR1-interacting protein 1 122 118

P17426 AP-2 complex subunit alpha-1 107 120

P61161 Actin-related protein 2 104 101

P84084 ADP-ribosylation factor 5 75 53

P17427 AP-2 complex subunit alpha-2 74 87

O08788 Dynactin subunit 1 45 29

Q6IRU5 Clathrin light chain B 36 80

B1AWD9 Clathrin light chain A 33 44

Q9R0Q6 Actin-related protein 2/3 complex subunit 1A 33 29

F7D6E8 Glyceraldehyde-3-phosphate dehydrogenase 27 21

P61205 ADP-ribosylation factor 3 25 54

Q641P0 Actin-related protein 3B 22 21

Q9Z0Y1 Dynactin subunit 3 16 6

P46638 Ras-related protein Rab-11B 11 25

Cytoplasmic dynein 1 light intermediate chain Q6PDL0 10 11 2 P62331 ADP-ribosylation factor 6 8 3

Q9CQW2 ADP-ribosylation factor-like protein 8B 7 10

P84096 Rho-related GTP-binding protein RhoG 5 8

Q9CPW4 Actin-related protein 2/3 complex subunit 5 5 7

O88456 Calpain small subunit 1 5 2

199

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Q8BGR6 ADP-ribosylation factor-like protein 15 3 5

Hypoxia response via HIF activation (P00030)

E9QAN8 Protein Pik3c2b 2 2

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3

Inflammation mediated by chemokine and cytokine signaling pathway (P00031)

P68033 Actin, alpha cardiac muscle 1 886 238

P63260 Actin, cytoplasmic 2 638 328

Q8BFZ3 Beta-actin-like protein 2 301 316

P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

P63085 Mitogen-activated protein kinase 1 57 64

Q9JM76 Actin-related protein 2/3 complex subunit 3 52 42

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 P59999 Actin-related protein 2/3 complex subunit 4 44 47

Q9CVB6 Actin-related protein 2/3 complex subunit 2 41 31

Q9R0Q6 Actin-related protein 2/3 complex subunit 1A 33 29

Q80X19 Collagen alpha-1(XIV) chain 30 19

E9PYM9 Rho-associated protein kinase 2 24 16

P68404 Protein kinase C beta type 24 13

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 PTK2 protein tyrosine kinase 2 beta, isoform G3X8V1 16 15 CRA_a cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta P84096 Rho-related GTP-binding protein RhoG 5 8

Q9CPW4 Actin-related protein 2/3 complex subunit 5 5 7

Q8K394 Inactive phospholipase C-like protein 2 4 3

Q8VDD5 Myosin-9 3 3

E9Q753 GTPase NRas 3 2

Q69ZX3 MKIAA0866 protein 3 7

Insulin/IGF pathway-mitogen activated protein kinase kinase/MAP kinase cascade (P00032) Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 P63085 Mitogen-activated protein kinase 1 57 64

Insulin/IGF pathway-protein kinase B signaling cascade (P00033)

E9QAN8 Protein Pik3c2b 2 2

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3

200

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Q2NL51 Glycogen synthase kinase-3 alpha 2 2

Integrin signalling pathway (P00034)

P63260 Actin, cytoplasmic 2 638 328

Q8BFZ3 Beta-actin-like protein 2 301 316

G5E8B8 Anastellin 129 73

Q61292 Laminin subunit beta-2 102 49

P84084 ADP-ribosylation factor 5 75 53

Q8BTM8 Filamin-A 74 58

P63085 Mitogen-activated protein kinase 1 57 64

Q9JM76 Actin-related protein 2/3 complex subunit 3 52 42

F8VQJ3 Laminin subunit gamma-1 48 25

Q9CVB6 Actin-related protein 2/3 complex subunit 2 41 31

G5E8S5 Dystrobrevin alpha, isoform CRA_e 37 28

Q9R0Q6 Actin-related protein 2/3 complex subunit 1A 33 29

Q80X19 Collagen alpha-1(XIV) chain 30 19

P61205 ADP-ribosylation factor 3 25 54

P57780 Alpha-actinin-4 24 15

Q7TPR4 Alpha-actinin-1 23 10

P97927 Laminin subunit alpha-4 22 13

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 F8VQ43 Laminin subunit alpha-2 16 6

ADP-ribosylation factor GTPase-activating Q9EPJ9 16 23 protein 1 PTK2 protein tyrosine kinase 2 beta, isoform G3X8V1 16 15 CRA_a E9QMQ8 Focal adhesion kinase 1 13 20

P62331 ADP-ribosylation factor 6 8 3

P61226 Ras-related protein Rap-2b 7 6

Q80ZJ1 Ras-related protein Rap-2a 7 4

P62746 Rho-related GTP-binding protein RhoB 5 14

Q9CPW4 Actin-related protein 2/3 complex subunit 5 5 7

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 Q8BGR6 ADP-ribosylation factor-like protein 15 3 5

E9Q753 GTPase NRas 3 2

E9QAN8 Protein Pik3c2b 2 2

O55222 Integrin-linked protein kinase 2 2

201

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Interferon-gamma signaling pathway (P00035)

Tyrosine-protein phosphatase non-receptor P35235 12 8 type 11 P63085 Mitogen-activated protein kinase 1 57 64

Interleukin signaling pathway (P00036)

Q2NL51 Glycogen synthase kinase-3 alpha 2 2

P63085 Mitogen-activated protein kinase 1 57 64

E9Q753 GTPase NRas 3 2

Ionotropic glutamate receptor pathway (P00037)

P60879 Synaptosomal-associated protein 25 68 112

Q3TXX4 Vesicular glutamate transporter 1 6 14

P43006 Excitatory amino acid transporter 2 27 87

P46460 Vesicle-fusing ATPase 308 397

Mannose metabolism (P02752)

Q922H4 Mannose-1-phosphate guanyltransferase alpha 15 13

Q8BTZ7 Mannose-1-phosphate guanyltransferase beta 7 1

Metabotropic glutamate receptor group I pathway (P00041)

P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P68404 Protein kinase C beta type 24 13

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha

Metabotropic glutamate receptor group II pathway (P00040)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 P60879 Synaptosomal-associated protein 25 68 112

cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta P61264 Syntaxin-1B 25 70

Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha Guanine nucleotide-binding protein subunit P62881 14 13 beta-5

202

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit

Metabotropic glutamate receptor group III pathway (P00039)

P60879 Synaptosomal-associated protein 25 68 112

Q3TXX4 Vesicular glutamate transporter 1 6 14

cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta P43006 Excitatory amino acid transporter 2 27 87

P61264 Syntaxin-1B 25 70

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit

Methylmalonyl pathway (P02755)

P16332 Methylmalonyl-CoA mutase, mitochondrial 2 5

Propionyl-CoA carboxylase beta chain, Q99MN9 11 4 mitochondrial

Muscarinic acetylcholine receptor 1 and 3 signaling pathway (P00042)

P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P61264 Syntaxin-1B 25 70

P68404 Protein kinase C beta type 24 13

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11

203

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5

Muscarinic acetylcholine receptor 2 and 4 signaling pathway (P00043)

Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha

Nicotine pharmacodynamics pathway (P06587)

Q8BTM8 Filamin-A 74 58

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 Serine/threonine-protein phosphatase PP1-beta P62141 13 11 catalytic subunit cAMP-dependent protein kinase catalytic P68181 8 18 subunit beta P49615 Cyclin-dependent kinase 5 8 19

Serine/threonine-protein phosphatase PP1- P62137 4 4 alpha catalytic subunit

Nicotinic acetylcholine receptor signaling pathway (P00044)

P68033 Actin, alpha cardiac muscle 1 886 238

P63260 Actin, cytoplasmic 2 638 328

204

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Q8BFZ3 Beta-actin-like protein 2 301 316

P60879 Synaptosomal-associated protein 25 68 112

P61164 Alpha-centractin 36 94

Sodium- and chloride-dependent GABA P31650 36 94 transporter 3 P61264 Syntaxin-1B 25 70

Cutaneous T-cell lymphoma-associated H3BJS0 8 1 antigen 5 homolog Q8VDD5 Myosin-9 3 3

Q69ZX3 MKIAA0866 protein 3 7

O-antigen biosynthesis (P02757)

Q99LB6 Methionine adenosyltransferase 2 subunit beta 9 9

Opioid prodynorphin pathway (P05916 )

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2

Opioid proenkephalin pathway (P05915)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 P60879 Synaptosomal-associated protein 25 68 112

Opioid proopiomelanocortin pathway (P05917)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 P60879 Synaptosomal-associated protein 25 68 112

205

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Oxytocin receptor mediated signaling pathway (P04391)

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 P60879 Synaptosomal-associated protein 25 68 112

Q8K394 Inactive phospholipase C-like protein 2 4 3

PDGF signaling pathway (P00047)

P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

Q9Z268 RasGAP-activating-like protein 1 60 84

P63085 Mitogen-activated protein kinase 1 57 64

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 P46638 Ras-related protein Rab-11B 11 25

Q8C0D4 Rho GTPase-activating protein 12 11 11

Q7TT37 Elongator complex protein 1 10 1

O35864 COP9 signalosome complex subunit 5 9 9

P62746 Rho-related GTP-binding protein RhoB 5 14

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 E9Q753 GTPase NRas 3 2

Q2NL51 Glycogen synthase kinase-3 alpha 2 2

PI3 kinase pathway (P00048)

Guanine nucleotide-binding protein G(i) B2RSH2 43 68 subunit alpha-1 Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11

206

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Q2NL51 Glycogen synthase kinase-3 alpha 2 2

Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha E9Q753 GTPase NRas 3 2

Guanine nucleotide-binding protein G(i) P08752 44 48 subunit alpha-2 P63101 14-3-3 protein zeta/delta 161 296

Parkinson disease (P00049)

P63017 Heat shock cognate 71 kDa protein 776 783

P17156 Heat shock-related 70 kDa protein 2 384 390

Q7TSZ0 Heat shock protein 9 243 271

P63101 14-3-3 protein zeta/delta 161 296

P17879 Heat shock 70 kDa protein 1B 127 207

P62259 14-3-3 protein epsilon 106 183

P68510 14-3-3 protein eta 90 139

P63085 Mitogen-activated protein kinase 1 57 64

O70456 14-3-3 protein sigma 41 24

B7ZNM7 Sept5 protein 19 51

Ubiquitin carboxyl-terminal hydrolase isozyme Q9R0P9 9 25 L1 Q9QUM9 Proteasome subunit alpha type-6 8 9

P28661 Septin-4 7 18

NADH dehydrogenase [ubiquinone] Q9D6J6 3 11 flavoprotein 2, mitochondrial O54833 Casein kinase II subunit alpha' 3 4

P68037 Ubiquitin-conjugating enzyme E2 L3 2 1

P42208 Septin-2 1 2

Pentose phosphate pathway (P02762)

Q93092 Transaldolase 3 3

P17710 Hexokinase-1 80 137

Phenylalanine biosynthesis (P02765)

P05202 Aspartate aminotransferase, mitochondrial 163 221

P05201 Aspartate aminotransferase, cytoplasmic 64 131

Phenylethylamine degradation (P02766)

Cytosolic 10-formyltetrahydrofolate Q8R0Y6 99 79 dehydrogenase

207

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

P47738 Aldehyde dehydrogenase, mitochondrial 61 55

Plasminogen activating cascade (P00050)

E9PV24 Protein Fga 125 152

Proline biosynthesis (P02768)

Q9DCC4 Pyrroline-5-carboxylate reductase 3 5 5

Pyridoxal phosphate salvage pathway (P02770)

Q8K183 Pyridoxal kinase 26 20

Pyrimidine Metabolism (P02771)

O08553 Dihydropyrimidinase-related protein 2 484 520

O35098 Dihydropyrimidinase-related protein 4 47 25

Q9EQF6 Dihydropyrimidinase-related protein 5 42 39

4-aminobutyrate aminotransferase, P61922 24 52 mitochondrial Methylmalonate-semialdehyde dehydrogenase Q9EQ20 18 16 [acylating], mitochondrial

Pyruvate metabolism (P02772)

E9QPD7 Protein Pcx 146 121

P52480 Pyruvate kinase isozymes M1/M2 112 189

Q9CZU6 Citrate synthase, mitochondrial 69 92

P14152 Malate dehydrogenase, cytoplasmic 43 86

Q80X68 Citrate synthase 34 31

Succinyl-CoA ligase [ADP/GDP-forming] Q9WUM5 21 25 subunit alpha, mitochondrial Q99KE1 NAD-dependent malic enzyme, mitochondrial 6 4

Ras Pathway (P04393)

P63085 Mitogen-activated protein kinase 1 57 64

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 P62746 Rho-related GTP-binding protein RhoB 5 14

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 E9Q753 GTPase NRas 3 2

Q2NL51 Glycogen synthase kinase-3 alpha 2 2

S adenosyl methionine biosynthesis (P02773)

Q3THS6 S-adenosylmethionine synthase isoform type-2 13 9

Salvage pyrimidine ribonucleotides (P02775)

Q9WTP7 GTP:AMP phosphotransferase, mitochondrial 7 10

E9PZF0 Nucleoside diphosphate kinase 2 3

208

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Serine glycine biosynthesis (P02776)

Q61753 D-3-phosphoglycerate dehydrogenase 29 40

Q9CZN7 Serine hydroxymethyltransferase 2 6

Succinate to proprionate conversion (P02777)

Propionyl-CoA carboxylase beta chain, Q99MN9 11 4 mitochondrial P16332 Methylmalonyl-CoA mutase, mitochondrial 2 5

Synaptic vesicle trafficking (P05734)

P46460 Vesicle-fusing ATPase 308 397

O08599 Syntaxin-binding protein 1 157 220

O88935 Synapsin-1 98 210

P63011 Ras-related protein Rab-3A 87 184

P60879 Synaptosomal-associated protein 25 68 112

Q64332 Synapsin-2 58 96

P61264 Syntaxin-1B 25 70

T cell activation (P00053)

Serine/threonine-protein phosphatase 2B P48453 93 100 catalytic subunit beta isoform P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

P63085 Mitogen-activated protein kinase 1 57 64

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 E9Q753 GTPase NRas 3 2

TCA cycle (P00051)

Q99KI0 Aconitate hydratase, mitochondrial 386 460

P97807 Fumarate hydratase, mitochondrial 76 78

Q9CZU6 Citrate synthase, mitochondrial 69 92

E9Q7L0 Protein Ogdhl 59 66

Q60597 2-oxoglutarate dehydrogenase, mitochondrial 44 45

P14152 Malate dehydrogenase, cytoplasmic 43 86

Q80X68 Citrate synthase 34 31

Isocitrate dehydrogenase [NADP], P54071 24 31 mitochondrial Succinyl-CoA ligase [ADP/GDP-forming] Q9WUM5 21 25 subunit alpha, mitochondrial Pyruvate dehydrogenase [lipoamide]] kinase Q8BFP9 13 8 isozyme 1, mitochondrial

209

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed [Pyruvate dehydrogenase [lipoamide]] kinase Q9JK42 11 14 isozyme 2, mitochondrial

TGF-beta signaling pathway (P00052)

P63085 Mitogen-activated protein kinase 1 57 64

TGF-beta-activated kinase 1 and MAP3K7- Q8CF89 7 7 binding protein 1 E9Q753 GTPase NRas 3 2

Q62432 Mothers against decapentaplegic homolog 2 2 2

Thyrotropin-releasing hormone receptor signaling pathway (P04394)

P60879 Synaptosomal-associated protein 25 68 112

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P68404 Protein kinase C beta type 24 13

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 Guanine nucleotide-binding protein subunit P62881 14 13 beta-5 Q8K394 Inactive phospholipase C-like protein 2 4 3

Toll receptor signaling pathway (P00054)

P63085 Mitogen-activated protein kinase 1 57 64

Dual specificity mitogen-activated protein P31938 17 15 kinase kinase 1 TGF-beta-activated kinase 1 and MAP3K7- Q8CF89 7 7 binding protein 1

Transcription regulation by bZIP transcription factor (P00055)

DNA-directed RNA polymerase II subunit E9Q898 12 9 RPB3 cAMP-dependent protein kinase type II-beta P31324 12 6 regulatory subunit DNA-directed RNA polymerases I, II, and III Q923G2 5 4 subunit RPABC3 Protein kinase, cAMP dependent regulatory, Q8K1M3 5 13 type II alpha

Tyrosine biosynthesis (P02784)

P05202 Aspartate aminotransferase, mitochondrial 163 221

P05201 Aspartate aminotransferase, cytoplasmic 64 131

Ubiquitin proteasome pathway (P00060)

Eukaryotic translation initiation factor 3 Q9DCH4 14 13 subunit F

210

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

26S proteasome non-ATPase regulatory Q9CR00 7 5 subunit 9 P68037 Ubiquitin-conjugating enzyme E2 L3 2 1

VEGF signaling pathway (P00056)

P63085 Mitogen-activated protein kinase 1 57 64

P68404 Protein kinase C beta type 24 13

E9QMQ8 Focal adhesion kinase 1 13 20

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 E9Q753 GTPase NRas 3 2

E9QAN8 Protein Pik3c2b 2 2

Vitamin B6 metabolism (P02787)

Q8K183 Pyridoxal kinase 26 20

Wnt signaling pathway (P00057)

Q3TKT4 Transcription activator BRG1 1388 1148

Probable global transcription activator H3BLH0 1086 1432 SNF2L2 P68033 Actin, alpha cardiac muscle 1 886 238

P97496 SWI/SNF complex subunit SMARCC1 578 581

SWI/SNF-related matrix-associated actin- Q61466 dependent regulator of chromatin subfamily D 520 573

member 1 SWI/SNF-related matrix-associated actin- Q6P9Z1 dependent regulator of chromatin subfamily D 466 452

member 3 SWI/SNF-related matrix-associated actin- Q99JR8 dependent regulator of chromatin subfamily D 223 247

member 2 Q02248 Catenin beta-1 94 78

Serine/threonine-protein phosphatase 2B P48453 93 100 catalytic subunit beta isoform P11881 Inositol 1,4,5-trisphosphate receptor type 1 88 39

Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1 O88712 C-terminal-binding protein 1 43 48

P26231 Catenin alpha-1 31 15

Guanine nucleotide-binding protein G(q) P21279 28 49 subunit alpha P68404 Protein kinase C beta type 24 13

Q63810 Calcineurin subunit B type 1 20 28

211

Spectral counts Uniprot Pathway Protein name Nicotine ID Control exposed

Guanine nucleotide-binding protein subunit P21278 19 29 alpha-11 Serine/threonine-protein phosphatase 2A P62715 9 5 catalytic subunit beta isoform P55288 Cadherin-11 8 6

P70288 Histone deacetylase 2 3 2

O54833 Casein kinase II subunit alpha 3 4

Q2NL51 Glycogen synthase kinase-3 alpha 2 2

p38 MAPK pathway (P05918)

TGF-beta-activated kinase 1 and MAP3K7- Q8CF89 7 7 binding protein 1 TGF-beta-activated kinase 1 and MAP3K7- Q99K90 5 3 binding protein 2

p53 pathway by glucose deprivation (P04397)

Serine/threonine-protein phosphatase 2A P62715 9 5 catalytic subunit beta isoform 5'-AMP-activated protein kinase subunit Q91WG5 1 5 gamma-2

p53 pathway feedback loops 2 (P04398)

Q02248 Catenin beta-1 94 78

Serine/threonine-protein phosphatase 2A P62715 9 5 catalytic subunit beta isoform Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 E9Q753 GTPase NRas 3 2

E9QAN8 Protein Pik3c2b 2 2

p53 pathway (P00059)

O70456 14-3-3 protein sigma 41 24

Serine/threonine-protein phosphatase 2A P62715 9 5 catalytic subunit beta isoform P70288 Histone deacetylase 2 3 2

Phosphatidylinositol 3-kinase catalytic subunit Q6PF93 3 2 type 3 Q8VDQ8 NAD-dependent deacetylase sirtuin-2 2 6

E9QAN8 Protein Pik3c2b 2 2

212

Discussion

Comparison of Current Proteomic Studies to Previous Related Proteomic Studies

Variability instrumentation techniques, peptide identification strategies, and database populations make comparisons with previous proteomic studies challenging.

Only two proteomic studies have examined the effects of nicotine exposure on neural tissue in a rat model (J. Wang et al., 2011; Yeom, Shim, Lee, & Hahm, 2005). Both of these studies employed MALDI-TOF mass spectrometry followed by peptide fingerprinting instead of MS/MS. Our study did not demonstrate any nicotine-induced proteins in common with the dataset generated by Yeom et al.; however, 12 proteins in our current dataset were also previously identified by Hwang et al. Each of these proteins were observed in both the nicotine-exposed and control datasets (Pyruvate kinase, muscle, Malate dehydrogenase, Mitochondrial, N-Ethylmaleimide sensitive fusion protein, Pyruvate dehydrogenase, (lipoamide) β-both, Similar to N- ethylmaleimide-sensitive factor-attachment protein β, phosphatase 2a catalytic subunit

α, Protein Heat shock protein 1, Dynamin 1, Dihydrolipoamide dehydrogenas, NADH dehydrogenase (ubiquinone) Fe-S protein, Glutamate dehydrogenase 1, unc-18 protein homolog, and ATP synthase). Although none of the proteins identified by Hwang et al. demonstrated any statistically significant change protein regulation in our datasets, one subtype of ATP synthase demonstrated up-regulation after nicotine-exposure

(ATP synthase subunit α, mitochondrial , +1.753 fold increase). Here, we present the largest dataset of identifications and employ the most technologically advanced instrumentation yet in a nicotine exposure neuroproteomics study. The MALDI-TOF approach has a large drawback in terms of ability to generate spectra rapidly and

213 analyze proteins based on peptide sequence compared to ESI-LTQ-Orbitrap instrumentation.

Correlation between Previous β-Containing nAChR Proteomic Study

Kabbani et al. conducted a proteomic study in 2007 using MALDI-TOF-TOF analysis of proteins that interacted with the M3–M4 loop of β2 containing nAChRs

(Kabbani, Woll, Levenson, Lindstrom, & Changeux, 2007). They identified 6 proteins associated with the cytoplasmic loop. Among those proteins, α-soluble NSF attachment protein was found only in nicotine-exposed datasets while Dynamin-1 was found in both the nicotine-exposed and control datasets. These two proteins may represent a common cytoplasmic interacting motif or a related intracellular communication pathway between the α7 nAChR and β2 containing nAChRs. The association between the interaction of both of these proteins with the cytoplasmic loop of the β2 subunit likely means that they interact directly with the cytoplasmic loop of the α7 nAChR as well.

Correlation between Previous α7 nAChR based Proteomic Studies

Of the 55 potential α7 nAChR interacting proteins identified in Paulo et al., 33 were re-identified here. Twenty-seven of these proteins were identified in both nicotine-exposed and control datasets; 5 were found only in nicotine-exposed datasets while and 1 was found only in control datasets, Ankyrin-2. The 5 identified proteins that were unique to the nicotine-exposed group were: Myristoylated alanine-rich C- kinase substrate , Synaptic vesicle membrane protein VAT-1 homolog-like ,

Clathrin-coated vesicle/synaptic vesicle proton pump 116 kDa subunit , Neuron- specific antigen HPC-1, and Neuronal tropomodulin . Proteins common to both the nicotine-exposed and control datasets included these 26 proteins: Adenylyl cyclase-

214 associated protein 2, AHD-M1, Glutathione S-transferase GT8.7, Adapter-related protein complex 3 subunit β-2, 22 kDa neuronal tissue-enriched acidic protein-both,

58 kDa glucose-regulated protein, Fascin, Synaptogyrin-1, 14-3-3 protein eta, 14-3-3 protein sigma, Heat shock 84 kDa, Deubiquitinating enzyme 5, Calmodulin, cAMP- dependent protein kinase catalytic subunit β, 91 kDa synaptosomal-associated protein,

Contactin-1, Adenylate cyclase-inhibiting G α protein, Guanine nucleotide-binding protein G(o) subunit α, Guanine nucleotide-binding protein α-q, Axonal membrane protein GAP-43, Protein kinase C gamma type, Protein phosphatase 1 regulatory subunit 22, Ras-related protein Rab-2A, Serine/threonine-protein phosphatase PP1-α catalytic subunit, Janusin, and Neuronal protein NP25. Two of the proteins found in both the nicotine and control datasets demonstrated up-regulation. Guanine nucleotide-binding protein G(o) subunit α demonstrated a nicotine induced up regulation of +0.672 while 22 kDa neuronal tissue-enriched acidic protein demonstrated an up-regulation of +1.335.

Although both Paulo et al. and this current study focused on isolating α7 nAChR complexes using Bgtx affinity, the methodology in this current study was refined to exclude nonspecific binding proteins. A glycine conjugated sepharose bead preclearing step was introduced to remove proteins with nonspecific binding to the α- bungarotoxin affinity beads. A high salt elution (2 M NaCl) was performed on the

Bgtx affinity beads post isolation to remove weakly associated proteins. The more stringent and exclusionary preparatory method employed here strengthens the likelihood that the 33 proteins these two studies identified in common are bona fide members of the α7 nAChR interacting complex.

215

Quantitative Proteomic Studies

Proteins Downregulated by Nicotine Exposure

ACC-α (BF=27.07, fc=-0.791) is short for acetyl-CoA carboxylase, a rate- limiting protein in fatty acid synthesis. This protein has previously been associated with decreased activity in response to chronic nicotine exposure (An et al., 2007).

Nicotine exposure induces a catabolic state in cells that induces lipolysis and inhibits fatty acid synthesis by phosphorylation of acetyl-CoA carboxylase. This observation supports finding that ACC-α would be downregulated (negative fold change) in response to chronic nicotine exposure.

Epiplakin (BF=102.684, fc=-0.699) is a protein present in epithelial cells. For example, it is expressed on the surface of keratinocytes as they organize collagen to create a scar during wound healing. It was hypothesized that epiplakin knockout mice would have difficulty with wound healing (Ishikawa et al., 2010); however, these mice paradoxically exhibit faster rates of epithelial migration and increased wound healing

(Goto et al., 2006). An association between nicotine and epiplakin has not been reported previous, but there is an association with α7-mediated signaling through epithelial cells (Egleton, Brown, & Dasgupta, 2009). The physiological relevance of nicotine downregulating epiplakin may be related to nicotine’s ameliorative effects on the course of ulcerative colitis. Ulcerative colitis induces epithelial damage in the gut through inflammation. Nicotine has been shown to improve barrier function in cultured intestinal epithelial cells through an α7 nAChR dependent pathway

(Costantini et al., 2012). It is possible that nicotine induced downregulation of epiplakin through the α7 nAChR is one of the mechanisms of accelerated intestinal epithelial healing in ulcerative colitis. 216

Lamin A (BF=20.954, fc=-0.663) is a nuclear protein that has never been previously associated with nicotine. It is pathologically linked to multiple lipodystrophies as well as Hutchinson Guilford disease. They downregulation seen here may result from nicotine induced reduction in fatty acid synthesis and increase in the catabolic state (Ruiz de Eguino et al., 2012).

Laminin subunit β-2 (BF=125.031, fc=-0.602) is an extracellular matrix protein associated with basement membranes. It is a candidate protein biomarker for

Parkinson’s disease (Grunblatt et al., 2010). Although not an exact match, Laminin receptor 1 was found to be downregulated in the Hwang et al. nicotine exposure study from 2006 as well.

Inositol 1,4,5-trisphosphate receptor type 1 (BF=288.33, fc=-0.642) is involved in calcium based signaling in several tissue types and has been linked to the physiology of the α7 nAChR previously (Suzuki et al., 2006). The activation of the α7 nAChR causes it to increase intracellular calcium and activate second messenger systems like PI3-kinase/AKT pathway, activate transcriptional systems like CREB as well as initiating proteolytic processes like the cleaving of β-amyloid into the nontoxic variant (Gahring & Rogers, 2005). The microglial α7 nAChRs are coupled directly to

PLC activation and CICR from IP3 sensitive calcium stores (Suzuki et al., 2006).

Down regulation may be a compensatory mechanism to protect against calcium toxicity in the face of the regular activation of α7 nAChRs by nicotine in chronic exposure models.

Tcf20 protein (BF=16.535, fc=-0.649) stands for transcription factor 20. It has never been associated with nicotine previously (Xiang et al., 2012). Its function is not

217 entirely defined but its downregulation indicates that chronic nicotine exposure would negatively impact the genes that Tcf20 protein regulates.

Carbamoyl-phosphate synthetase aspartate transcarbamylase, and dihydroorotase (BF=16.645, fc=-0.626) is a protein involved in the de novo synthesis of pyrimidine nucleotides (Ye, Wang, Zhang, Lu, & Yan, 2012). Although this protein has never been directly linked to nicotine previously, a decrease in pyrimidine nucleotide production has been seen in some Pseudomonas species that occurs in response to higher doses of nicotine; lower nicotine doses stimulate pyrimidine synthesis (Ye et al., 2012).

Proteins Up-regulated by Nicotine Exposure

Calcium signaling

Many of the proteins up-regulated in response to nicotine exposure have relationships to calcium signaling. Calretinin (BF=17.443, fc=+0.632), is a calcium binding protein involved in calcium signaling. Previously it has been shown to be up- regulated in response to chronic nicotine administration in a rat model (Liu, Mohila,

Gong, Govindarajan, & Onn, 2005). Calcium/calmodulin-dependent protein kinase type II subunit delta (CaMKII) (BF=17.1, fc=+0.698), another protein involved with calcium signaling, was found to be up-regulated as well. This is ignificant because

CaMKII is has been shown to play a role in long-term potentiation. Observation of its up-regulation here represents a link between the α7 nAChR, nicotine, and the cognitive enhancements seen in behavioral tests. Previous studies have demonstrated that nicotine increased CaMKII activity and phosphorylated CREB in the ventral tegmental area, nucleus accumbens, and the amygdala (Jackson, Walters, & Damaj,

218

2009). CaMKII has also been found to interact with β2 containing nAChRs as well

(Jackson et al., 2009).

Protein NipSnap homolog 1 (BF=12.162, fc= +0.585) Nipsnap1 is primarily an auxiliary protein believed to be involved in vesicular transport and regulation of L- type calcium channel activity. Closely related Nipsnap2 is directly involved with

CREB phosphorylation, a key step in long-term potentiation (Brittain, Wang, Wilson,

& Khanna, 2012)). Activation of the α7 nAChR is one initiating pathway of CREB phosphorylation through calcium dependent signaling (Gahring & Rogers, 2005). Up- regulation of Nipsnap1 could be related to increased calcium signaling by the α7 nAChR and may be related to increased release of neurotransmitters. Activation of presynaptic α7 nAChRs leads to the release of multiple neurotransmitters like norepinephrine and serotonin. Up-regulation of Protein NipSnap homolog 1 could be related to increases in this vesicular transport (Martin, de Fiebre, & de Fiebre, 2004).

Clathrin light chain B (BF=18.618, fc= +0.594) is a major component of vesicles and is involved in calcium signaling (Zhao et al., 2009). Syntaxin-1B (BF=21.21, fc=+0.715) is a SNARE protein associated with the fusion of synaptic vesicles and its up regulation could be related to an increase in neurotransmission (Zhou et al., 2012).

FK506 binding protein 1a (BF=18.351, fc=+0.901) is also known as the mammalian target of Rapamycin (mTor) and is well known to be involved with cellular proliferation and survival. It is also part of a calcium-signaling network and has been demonstrated previously to interact with both the α7 nAChR and

PPAR β/delta and has been linked to the pathology of Alzheimer’s disease (Sun et al.,

2009). Nicotine has been shown to activate α7 nAChRs and increase mTor signaling

219 leading to the proliferation of non-small cell lung carcinoma (Sun et al., 2009). This pathway would link nicotine exposure to the direct pathophysiology of non-small cell lung cancer. The most potent carcinogen associated with smoking is a chemical derivative of nicotine, NNK, which activates α7 nAChRs on the surface of epithelial cells of the lung and promotes pathological cell survival (Shen et al., 2012). This α7 based signaling has been shown to activate c-Src, PKCi and FAK all of which induce cellular survival and enhanced migration and invasion (Shen et al., 2012). Part of the

α7 nAChR signaling that promotes cellular survival may be the up-regulation of mTOR. Ras-related protein Rab-33B (BF=35.097, fc=+1.193) and Ras-related protein Rab-3A (BF=121.651, fc=+0.638) are small G-proteins in the Ras family.

Signaling through this pathway is associated with the α7 nAChR (Chernyavsky,

Arredondo, Qian, Galitovskiy, & Grando, 2009). Ras-related protein Rab-3A is involved in calcium signaling and is a target of CaMKII. In combination with the increases in other observed calcium signaling proteins up-regulation of RAB-3A fits into the pattern (Sahyoun, McDonald, Farrell, & Lapetina, 1991). It may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release

(Sahyoun et al., 1991). 14-3-3 protein zeta/delta (BF=97.657, fc=+0.609) is a modulatory protein that interacts with kinases and phosphatases. Guanine nucleotide- binding protein G(o) subunit α (BF=3069.84, fc=0.672) is part of a membrane bound G-coupled protein receptor and is highly abundant in nervous tissue and adrenal cortex and medulla (Lang & Costa, 1989).

p39 (BF=11.669, fc=+0.846) is the activator of cyclin dependent kinase 5. It is believed to be involved in the pathenogenesis of Alzheimer’s disease as well as

220 normal neuronal development (Shukla, Skuntz, & Pant, 2012). Phosphorylation of tau and neurofiliments lead to paired helical filiments and neurofibrillary tangles. Both of these proteins are phosphorylated by cyclin dependent kinase 5. Cyclin dependent kinase 5 is modulated by p39. When a neuron enters a stress state p39 is cleaved by calpain and becomes a truncated protein that can no longer adequately modulate cyclin dependent kinase 5 leading to pathological hyperphosphorylation of cytoskeletal proteins and to neurodegeneration (Shukla et al., 2012). Cdk5 is a potential therapeutic target to treat Alzheimer’s disease. The α7 nAChR is associated with neuroprotection against Alzheimer’s disease. The ability of α7 nAChR activation to up-regulate p39, a major regulatory protein for cyclin dependent kinase 5, may be a relevant physiological mechanism for this effect.

Structural Proteins

Neurofiliments are the principal cytoskeletal proteins of neurons. Several were shown to be up-regulated by chronic nicotine exposure. Many are involved in the regulation and formation of synapses indicating that the α7 nAChR may be involved in nicotine induced synapse formation including: 160 kDa neurofilament protein

(BF=33.124, fc=+0.589), 68 kDa neurofilament protein (BF=116.915, fc=+0.623),

66 kDa neurofilament protein (BF=2709.558, fc=+0.879). The 68kDa neurofiliment protein has been previously associated with nicotine. It was downregulated with nicotine exposure in the ventral tegmental area in rats (Sbarbati et al., 2002). The up- regulation of these 3 neurofiliment proteins might indicate changes to the cytoskeletal structures that correlate to the formation and creation of new synapses. It is possible that in the VTA these proteins are downregulated but globally across the CNS they experience up-regulation. Detecting this could be achieved by limiting the brain tissue 221 being analyzed to specific isolated regions rather than whole brain tissue. Other proteins in this dataset are related to synapse formation as well. Synapsin I

(BF=19320.81, fc=+0.704) is also believed to be involved in the regulation of axonal properties and synapse formation (Jackson et al., 2009). Calcium dependent pathways and CaMKII lead to phosphorylation of this protein (Jackson et al., 2009). An up- regulation of this protein is consistent with the other up-regulated calcium signaling proteins seen in this dataset. Synaptophysin (BF=52.654, fc=+0.675) is a synaptic vessel glycoprotein that participates in synaptic function and transmission. It has previously been associated with nicotine in a study involving prenatal nicotine exposure (Parameshwaran et al., 2012). In utero nicotine exposure has been linked to increased anxiety, difficulty with auditory processing, depression, and ADHD (Horst et al., 2012; Parameshwaran et al., 2012). Pups exposed to nicotine during prenatal development demonstrated decreases in synaptic plasticity and long-term potentiation, basal synaptic transmission, and AMPA receptor-mediated synaptic currents

(Parameshwaran et al., 2012). These deficits were directly correlated to a reduction in a number of proteins one of which was synaptophysin (Parameshwaran et al., 2012). If decreases in synaptophysin are related to decreases in cognitive function perhaps an up-regulation of synaptophysin could lead to enhanced cognitive performance

Synaptogyrin-1 (BF=95780.78, fc=+0.833) is an integral protein in synaptic vesicles and is believed to be involved in synaptic plasticity. The gene that encodes it is believed to be a candidate gene for schizophrenia (Cheng & Chen, 2007;

Iatropoulos et al., 2009). The α7 nAChR is also believed to be involved in the pathophysiology of schizophrenia’s sensory gating deficits (Sharma &

222

Vijayaraghavan, 2008). Schizophrenic patients are known to smoke to improve some of these deficits. It is possible that an up-regulation of synaptogyrin-1 is a downstream effector in this regard. Microtubule-associated proteins (BF=1379.483, fc=+0.755) are proteins that interact with tubulin and promote its polymerization. They have been previously connected to the α7 nAChR and the PI3K-Akt signaling pathway (Huang et al., 2012). Colchicine is a medication that inhibits microtubule formation and is used in the treatment of acute gout. One of the side effects of colchicine is neuropathy associated with deficiencies of microtubule related transport in the axon. Colchicine also has the ability to induce apoptosis in neurons. The apoptotic effects of colchicine can be thwarted through activation of the α7 nAChR (Huang et al., 2012).

22 kDa neuronal tissue-enriched acidic protein (NAP-22 or Brain acid soluble protein 1 (BF=1497.446, fc=+1.335) is a membrane bound protein that regulates the activity of synaptojanin and is heavily involved endocytosis of synaptic vesicles. NAP-22 is involved in calcium signaling and binds calmodulin. NAP-22 inhibits the phosphatase activity of synaptojanin that is responsible for uncoating vesicles (Takaichi et al., 2012). Nicotinic acetylcholine receptors, especially β2 containing receptors are up-regulated in response to nicotine exposure. The mechanism of their up-regulation is often debated but is not believed to be due to an increase in subunit expression because their respective mRNA levels do not increase in response to nicotine. One hypothesis for nAChR up-regulation is that their rate of removal by endocytosis is decreased (Govind, Vezina, & Green, 2009). The up- regulation of NAP-22 would serve to strengthen this hypothesis because its increased activity could decrease endocytosis by preventing the synaptojanin from uncoating

223 vesicles. The up-regulation of β2 containing nAChRs in response to chronic nicotine could possibly be due to signaling pathways initiated by α7 nAChRs.

Proteins involved in Ion and SmallMolecule Transport

Endomembrane proton pump 58 kDa subunit (BF=67.749, fc=+0.617) is a mouse vacuolar-type proton-translocating ATPase that acidifies vesicles (Sun-Wada,

Murakami, Nakai, Wada, & Futai, 2001). Its up-regulation could be linked to increased vesicular transport. Sodium- and chloride-dependent GABA transporter

3 (BF=743.863, fc=0.787) may be up-regulated due to increased need for GABA and increased GABAergic neurotransmission. Sodium/potassium-dependent ATPase subunit beta-1 (BF=20.773, fc=0.607) expression has been previously studied in chronic nicotine exposure studies (L. Wang, McComb, Weiss, McDonough, &

Zlokovic, 1994). Nicotine exposure was found to reduce the expression of the α2 sodium pump subunit but not affect the expression of the α1 or β1 subunits in vascular tissue (L. Wang et al., 1994). It is suggested that chronic exposure to nicotine reduces expression of functional Na/K-ATPase at the blood-brain barrier and brain by downregulating the α-isoform (L. Wang et al., 1994). Nicotine was shown to decrease cerebromicrovascular and brain Na/K ATPase enzymatic activities by 22% and 17%

(L. Wang et al., 1994). It is possible use of a mouse rather than a rat model or the use of whole brain tissue rather than selected brain regions may contribute to the differences observed in our study compared to those previously published.

Protein Synthesis and Gene Regulation

There were numerous histone-associated proteins found to be upregulated: H1

VAR.4 (BF=60.523, fc=0.845), H1 VAR.2 (BF=333.292, fc=0.962), and Histone H4

(BF=11150.29, fc=1.145). Histones control the confirmation of DNA and the ability

224 for transcription factors to access specific genes. Histone modifying proteins such as histone deacetylase have been shown to play a role in establishing conditioned place preference to nicotine in rodent models (Pastor, Host, Zwiller, & Bernabeu, 2011).

Histone acetylation has a relaxing effect on the histones and opens up DNA to transcription factor binding. Histone deacetylation would, therefore, serve to prevent the binding of transcription factors like the CREB binding protein which is associated with regulating genes responsible for synaptic plasticity and addiction (Pastor et al.,

2011). The inhibition of histone deacetylase is shown to improve memory and promote synaptic plasticity (Pastor et al., 2011). Nicotine may serve to increase the activity of histone deacetylase because the inhibition of this enzyme reduced conditioned place preference for nicotine (Pastor et al., 2011). Histone deacetylase was a protein identified as unique to the nicotine xposed data sets.

Metabolism

A number of proteins involved with ATP production or catabolism whose up- regulation accommodates the physiologic need for increased energy demands and catabolic state induced in a cell by nicotine exposure were observed. ( S)-3-amino-2- methylpropionate transaminase (BF=41.904, fc=+0.589) is a protein involved in the catabolism of branched chain amino acids (Tamaki, Sakata, & Matsuda, 2000). It is possibly up-regulated due to the catabolic state that nicotine exposure induces.

Catabolized proteins target the Kreb’s cycle to compensate for increased work demands of the cell. Glutamate/H(+) symporter 1 (BF=53.162, fc=+0.712) is a mitochondrial glutamate transporter whose up-regulation would lead to increased glutamate catabolism or export from the mitochondria after the breakdown of other

225 amino acids (Fiermonte et al., 2002). Other up-regulated mitochondrial proteins involved in energy metabolism are ATP synthase subunit α, mitochondrial

(BF=1046.91, fc=+0.753), ATP synthase subunit beta, mitochondrial

(BF=18324.58, fc=+1.037), Cytochrome c, somatic (BF=112.004,fc=+1.126) and

Phosphate carrier protein, mitochondrial (BF=25.399, fc=+0.965). The α7 nAChR has been shown to be expressed on the membranes of mitochondria where they have a role in decreasing the release of cytochrome c from the mitochondria (Gergalova et al.,

2012). Cytochrome c accumulation in the cytoplasm leads to the caspase activated apoptosis (Gergalova et al., 2012; Yu, Mechawar, Krantic, & Quirion, 2011). This mechanism of cellular protection is also relevant in nicotine-mediated neuroprotection from the toxicity of pathological β-amyloid, which involves α7 nAChR activation and

PI3K/AKT signaling (Yu et al., 2011). Nicotine is lipophilic and may activate intracellular α7 nAChRs in the mitochondrial membrane as well as those expressed in the plasma membrane. Although the physiological role for the α7 nAChR involves decreasing levels of cytoplasmic cytochrome c, it is up-regulated in this study. The α7 nAChR complexes were isolated from a homogenate that is a non-physiological state, so the regulation of cytochrome c could be differentially regulated in tissues that are combined in a homogenate. It is also possible that while the α7 nAChR may act as a gatekeeper for cytochrome c, it may not regulate its transcription.

Analysis of Qualitative Data

Pathways with Participating Proteins Identified only in Nicotine Exposed Datasets

The Vasopressin synthesis pathway, Fas signaling pathway, Adenine and hypoxanthine salvage pathway and cholesterol synthesis pathways were identified to be specific to the proteins unique to the nicotine-exposed dataset. The proteins 226 participating in those pathways were unique to them as well and did not participate in other identified pathways. Carboxypeptidase E was identified as being part of the vasopressin synthesis pathway. Nicotine has previously been shown to elevate vasopressin levels, which could be due to its increased synthesis (Maity, Biswas, Roy,

Banerjee, & Bandyopadhyay, 2003). Vasopressin may be related to nicotine’s anxiolytic effects as it activates the secretion of ACTH and corticosterone (Lutfy et al.,

2012). Increased plasma vasopressin levels are believed to increase the risk of peptic ulcer associated with smoking (Maity et al., 2003). The Fas signaling pathway had 2 participating proteins, polypolymerase family member 1 and Lamin B1. The Fas signaling pathway is one of the chief regulators of cellular apoptosis (Hiramoto et al.,

2008). The activation of the α7 nAChR has previously linked to the inhibition of FAS induced apoptosis in hepatocytes through signaling in the vagus nerve (Hiramoto et al., 2008). The cholesterol synthesis pathway had one identified participant HMG-

CoA Synthase. It is possible that a nicotine stimulated cholesterol synthesis pathway could be related to increased modification of proteins with lipophilic moieties due to increased metabolic and synaptic activity from nicotine exposure.

Pathways with Participating Proteins Identified only in Control Datasets

The Notch signaling pathway, oxidative stress response pathway, an mRNA splicing pathway, and the JAK/STAT pathway were identified to be specific to the proteins unique to the control dataset. In the cases of the Notch signaling pathway and the mRNA splicing pathway the identified proteins participated only in those pathways. The Notch signaling pathway had 3 identified proteins, protein numb homolog 1, numb like protein, and α 16 fucosyl transferase. The Notch pathway is involved with differentiation of several cell lines and signaling through it has been 227 shown to be up-regulated by prenatal nicotine exposure (Liszewski et al., 2012). The

Notch pathway is also associated with the pathophysiology of Buerger’s disease, a severe vasculitis of small and medium arteries that is seen almost exclusively in smokers (Tamai et al., 2013). The oxidative stress pathway had one protein associated with it, MAPK 14, while the JAK/STAT pathway had 2 associated proteins, MAPK

14 and Tyrosine protein kinase (JAK2). JAK2 was present only in control datasets, but the 6 other pathways that it participated in were present in proteins common to the nicotine-exposed and control datasets. The same was true for the 14 other pathways that MAPK 14 was associated with. The α7 nAChR is associated with decreasing oxidative stress through the transcriptional up-regulation of heme oxygenase, a protein that combats oxidative stress (Parada et al., 2013). Activation of the α7 nAChR is also associated with signaling through the JAK/STAT pathway and is believed to be a mechanism for its role in neuroprotection. Activation of the α7 nAChR is believed to stimulate phosphatidylinositol 3-kinase and Akt through JAK2 leading to neuroprotection (Martin et al., 2004; Shaw, Bencherif, & Marrero, 2002).

Pathways with Participating Proteins Identified in both Nicotine Exposed and Control Datasets

The pathway analysis of proteins that are seen in both the nicotine-exposed and control datasets cannot be used to make conclusions about the quantitative effects of nicotine on pathway activity. The spectral counts for proteins identified to be within a pathway do not demonstrate significant uniform increases or decreases even when one protein within the identified pathway has shown statistically significant fold change.

Almost all of the pathway components for both the ATP synthesis and Synaptic vesicle trafficking pathways demonstrate statistically significant up-regulation in

228 response to nicotine exposure. These two could be functionally related as increases in synaptic activity would require increased ATP production and metabolism. Many of the pathways and the proteins identified by the PANTHER database are related to the physiology of the α7 nAChR and the physiological effects of nicotine. The fact that the majority of proteins in this dataset are identified in all 6 data sets (C1, C2, C3, N1,

N2, N3) gives their presence additional validity. Based on the identities of the proteins seen in both the nicotine and control samples PANTHER identified 108 total pathways that were comprised of 224 different proteins. 33 of the 224 proteins can be linked to the α7 nAChR by previous biochemical experimentation, predicted association with the cytoplasmic loop of α7 nAChR and the previous proteomic studies of the α7 nAChR. The identified proteins participate in many different

PANTHER identified pathways and the 33 α7 nAChR associated proteins contribute to 73 of the 108 pathways. The multiple pathways that the α7 nAChR associated proteins participate in could represent a potentially large network of α7 nAChR related signaling.

Analysis of α7 nAChR related proteins appearing in multiple pathways

There were multiple G coupled protein receptor related proteins that were previously associated with the α7 nAChR: G(i) subunit α-2, nucleotide-binding protein, G(q) subunit α, and Guanine nucleotide-binding protein G(o) subunit α were identified in previous proteomic studies of the α7 nAChR (Paulo, Brucker, & Hawrot,

2009). Guanine nucleotide-binding protein G(o) subunit α was found to be up regulated in response to nicotine exposure in this current quantitative proteomic study indicating the possibility of increased activity of pathways that it is involved in. The

229 small G-protein Ras-related protein Rab-3A was found to be up-regulated in this current study.

The 14-3-3 are involved in a wide variety of cellular signaling pathways including cellular responses, cell cycle progression, DNA damage checkpoints (J.

Wang et al., 2011). They interact with small G-proteins like Raf as well as protein kinase C (J. Wang et al., 2011). 14-3-3 protein sigma and 14-3-3 protein eta were identified with the α7 nAChR in the α7 nAChR focused proteomic study (Paulo et al.,

2009). 14-3-3 protein zeta/delta was found to be up regulated in response to nicotine exposure in this current proteomic study.

There were many α7 nAChR related kinases that appeared multiple times in these pathways. Protein kinase A (cAMP-dependent protein kinase catalytic subunit beta, protein kinase, cAMP dependent regulatory, type II α), glycogen synthase kinase-3 α, and Casein kinase II were found to have interaction sites on the cytoplasmic loop of the α7 nAChR. Protein kinase C is frequently associated with calcium signaling and α7 nAChR activation (Shen et al., 2012). Other kinases related to calcium signaling and cell survival like protein Phosphatidylinositol 3-kinases

(Protein Pik3c2b, Phosphatidylinositol 3-kinase catalytic subunit type 3), the inositol

1,4,5-trisphosphate receptor type 1 was found to be downregulated in response to nicotine exposure in this current proteomic study (Gahring & Rogers, 2005; Hancock,

Canetta, Role, & Talmage, 2008). There was several mitogen activated protein kinases

(MAPKs) associated identified in these pathways. The cytoplasmic loop of the α7 nAChR has docking sites for MAPKs, both dual specificity MAPK and MAPK 1 were identified in both nicotine and control samples. MAPK1 was identified the α7 nAChR

230 proteomic study in Chapter 2. Many MAPKs have been associated with α7 nAChR activation (Egleton et al., 2009).

There were multiple serine/threonine-protein phosphatases (serine/threonine- protein phosphatase PP1-beta catalytic subunit α, Serine/threonine-protein phosphatase PP1-α, Serine/threonine-protein phosphatase 2A) that were previously identified in proteomic studies (Paulo et al., 2009). Activation of the α7 nAChR may activate phosphatases downstream. There is also evidence that phosphates activity increases the duration of desensitization that rapidly follows the activation of α7 nAChRs (Picciotto et al., 2001).

There were numerous structural proteins that were found to be common to these pathways as well. Actin, α cardiac muscle 1 was found to be downregulated in response to nicotine exposure in this current study while clathrin light chain B, syntaxin-1B, and Sodium- and chloride-dependent GABA transporter 3 were found to be up regulated. AP-2 complex subunit α-1, AP-2 complex subunit α-2 were both found to be related to the structural analysis of the α7 nAChR cytoplasmic loop in regards to cell cycle related destruction. Excitatory amino acid transporter 2 was identified in previous α7 nAChR proteomic studies in this dissertation (Chapter 2).

There were numerous metabolic proteins as well, fumarate hydratase, mitochondrial pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial, and pyruvate dehydrogenase [lipoamide]] kinase isozyme 2, mitochondrial were identified in the previous proteomic studies Paulo et al. and in Chapter 2 of this dissertation. Some were found to be up regulated in response to nicotine exposure:

MCG115977; ATP synthase subunit α, mitochondrial; ATP synthase subunit beta,

231 mitochondrial; and 4-aminobutyrate aminotransferase, mitochondrial. The up regulation of these proteins reflects an increased metabolic rate induced by nicotine and an increase in amino acid catabolism. Their association with the α7 nAChR could be nonspecific and driven by their high abundance or a more direct interaction with α7 nAChRs in the mitochondrial membrane (Gergalova et al., 2012).

Interaction with other signaling pathways

There were numerous common intermediates between the α7 associated proteins detected in this study and other receptor based neurotransmitter systems like histamine, α and beta adrenergic, ionotropic and metabotropic glutamate receptors, serotonin receptors, dopamine receptors, cannabinoid receptors, GABA B receptors, and oxytocin receptors. Most of the common intermediates were the identified G proteins, protein kinase A, and protein kinase C. The common intermediates to many of these pathways might indicate a significant degree of cross communication between receptor systems and allow intracellular modulation of each other’s pathways. This type of cross communication and potential modulation may explain how the physiology of some receptors are less obvious in physiology like the α7 nAChR’s role in nicotine abuse. Activation of the α7 nAChR may be able to modulate the effects of multiple other receptor systems and in turn they may compensate for it physiologicals role in knockout models.

The endocrine system and α7 nAChRs

Nicotine is well known to have effects on almost every hormone in the body

(Kapoor & Jones, 2005). Several common intermediates were found with the cortocotropin releasing factor receptor signaling pathway which may indicate a relationship between α7 nAChR signaling and the release of cortsiol. Acute nicotine

232 administration elevates plasma ACTH and corticosterone in laboratory animals (Lutfy et al., 2012; Xue et al., 2010). Corticotropin releasing hormone stimulates the release of adrenocorticotropic hormone (ACTH) from the pituitary gland, leading to increases in cortisol secretion from the adrenal gland (Lutfy et al., 2012). In humans, nicotine has been shown to suppress the gonadotropin, luteinizing hormone (LH) in males.

This effect is not present in females (Jana, Samanta, & De, 2010). The gonadotropin releasing hormone receptor pathway has common intermediates identified in both nicotine-exposed and control datasets that might indicate that is shares signaling partners with the α7 nAChR. This decreased LH secretion and lowered serum testosterone were replicated in laboratory animals exposed to nicotine (Jana et al.,

2010). This connection represents the possibility of α7 nAChR signaling leading to decreased LH secretion possibly by inhibiting release of gonadotropin releasing hormone. Another hypothesis is that elevated cortisol levels have some degree of negative feedback on gonadotropin release (Jana et al., 2010). Angiotensin II is one of the effector molecules in the mineralocortocoid pathway. An Angiotensin II signaling pathway was also found to have common intermediates with those present in the nicotine-exposed and control datasets. Activation of the α7 nAChR leads to increased signaling through the phosphatidylinositol 3-kinase and Akt pathways via JAK2

(Shaw et al., 2002). Angiotensin II, through the activation of the AT(2) receptor can block the progression of this α7 dependent pathway by inhibiting the actions of JAK2

(Shaw et al., 2002).

Vascular proliferation and α7 nAChRs

Nicotine has been noted to have proliferative effect on many cell types including endothelial cells through α7 nAChR dependent mechanisms that lead to 233 activation of calcium signaling pathways (Egleton et al., 2009). Many of the pathways identified intersect with the α7 nAChR’s role in vasculogenesis, the angiogenesis :

EGF receptor signaling pathway, VEGF signaling pathway, p38 MAPK pathway, PI3 kinase pathway, and hypoxia response via HIF activation pathways (Egleton et al.,

2009). These pathways may be common in many tissues where the α7 nAChR has been shown to have a proliferative effect.

Immune System and α7 nAChRs

Many of the identified pathways are related to the immune system. The α7 nAChR has a role in decreasing inflammation and modulating the immune system

(Filippini, Cesario, Fini, Locatelli, & Rutella, 2012). Nicotine has been known to affect the function of T-Cells, B-cells, dendritic cells (Filippini et al., 2012). The nonneuronal nAChRs are believed to signal heavily through the JAK/STAT pathway

(Filippini et al., 2012). Detection of these pathways could have resulted from isolation of α7 nAChRs on microglia or other immune cells that were present in the whole brain homogenate. Both B-Cell and T-cell activation pathways were found to have common intermediates with proteins that had previously been associated with α7 nAChRs. The identification of common α7 nAChR intermediates in the toll receptor signaling pathway , interleukin signaling pathway, and interferon-gamma signaling pathway are relevant as well. Lipopolysacchride has been shown to induce the release of cytokines by interacting with the Toll-like receptor. This is a major event in the pathogenesis of septic shock. Nicotine inhibits lipopolysaccharide induced TNF α release in rat microglia (Egleton et al., 2009). The activation of α7 nAChRs leads to a suppression of JNK and p38 MAPK that regulate the posttranscriptional steps of TNF release

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(Suzuki et al., 2006). The microglial α7 nAChRs are coupled to phospholipase C activation and CICR from IP3 sensitive calcium stores (Suzuki et al., 2006).

Opioid Signaling and the α7 nAChR

The Opioid prodynorphin pathway, Opioid proenkephalin pathway, and the

Opioid proopiomelanocortin pathway were all found to have intermediates in common with proteins found in this study and those previously associated with the α7 nAChR.

Nicotine is noted to have analgesic effects principally due to the activation of α7 nAChRs (Costa, Motta, Manjavachi, Cola, & Calixto, 2012). One of the withdrawal effects of nicotine mediated by the α7 nAChRs are the hyperalgesic effects, this would indicate that the α7 receptor had antinociceptive properties when activated (Costa et al., 2012). Nicotine is noted to alleviate visceral pain. Opiates are not effective at treating this type of pain, suggesting that α7 nAChR mediated antinociceptive mechanism may be different, but once activated it may also activate some opioidergic pathways. Previous evidence has suggested that nicotine’s behavioral effects involve opioidergic signaling. It has been reported that β-endorphin, met-enkephalin and dynorphin secretion are altered after acute or chronic nicotine treatment and during nicotine withdrawal (Hadjiconstantinou & Neff, 2011).

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CHAPTER 5: DISCUSSION AND FUTURE DIRECTIONS

Challenges in Detection of α7 nAChR Peptides

One of the primary themes of this dissertation is the use of Bgtx as a probe to both detect and isolate the α7 nAChR from in vivo tissue sources. The immunologic probing for the α7 nAChR discussed in Chapter 2 and the radiologic 125 I-Bgtx binding assays performed on Bgtx conjugated affinity beads post isolation demonstrate the ability to isolate the α7 nAChR from whole brain tissue. Despite the fact that the α7 nAChR is being enriched for, none of its peptides have ever been identified in any of the proteomics studies employing LC-ESI-LTQ or LC-ESI-LTQ-Orbitrap instrumentation. When a protein is being enriched for it should be the most abundant in a sample. A refined experimental design was developed, as described in Chapter 4, in order to minimize non-specific binding of proteins that could bind to the Bgtx affinity beads. These additional steps included preclearing with glycine-conjugated sepharose beads and post isolation high salt elution with 2 M NaCl. However, after these attempts to eliminate background proteins no α7 nAChR peptides were detected.

Perhaps the most obvious explanation is that the peptides are not being detected by the instrumentation or similarly they are identified but assigned incorrectly by the analysis algorithm (SEQUEST, MASCOT, etc). It is also possible that relatively few α7 nAChR peptides are generated by in-gel tryptic digestion and thus are less abundant than other proteins in the sample. This scenario would result in a

242 detection bias by the instrument since more abundant peptides are selected for and ion abundance informs peptide selection for tandem mass spectrometry. A third hypothesis is that the peptides generated may be more hydrophobic due to the 4 transmembrane regions of each subunit and this hydrophobicity leads to retention on the reverse phase LC column and prevents detection by the instrument. One of the advantages to using trypsin is that it cleaves after carboxyl side of arginine and lysine residues so every resulting peptide in the digest is guaranteed a basic residue. The method of ionization in ESI is based on protonation of peptides; peptides with multiple basic residues will have an ionization advantage over peptides that are more hydrophobic. Hydrophobic peptides resulting from a membrane protein like the α7 nAChR may not be easily detected because of poorer ionization ability than other in their mixture.

Only one α7 nAChR peptide was identified after an in-gel chymotryptic digest using MALDI-TOF and post source decay (Paulo, Brucker, & Hawrot, 2009). The mechanism of ionization in MALDI is not dependent on protonation from the solvent, so it is more tolerant of hydrophobic peptides than ESI. Post source decay involves the unassisted fragmentation of a peptide after it has been ionized. As a result although y and b ions predominate the spectra, there are also a, c, x, and y ions. It is much less reproducible and effective than the collision induced dissociation tandem mass spectrometry methods of the LTQ based instrumentation. Chymotrypsin is much more tolerant of hydrophobic domains compared to trypsin. The transmembrane regions of the α7 may also expose fewer arginine and lysine residues for trypsin to cleave after the carboxy-terminus. The in-gel digestions with chymotrypsin were only effective

243 one time; α7 nAChR peptide generation with this technique was not reproducible.

Although four transmembrane regions are present, more than half of the amino acids in the primary structure are devoted to extracellular regions. 207 amino acids are present in the N-terminal extracellular region, and 151 amino acids are present in the cytoplasmic loop. It is possible that the in-gel nature of the digestions leads to the development of a conformational change in the α7 nAChR that obscures these potentially accessible amino acid regions.

After SDS-PAGE is completed, the proteins within the polyacrylamide gel matrix are fixed with acetic acid. The matrix of the polyacrylamide gel could restrict some of the motion of proteases like trypsin and prevent regions of the α7 nAChR from being digested. The fixing procedure could also alter the confirmation of the α7 nAChR so that its digestible regions are less accessible. The nature of the in-gel tryptic digestion could be a reason that fewer α7 nAChR peptides are seen. The α7 nAChRs are isolated from in vivo tissue sources through detergent solubilization of the membranes that they are embedded in. Detergent is detrimental to the quality of spectra, so it must be eliminated from samples before they are introduced into the instrument. The advantage of the in-gel digest is that the detergents can be washed out of the proteins that are trapped in the gel matrix. These washing steps are essential to eliminating detergent to ensure high quality spectra. Since the α7 nAChR is isolated with detergent, it may be a necessary component to maintaining a more native confirmation. The washing steps eliminate this detergent and may cause the protein to precipitate or shift to a confirmation where the hydrophilic portions of the protein are less exposed and accessible to trypsin.

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Database searching algorithms like SEQUEST and MASCOT do not analyze spectra by biochemical means but rather assign scores based on the correlation to predicted spectra. MASCOT employs a more probabilistic model to identify spectra that are more likely to form whereas SEQUEST assigns a score based on any predicted spectrum regardless of the probability that it would exist (Steen & Mann,

2004). It is possible that modifications made as a result of acrylamide adduction or posttranslational modifications that are present in the α7 nAChR may alter the masses of the peptides and generate spectra that are not predicted and assigned to another protein.

One potential methodology for α7 nAChR peptide detection is to employ instrumentation like MALDI-TOF-TOF or MALDI-Orbitrap. This strategy would combine the hydrophobic friendly MALDI ionization technique with the potential for collision induced dissociation and algorithm assisted protein database analysis

(Rietschel et al., 2009). MALDI produces only singly charged ions that limit some of the effectiveness of collision-induced dissociation in creating product ions (Rietschel et al., 2009). MALDI-Orbitrap instrumentation does exist and may proved to be the right instrumentation with the capability of performing electron transfer dissociation

(ETD) (Frese et al., 2013). ETD creates specific c and z ions as opposed to the predominant y and b ions generated through collision induced dissociation.

Posttranslational modifications are left intact as well (Frese et al., 2013). ETD is a very robust method of generating product ions that could overcome the poorer fragmentation abilities of singly charged ions.

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Chymotrypsin was only effective at generating α7 nAChR peptides once, but the use of multiple proteases to increase peptide generation is a technique that can be employed and may effectively increase the number of α7nAChR peptides (Bian et al.,

2012). The use of the protease Glu-C, which cleaves after glutamate and aspartate, is employed as an initial protease to break down proteins before a tryptic digest is performed. It has been shown to increase the number of peptides identified (Bian et al., 2012). This method of dual digestion could be used to improve peptide formation in α7 nAChRs.

The polyacrylamide matrix is very important in the in-gel tryptic digestion of membrane proteins. Detergents are often necessary solubilizing agents in the isolation of membrane proteins. While detergents are useful in isolating these proteins they are detrimental to their analysis by mass spectrometry. The polyacryamide gel matrix acts as a sieve in the in-gel tryptic digestion protocol and holds the proteins in place as the impurities, like detergents, are washed away. In that respect the gel matrix is essential to membrane proteomics. Unfortunately the in-gel digestion process is less efficient than in-solution digestion.

The reproducibility of spectral counts between replicates of a dataset in

Chapter 4 was very good using the in-gel tryptic digestion approach, especially in the control samples where the average R 2 value for spectral count variability was 0.93.

However, this protocol involved extensive amounts of handling and 192 individual in- gel tryptic digests. Sample consistency is a very important aspect of quantitative proteomics and a large number of handling steps increases variability in samples. An in-solution digest would be preferable in many ways, because it is significantly less

246 labor intensive and requires vastly fewer experimental steps. In the case of these samples, there would have only been 6 in-solution digests as opposed to 192 in-gel digests. In solution digest would result in a more efficient generation of peptides as well as greater reproducibility. The filter-aided sample preparation (FASP) technique is a new method being used for membrane proteomics that has the cleaning potential of an in-gel digestion coupled with the efficiency and reproducibility of an in-solution digest (Yu et al., 2012). In the FASP technique a microcentrifuge spin cup filter is employed with a pore size that is small enough for peptides to pass through but not proteins (Yu et al., 2012). The microcentrifuge spin cup filter acts as a barrier during several washing steps to remove detergent and impurities. This is followed by an in solution digest and centrifugation to collect the peptides (Yu et al., 2012). This protocol could be an excellent addition to future quantitative proteomic experiments.

Refining the α7 nAChR interactome by sample selection

Murine whole brain tissue was used as the source of α7 nAChRs in all studies in this dissertation. The α7 nAChR is abundant in whole brain tissue and is present in virtually all CNS regions but in different proportions with the most abundant being in the hippocampus (Picciotto et al., 2001; Sharma & Vijayaraghavan, 2008). Because the α7 nAChR is a “neuronal nAChR” most of the implied worth of studying its interactome is to gain a greater understanding of its role in neurophysiology. The presence of α7 nAChRs in almost every tissue source within the brain including astrocytes, microglia and endothelial cells makes this a challenge (Egleton, Brown, &

Dasgupta, 2009).

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The only tissue refinement used in the studies in this dissertation was to focus only on membrane portions before the α7 nAChR complexes were isolated with Bgtx affinity beads. After the whole brain tissues were homogenized the soluble proteins were separated from the membrane proteins by ultracentrifugation and aspirated. The membrane portions were then solubilized in a detergent-containing buffer and ultracentrifugation was used again to separate the insoluble portions of the membrane.

This type of methodology selects for the membrane-associated proteins in every tissue in the brain. This would give a mix of α7 interacting proteins present in different brain regions and in different tissues as well as some potential artificial interactions that could occur once the cells were homogenized. Even if only neurons were analyzed the

α7 nAChR is heterogeneously localized on the cell membrane depending on which region of the brain the neurons are in (Sharma & Vijayaraghavan, 2008).

The α7 nAChR can be located either pre or post synaptically where it mediates different functions. Besides different locations on the plasma membrane, α7 nAChRs are found in the membranes of organelles where they serve functional roles

(Gergalova et al., 2012). For example, α7 nAChRs are present in the endoplasmic reticulum for assembly and in different vesicles for trafficking all of which could possess different protein interacting partners (Sharma & Vijayaraghavan, 2008).

Eliminating background proteins and tissues is essential to obtaining a focused interactome. Background proteins can easily dominate low abundance proteins in proteomic studies and obscure their detection (Abul-Husn & Devi, 2006). There are numerous strategies at both the gross and microscopic scale to achieve this type of separation.

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The post isolation, on-bead 125 I-Bgtx binding assays detailed in Chapter 3 seem to be an excellent tool to detect and quantify the amount of α7 nAChRs isolated from different selected anatomical and subcellular regions in order to titrate tissue needs for an experiment. Targeted gross dissection is also an excellent way to enrich for different CNS regions known to be involved in specific pathways, functionalities or pathologies involving the α7 nAChR. To increase the precision of the dissection, laser capture microdissection could also be tried (Abul-Husn & Devi, 2006). This is a frequently used technique in neuroproteomics that is capable of targeting specific populations of neurons from frozen tissue (Abul-Husn & Devi, 2006). The whole brain tissue used in the studies in this dissertation was frequently stored at -80 o C for several months before use and did not have evidence of significant loss of specific

125 I-Bgtx binding in post-isolation on bead 125 I-Bgtx binding assays. The laser capture microdissection technique could be performed to isolate the hippocampus, striatum, and VTA regions from mice exposed to nicotine on a chronic basis to compare changes in α7 nAChR interacting proteins by label free proteomic study similar to that described in Chapter 4.

The nicotine exposure studies on rats by Hwang et al. indicated that nicotine differentially expressed and that the expression levels of proteins depended on which region of the brain was being studied (Hwang & Li, 2006). A more focused gross anatomical dissection may yield different expression changes for interacting partners of the α7 nAChR based on anatomical location. Dissection by sequential coronal sections followed by punch biopsy would be another effective dissection technique

(Hwang & Li, 2006). More refined gross dissections prior to α7 nAChR isolation may

249 be an effective way to refine the α7 nAChR interactome, but it is possible to isolate different intracellular portions of isolated cell populations as well. Synaptosomes are neuronal cellular structures that contain the presynaptic nerve terminal, synaptic vesicles, mitochondria, the postsynaptic density (PSD) and parts of the post synaptic membrane (Abul-Husn & Devi, 2006). Synaptosomes can be isolated from brain tissue homogenates with sucrose gradients (Abul-Husn & Devi, 2006). Isolating α7 nAChRs from synaptosomes of dissected brain regions would allow for a highly targeted proteomic study of its interacting partners. Other subcellular structures are capable of being isolated as well. Other neuroproteomic studies have focused on synaptic membranes, PSDs, synaptic vesicles, and the presynapse (Abul-Husn & Devi, 2006).

It is possible to isolate other organelles from neurons like mitochondria using subcellular fractionation techniques (Kristian, 2010). Organelle isolation techniques could be useful to further invetigation of α7 nAChRs in specific intracellular compartments. The amount of the α7 nAChR in these tissues would need to be quantified by 125 I-Bgtx binding assay to determine the amount of tissue required for a proteomics experiment.

Utility of Qualitative Proteomic Data

Qualitative proteomic data can be difficult to interpret and may represent the identification of thousands of proteins in a single dataset. The role of the identified proteins is not always immediately obvious. In the case of the studies performed in this dissertation many of the proteins included in the datasets are structural or have roles in basic metabolism which is not uncommon for a proteomic study (Wang, Yuan,

& Li, 2011). These proteins might be closely related to the function of the α7 nAChR,

250 or they could be highly abundant background proteins that are isolated nonspecifically. In many cases these proteins serve as targets for future experimental study or points of connection for other proteomic or biochemical studies. In this dissertation the value of the hypothesis generating approach was seen in Chapter 2 with the exploration of the relationship of GNB3 and the α7 nAChR.

GNB3 appeared in 3 out of 4 datasets that represented Bgtx isolates from wildtype detergent solubilized membrane extracts (DSME), and it was never identified in a Bgtx isolates from an α7 nAChR knockout mouse (KO). Without the consistency of its identification with the α7 nAChR there never would have been a reason to suspect that it could be related to the α7 nAChR. Western blotting studies to detect

GNB3 demonstrated that it was less abundant in the DSME of KO mice than in

DSME from WT mice indicating that without the α7 nAChR its expression was decreased. GNB3 has an allelic variant, GNB3 825T, which contains a gain-of- function mutation and is linked to essential hypertension in many populations and is suspected as a candidate gene (Bae et al., 2007). The α7 KO mice demonstrated decreased baroreceptor reflexes indicating some deficit of the sympathetic nervous system. The precise relationship between the α7 nAChR and GNB3 requires further exploration. A combination of proteomic and biochemical data as well as literature review indicates the possibility of a functional relationship between these two previously unassociated proteins.

The most important aspect of the qualitative proteomic data may be consistency from replicate to replicate. In this case the proteomic data gathered in

Chapter 2 to gain further understanding of the interaction partners of the α7 nAChR in

251 its native state and in Chapter 4 to understand changes in interacting partners that occur with chronic nicotine exposure may serve as a point of reference for future biochemical studies to create a hypothesis or correlate results and future proteomic studies to establish consistency of identifications. Qualitative studies emphasize

“ruling” proteins “in” or “out” of a dataset based on whether or not they appear in a control model, in this case using a KO mouse. In affinity isolation protocols this

“ruling in and out model” operates under the assumption that the entire amount of a given protein in a cell is interacting with the target protein. An interactome constituent could interact with the target protein and be isolated with it in a complex and at the same time bind in nonspecific fashion to the affinity beads and be present in the control model as well. The nonspecific binding could occur as a property of itself, or it could be in a complex with another protein that binds to the affinity beads nonspecifically. Although it may be more of a well-defined model to rule proteins “in” and “out” based on their appearance in a negative control dataset, it might be better to shift to a “high association” and “low association” classification system. A control dataset is still valuable in these cases because if a protein is found in many replicates of a control dataset that decreases its probability of specifically being associated with an experimental dataset. Hypothetically, a protein could be identified in 10/10 experimental datasets but only 1/10 control datasets suggesting a higher association in the experimental model, but a more strict classification system would rule it out entirely because of its identification in the control dataset.

Although ruling “in” and “out” makes sense in an artificial system, it may not be a reliable way to approach proteomic data in realistic context. For example as

252 highlighted in Chapter, GLUR2 appeared in 2 out of the 4 datasets of proteins isolated from wild type whole brain DSME with Bgtx affinity and none of the datasets corresponding to the affinity isolation from KO DSME. Immunologic probing for

GLUR2 revealed that it was present in the Bgtx isolates from both wild type and KO datasets. Assuming that the antibody was specific, with enough replicates eventually

GLUR2 would have appeared in a KO dataset and would have been “ruled out”.

However, in other biochemical experiments the activation of the α7 nAChR has been shown to induce the expression of GLUR2 through the PI3k/Akt pathway (Tanaka,

Ohashi, Moue, & Kobayashi, 2012). Although that connection does not mean that the two proteins interact directly, it does imply a relationship, one that would likely have been missed if GLUR2 was excluded as an interactome member by dataset. The qualitative dataset for the nicotine exposure study is the largest neuroproteomic nicotine exposure dataset and represents a significant number of correlations and topics for future experimentation.

Quantitative Proteomic Studies

One of the most encouraging aspects of the quantitative data was the close correlation of spectral counts between replicates within a dataset. N1 was the only dataset that was divergent. The other two nicotine exposed datasets (N2, N3) and the control data sets (C1,C2,C3) had R 2 values that were above 0.90. This reflects that reproducible spectral count data can be generated form a protocol involving in-gel tryptic digests. The sample handling involved was quite extensive which increases the possibility of sample-to-sample variability within a dataset. Each dataset had 32 different in-gel tryptic digests, but the consistency between replicates was still high

253 overall. Label free quantitative proteomic studies that employ spectral counting are inexpensive and more facile to conduct than label based techniques especially when in vivo sources are used. A very compelling future proteomics study would be to compare the quantitative differences between Bgtx isolates from the DSME prepared from the whole brain and hippocampal tissues of WT and KO mice. The focus on the qualitative studies was what proteins were present or absent from the datasets. A significant number of proteins were identified in both datasets and a quantitative study could give an indication of which of those commonalities may be associated with the

α7 nAChR. This would generate more information than just involvement but also how protein expression is impacted by the loss of the α7 nAChR. This data could be confirmed by secondary quantitative analysis by western blot and densitrometry.

The quantitative nicotine exposure data revealed a number of physiologically relevant proteins. Almost every protein that was identified as up-regulated or downregulated had a prior connection to nicotine. Of all of the previous neuroproteomic investigations of nicotine exposure this study has the most advanced instrumentation and is the only quantitative mass spectrometry investigation. The other two published studies used densitrometry and mRNA levels (Hwang & Li, 2006;

Yeom, Shim, Lee, & Hahm, 2005). A future study could attempt to refine the quantitative study by using dissected brain regions rather than whole brain tissue. The study by Hwang et al. demonstrated quantitative changes that were inconsistent between neuroanatomical locations. The α7 nAChR is abundant in both the VTA and the hippocampus and those would be excellent target areas to investigate the changes in interacting proteins that are induced by nicotine.

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One of the issues with using nicotine in α7 nAChR focused proteomic investigation is the question of receptor activation. Serum levels of nicotine do not necessarily correlate to levels seen at the synapse. Measuring serum nicotine and cotinine levels can be used as additional data for the observed proteomic changes because nicotine induced proteomic changes have the potential of being dose dependent. An experiment to assess this dose dependence would be to perform a quantitative proteomic study of whole brain tissues of mice receiving regular drinking water, a low concentration nicotine water additive, and a high concentration nicotine water additive. The amount of water consumed per group of mice would be measured and nicotine and cotinine levels could be assessed at the middle and end of the exposure period. This study might indicate if protein up or down regulation is static, dose dependent, or variable. A behavioral test could be added as well to assess α7 nAChR associated behaviors during withdrawal. The α7 nAChR is associated with hyperalgesia during the withdrawal state (Grabus et al., 2005). The addition of the tail flick test to assess analgesia during nicotine administration and hyperalgesia of selected mice during a withdrawal period would reflect the activation of the α7 nAChR (Johansson et al., 2001).

The availability of α7 nAChR specific agonists and positive allosteric modulators present a unique opportunity to study α7 nAChR physiology. Nicotine activates other nAChRs as well as the α7 nAChRs. The use of α7 nAChR specific agonists and positive allosteric modulators are more targeted than nicotine (Pandya &

Yakel, 2013). A quantitative proteomic study using these agonists might present a more pure picture of α7 nAChR activation than nicotine. An interesting quantitative

255 proteomic experiment might be to compare the effects of nicotine to either an α7 specific agonist or a PAM on Bgtx isolated proteins from murine whole brain tissue or dissected hippocampal region DMSE. Dissected hippocampal tissue would be more informative, but a significant volume would be required for such studies. This may be more feasible in a rat model.

Inflammation, Neuropathic Pain, and Future α7 nAChR Experiments

The α7 nAChR is unique because many of its clinically relevant roles in physiology are not directly related to synaptic transmission. An extensive amount of research has been devoted to the α7 nAChR’s role in inflammation. Studying this pathway would be directly relevant to inflammatory physiology and alleviation of neuropathic pain

(Loram et al., 2012). The anti-inflammatory and anti-neuropathic pain properties of α7 nAChR activation may act through two closely related pathways both mechanisms are believed to result in the attenuation of cytokine production. Activation of glial cells in the spinal cord and astrocytes subsequently lead to increased cytokine production resulting in inflammation and damage of neural tissues. Αlpha-7 nAChR specific agonists were able to reverse neuropathic allodynia (Loram et al., 2012). The α7 nAChRs are expressed on macrophages, which are key mediators of inflammation.

They induce, modulate, and eventually resolve inflammation (Freitas, Ghosh, Ivy

Carroll, Lichtman, & Imad Damaj, 2013). Previous studies have demonstrated the importance of acetylcholine activating α7 nAChR on the surface of macrophages to down regulate the immune system by reducing the synthesis of proinflammatory cytokines and preventing tissue damage (Freitas et al., 2013). The spleen is a rich source of in vivo macrophages while in vitro macrophage models are readily available

256

(Lu et al., 2013). Due to their ability to provide a high and pure population of tissue, in vitro RAW264.7 cells (murine macrophage model cells) might be the preferred tissue to investigate (Lu et al., 2013). The post isolation on-bead 125 I-Bgtx binding assays would be an excellent way to assess presence and quantity of the α7 nAChR in these tissues to estimate tissue needs for a proteomic experiment. The effect of α7 nAChR specific agonists and α7 nAChR positive allosteric modulators would be more relevant in this case than nicotine as an agonist. A quantitative proteomic study could identify numerous interacting partners and give insight into the role the α7 nAChR has in their regulation. Identification of these interacting partners could lead to the development of highly targeted pharmacotherapies that would be in high demand. Neuropathic pain is extremely common and the current treatments have limited efficacy. Neuropathic pain is associated with the some of the most common diagnoses such as diabetes, strokes, and as a sequelae of resolved herpes infection (post herpetic neuralgia) (Eryilmaz,

Kocer, Kocaman, & Dikici, 2013; Nasare et al., 2013; O'Donnell et al., 2013).

Targeted and efficacious pharmacotherapy would be in high demand because neuropathic pain has significant impact on the quality of life of patients and can lead to secondary psychopathologies like depression.

Generation of Negative Control Models

The post incubation on-bead 125 I-Bgtx binding studies demonstrated the ability to create negative control models in tissues that do not have KO models through glycine conjugated sepharose beads or addition of MLA to the DSME prior to addition of

Bgtx affinity beads. This technique could be particularly useful in a study of human brain tissue where there are no KO models available. Post incubation on-bead 125 I-

257

Bgtx binding assays performed on human brain tissue yielded variable results. One study demonstrated a significant amount of specific binding but was never reproducible. Proteins are labile molecules that can degrade rapidly after death because the intracellular pH, enzymatic activities, alteration of post translational modifications, and protein confirmations can rapidly alter (Lull, Freeman, VanGuilder,

& Vrana, 2010). Studies of human brain tissue with pathology related to α7 nAChRs could be a first step in translational investigations. There is the possibility that the interactome detected would not reflect the typical in vivo interacome (Lull et al.,

2010). This is a concern for murine proteomic studies as well because the mice are asphyxiated with carbon dioxide before their whole brain tissues are removed. Despite the tissues being rapidly removed and immediately cooled with dry ice before being stored at -80 o C this process may affect the interacome. Many human postmortem brain tissue collections are not as optimal and tissues can be maintained at room temperature for varying amounts of time (Lull et al., 2010). The cause of death and length of storage time are variables as well leading to inconsistency among many human brain study datasets (Lull et al., 2010). Despite these challenges, the ability to generate negative control models would allow pathological samples from brains affected by α7 nAChR related pathologies (Alzheimer’s disease, Schizophrenia, and

Dementia with Lewy Bodies) to be the subject of α7 nAChR focused proteomic study.

258

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APPENDICES

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Table A.1: Proteins Identified Only in Nicotine-Exposed Samples

Spectral Uniprot Protein Name Counts ID N1 N2 N3 Putative uncharacterized protein F6TYB7 300 198 0 (blank) E9Q4N7 257 443 444 MCG1395 B2RSN3 233 0 0 (blank) E9QMG2 113 0 114 Trinucleotide repeat-containing gene 6C protein Q3UHC0 72 84 0 Ras-related protein Rab-3D P35276 66 0 0 Outer mitochondrial membrane protein porin 1 Q60932 58 14 0 Ras-related protein Rab-1A P62821 50 34 0 H1 VAR.1 P15864 47 0 0 Histone H2A A2AB79 46 0 30 Putative uncharacterized protein Q3UKW2 45 31 12 Myristoylated alanine-rich C-kinase substrate P26645 40 14 0 Superoxide dismutase [Cu-Zn] P08228 37 25 4 Electroneutral potassium-chloride cotransporter 2 Q91V14 37 8 0 G protein subunit beta-2 E9QKR0 32 19 0 RAB12, member RAS oncogene family A2CG35 31 0 0 65 kDa type II keratin Q99M73 30 34 55 Leukemia-associated gene protein P54227 30 3 0 Outer mitochondrial membrane protein porin 2 Q60930 29 11 0 Putative uncharacterized protein Q3TX38 27 5 0 Cytochrome c oxidase subunit IV isoform 1 A2RSV8 26 0 0 Calcium-binding mitochondrial carrier protein Q8BH59 25 0 0 Aralar1 Clathrin-coated vesicle/synaptic vesicle proton Q9Z1G4 24 3 0 pump 116 kDa subunit Complex I-75kD Q91VD9 24 1 0 Cytochrome c oxidase polypeptide VIc Q9CPQ1 22 5 0 Thymosin, beta 4, X chromosome A2AHI6 18 3 0 Cytochrome c oxidase polypeptide II P00405 17 0 0 MARCKS-like 1 B2KGE6 16 5 0 Cell death regulatory protein GRIM-19 Q9ERS2 16 4 0 Hippocalcin-like protein 1 P62748 16 1 0 Putative uncharacterized protein Gm5121 D3YW44 16 0 0 Guanine nucleotide-binding protein Q9DAS9 15 5 0 G(I)/G(S)/G(O) subunit gamma-12 G gamma-I P63213 14 8 2 H1 VAR.5 P43276 14 0 8

262

Spectral Uniprot Protein Name Counts ID N1 N2 N3 Proprotein convertase subtilisin/kexin type 1 B1AUA4 14 1 0 inhibitor Afadin E9Q9C3 13 10 20 Cyclophilin A E9Q1E3 13 12 0 Sideroflexin-3 Q91V61 13 1 0 (blank) E9Q455 13 0 0 ATP synthase subunit f, mitochondrial P56135 12 11 0 Calreticulin B2MWM9 12 0 0 Complex I-B18 Q9CR61 11 3 0 Tetraspanin-2 Q922J6 11 1 0 Putative uncharacterized protein G3X9Q2 11 0 0 Cytochrome c oxidase polypeptide Va P12787 11 0 0 Basic immunoglobulin superfamily P18572 11 0 0 Synaptotagmin II P46097 11 0 0 NEUG(55-78) P60761 11 0 0 ATP synthase subunit e, mitochondrial Q06185 11 0 0 SH3 domain-binding glutamic acid-rich-like Q91VW3 11 0 0 protein 3 Tpm1 protein B7ZNL3 10 0 6 Reticulon 1 A3QM89 10 0 0 Autocrine motility factor P06745 10 0 0 Claudin-11 Q60771 10 0 0 Neurocalcin-delta Q91X97 10 0 0 Complex I-51kD Q91YT0 9 0 0 DDAHI Q9CWS0 8 5 0 Complex I-B8 Q9CQ75 8 1 0 Mitochondrial carrier homolog 2 (C. elegans) Q9D050 8 0 0 B-cell/myeloid kinase F6UND7 7 5 10 B-cell receptor-associated protein 32 P67778 7 0 0 Dihydrolipoamide acetyltransferase component of Q8BMF4 7 0 0 pyruvate dehydrogenase complex Alpha-soluble NSF attachment protein Q9DB05 7 0 0 ATP synthase subunit d, mitochondrial Q9DCX2 7 0 0 Complex I-B14.5a Q9Z1P6 7 0 0 Aldehyde reductase Q540D7 6 1 2 Acyl-CoA-binding protein P31786 6 4 0 Cytochrome c oxidase subunit 7A2, mitochondrial P48771 6 3 0 PRA1 domain family 2 A2AEV6 6 0 0 (blank) E9PUL5 6 0 0 Mitochondrial inner membrane protein E9QLA0 6 0 0

263

Spectral Uniprot Protein Name Counts ID N1 N2 N3 B-cell receptor-associated protein BAP37 O35129 6 0 0 UPF0704 protein C6orf165 homolog Q8CDN9 6 0 0 Map3k7 protein Q923A8 5 3 8 Beta-MPP Q9CXT8 5 5 0 MCG50296 D3Z3Q9 5 3 0 MCG15083 E9PXU6 5 0 0 Lamin-B1 P14733 5 0 0 Calnexin P35564 5 0 0 Epidermal-type fatty acid-binding protein Q05816 5 0 0 Rab interacting lysosomal protein Q14AQ2 5 0 0 Myosin, heavy polypeptide 10, non-muscle Q3UH59 5 0 0 15 kDa phosphoprotein enriched in astrocytes Q62048 5 0 0 Up-regulated during skeletal muscle growth Q78IK2 5 0 0 protein 5 Sideroflexin-5 Q925N0 5 0 0 Core histone macro-H2A.1 Q9QZQ8 5 0 0 MKIAA0118 protein Q6A0C7 4 4 4 Vacuolar proton pump subunit D P57746 4 1 4 Paralemmin Q9Z0P4 4 1 0 Leucine rich repeat containing 8 family, member B B2RSI6 4 0 0 A6L P03930 4 0 0 Chromogranin-B P16014 4 0 0 Cytochrome c oxidase subunit 6B1 P56391 4 0 0 Ezrin-radixin-moesin-binding phosphoprotein 50 P70441 4 0 0 Cadherin-13 Q9WTR5 4 0 0 (blank) Q6ZQ29 3 4 0 (blank) G3X8R0 3 3 0 (blank) Q80W26 3 3 0 Beta-synuclein Q91ZZ3 3 1 0 Syntrophin acidic 1 A2AKD7 3 0 0 (blank) E9Q935 3 0 0 60S ribosomal protein L7 F6XI62 3 0 0 Neuron-specific antigen HPC-1 O35526 3 0 0 Contactin-associated protein 1 O54991 3 0 0 Complex I-13kD-A P52503 3 0 0 Protein transport protein Sec61 subunit-α isoform P61620 3 0 0 1 Ras-related protein Krev-1 P62835 3 0 0 ATP synthase-coupling factor 6, mitochondrial P97450 3 0 0 X-ray radiation resistance-associated protein 1 Q3U3V8 3 0 0

264

Spectral Uniprot Protein Name Counts ID N1 N2 N3 Leucine-rich repeat-containing protein KIAA1731 Q8BQ48 3 0 0 MCG67985 Q9CQB4 3 0 0 BTB/POZ domain-containing protein KCTD4 Q9D7X1 3 0 0 Developmentally-regulated brain protein Q9QXS6 3 0 0 N-acetyltransferase 10 Q9Z0P5 3 0 0 Cleavage stimulation factor, 3 pre-RNA, subunit 1 A2AP97 2 5 4 Putative uncharacterized protein Q540E6 2 5 4 Sorting nexin-27 Q3UHD6 2 3 2 ADP-ribosylation factor-like protein 10B Q8VEH3 2 3 2 COP9 signalosome complex subunit 7a Q9CZ04 2 3 2 Parvalbumin alpha P32848 2 4 0 GDP-D-mannose dehydratase Q8K0C9 2 0 4 Odd Oz/ten-m homolog 1 (Drosophila) A2ANM0 2 3 0 Coronin, actin binding protein 1B A2RS22 2 0 2 Limbic system-associated membrane protein Q8BLK3 2 0 2 Phosphatidate cytidylyltransferase A2AMQ4 2 1 0 Acid phosphatase 1, soluble Q4VAI2 2 1 0 MCG17262 B2RTB0 2 0 0 Dipeptidyl aminopeptidase-like protein 6 E9PWX1 2 0 0 (blank) E9PZQ0 2 0 0 (blank) E9Q309 2 0 0 (blank) E9Q8S0 2 0 0 Collagen alpha-1(XVIII) chain E9QPX1 2 0 0 Kryn F8WI17 2 0 0 Metallothionein-1 P02802 2 0 0 Sodium and chloride-dependent GABA transporter P31648 2 0 0 1 FXYD domain-containing ion transport regulator 7 P59648 2 0 0 Plexin-A1 P70206 2 0 0 Metalloendopeptidase homolog PEX P70669 2 0 0 Trafficking protein particle complex 1, isoform Q14BF8 2 0 0 CRA_b Mal, T-cell differentiation protein 2 Q2KHK7 2 0 0 MCG10089 Q4KL41 2 0 0 MKIAA4131 protein Q570Z5 2 0 0 Hydroxyacyl-coenzyme A dehydrogenase, Q61425 2 0 0 mitochondrial Membrane-associated progesterone receptor Q80UU9 2 0 0 component 2 Kindred of IgLON Q80Z24 2 0 0

265

Spectral Uniprot Protein Name Counts ID N1 N2 N3 TIP41-like protein Q8BH58 2 0 0 Malic enzyme 3 Q8BMF3 2 0 0 Nuclear membrane-binding protein Q8CGB3 2 0 0 Protein tweety homolog 1 Q9D3A9 2 0 0 Neuronal tropomodulin Q9JKK7 2 0 0 Hepatoma-derived growth factor-related protein 3 Q9JMG7 2 0 0 Gasdermin-1 Q9EST1 1 4 4 Endonuclease G-like 1 E9PZS5 1 1 4 H58 protein P40336 1 1 2 Excitatory amino acid transporter 1 P56564 1 0 8 Putative uncharacterized protein Q544R8 1 0 4 Novel ankyrin repeat domain containing protein A2ARS0 1 0 2 Aldehyde reductase G3UW73 1 0 2 120 kDa lysosomal membrane glycoprotein P11438 1 0 2 Alanine aminotransferase 1 Q566C3 1 0 2 (blank) Q8R4X3 1 0 2 Galactokinase Q9JIA6 1 0 2 Elongation factor 1-beta O70251 1 1 0 UPF0183 protein C16orf70 homolog Q922R1 1 1 0 Cob(I)alamin adenosyltransferase Q9D273 1 1 0 Neighbor of Brca1 gene 1 A1L329 1 0 0 Novel protein (C86695) A2A4E2 1 0 0 Kv channel interacting protein 3, calsenilin A2AHT3 1 0 0 Probable tubulin polyglutamylase TTLL9 A2APC3 1 0 0 MCG9726, isoform CRA_e A2AQ13 1 0 0 Protein kinase, interferon inducible double A2ATI5 1 0 0 stranded RNA dependent activator Proline-rich transmembrane protein 1 A2CG20 1 0 0 (blank) D3Z750 1 0 0 (blank) E9Q4E3 1 0 0 Protein FAM136A F7ATE7 1 0 0 Putative uncharacterized protein Pex5l F8SLQ3 1 0 0 Nuclear poly(A)-binding protein 1 G3UY42 1 0 0 (blank) G3UZJ1 1 0 0 Inositol 1,4,5-triphosphate receptor-associated G5E8V5 1 0 0 cGMP kinase substrate (blank) G5E8X1 1 0 0 Esterase 10 H3BKH6 1 0 0 Brain cyclic nucleotide-gated channel 2 O88703 1 0 0 Dystrophin P11531 1 0 0

266

Spectral Uniprot Protein Name Counts ID N1 N2 N3 High mobility group AT-hook protein 1 P17095 1 0 0 Cyclophilin B P24369 1 0 0 (blank) P33587 1 0 0 HpaII tiny fragments locus 9a protein P34022 1 0 0 Protein SOX-15 P43267 1 0 0 Cyclin-dependent kinase inhibitor 1B P46414 1 0 0 Nuclear receptor ROR-gamma P51450 1 0 0 Bcl-2-like protein 13 P59017 1 0 0 U6 snRNA-associated Sm-like protein LSm3 P62311 1 0 0 Carboxypeptidase E Q00493 1 0 0 Putative uncharacterized protein Q3UIG2 1 0 0 (blank) Q61114 1 0 0 Synaptonemal complex protein 1 Q62209 1 0 0 Bmi-1 upstream gene protein Q63829 1 0 0 CREB-regulated transcription coactivator 1 Q68ED7 1 0 0 Structural maintenance of chromosomes flexible Q6P5D8 1 0 0 hinge domain-containing protein 1 Platelet-derived growth factor receptor-like protein Q6PE55 1 0 0 Acidic leucine-rich EGF-like domain-containing Q71M36 1 0 0 brain protein Synaptic vesicle membrane protein VAT-1 Q80TB8 1 0 0 homolog-like Huntingtin-interacting protein 14 Q80TN5 1 0 0 MKIAA0719 protein Q80TT4 1 0 0 Carnitine O-palmitoyltransferase 1, brain isoform Q8BGD5 1 0 0 Tyrosine--tRNA ligase Q8BYL4 1 0 0 Carbonate dehydratase XII Q8CI85 1 0 0 Caspase recruitment domain-containing protein 11 Q8CIS0 1 0 0 Dimethylaniline monooxygenase [N-oxide- Q8K2I3 1 0 0 forming] 2 Metastasis-associated protein MTA1 Q8K4B0 1 0 0 Addicsin Q8R5J9 1 0 0 Ankyrin repeat, SAM and basic leucine zipper Q8VD46 1 0 0 domain-containing protein 1 (blank) Q925F3 1 0 0 Mediator complex subunit 1 Q925J9 1 0 0 Complex I-B15 Q9CQC7 1 0 0 Uncharacterized protein C5orf52 homolog Q9CR34 1 0 0 Tubulin polymerization-promoting protein family Q9CRB6 1 0 0 member 3

267

Spectral Uniprot Protein Name Counts ID N1 N2 N3 Coiled-coil-helix-coiled-coil-helix domain- Q9CRB9 1 0 0 containing protein 3, mitochondrial TATA box-binding protein-associated factor 1D Q9D4V4 1 0 0 Complex I-20kD Q9DC70 1 0 0 Alpha-globin transcription factor CP2 Q9ERA0 1 0 0 Cerebellin-3 Q9JHG0 1 0 0 Glycolipid transfer protein Q9JL62 1 0 0 Carboxy terminus of Hsp70-interacting protein Q9WUD1 1 0 0 Actin, aortic smooth muscle P62737 0 260 249 (blank) E9PYX4 0 75 63 Peroxiredoxin 6 Q6GT24 0 20 12 Spermatid perinuclear RNA binding protein A2BH81 0 4 10 Putative uncharacterized protein Lhfpl3 E9QN56 0 3 6 85 kDa nucleoporin Q8R480 0 4 4 (blank) Q6UJY2 0 1 4 Adaptor protein complex AP-1, gamma 1 subunit Q8CBB7 0 1 4 Epidermal TGase Q9JLF6 0 3 2 Cadherin-20 Q9Z0M3 0 1 4 Protein kinase C, epsilon B1B1C8 0 1 2 (blank) E9QNN9 0 1 2 Arginine/serine-rich coiled-coil protein 2 F8WHC5 0 1 2 Protein fat-free homolog Q3UVL4 0 1 2 Synaptosomal-associated 47 kDa protein Q8R570 0 1 2 Cytokeratin-14 Q61781 0 0 355 Alpha-tubulin 8 Q9JJZ2 0 0 59 RAB1, member RAS oncogene family Q5SW88 0 34 0 Small nuclear ribonucleoprotein Sm D2 P62317 0 0 28 Rab-13 P35283 0 0 18 Dihydrolipoyl dehydrogenase Q3TIE8 0 15 0 Histone H2B B2RTK3 0 0 14 Ataxin-2-like protein Q7TQH0 0 14 0 Arf-GAP with GTPase, ANK repeat and PH Q8BXK8 0 12 0 domain-containing protein 1 Aldehyde dehydrogenase family 1 member A1 P24549 0 0 10 (blank) E9Q8F2 0 8 0 Palladin Q9ET54 0 0 8 Putative uncharacterized protein Dsg4 D3YYT2 0 0 6 NHL repeat containing 2 F6ZR71 0 0 6 KH domain-containing, RNA-binding, signal Q9WU01 0 0 6 transduction-associated protein 2

268

Spectral Uniprot Protein Name Counts ID N1 N2 N3 Family with sequence similarity 92, member B B2RVK4 0 5 0 Aldehyde dehydrogenase family 1 member L2 Q8K009 0 5 0 (blank) A2ARZ3 0 0 4 Putative uncharacterized protein Gm5580 D3YU49 0 4 0 (blank) E9PX35 0 0 4 Importin alpha-S2 O35345 0 0 4 Calcium-activated neutral proteinase 1 O35350 0 0 4 5-AMP-activated protein kinase subunit gamma-1 O54950 0 0 4 67 kDa calelectrin P14824 0 0 4 3-hydroxy-3-methylglutaryl coenzyme A synthase P54869 0 0 4 Elongin 18 kDa subunit P62869 0 4 0 Casein kinase II subunit beta P67871 0 4 0 Echinoderm microtubule-associated protein-like 1 Q05BC3 0 0 4 (blank) Q60837 0 0 4 Hydrocephalus-inducing protein Q80W93 0 4 0 Protein unc-84 homolog B Q8BJS4 0 0 4 Putative uncharacterized protein Q8C5Z3 0 0 4 Oxysterol-binding protein Q8R2T7 0 0 4 F-box only protein 21 Q8VDH1 0 0 4 5-nucleotidase domain containing 2 Q91X76 0 0 4 ATPase WRNIP1 Q91XU0 0 0 4 (blank) Q921K2 0 4 0 Ashwin Q922M7 0 0 4 Growth arrest and DNA damage-inducible Q9CR59 0 0 4 proteins-interacting protein 1 Proteasome subunit alpha type-7-like Q9CWH6 0 4 0 Vacuolar protein sorting-associated protein 33A Q9D2N9 0 0 4 Inositol-3-phosphate synthase 1 Q9JHU9 0 0 4 Cyclophilin E Q9QZH3 0 0 4 Cullin-1 Q9WTX6 0 0 4 Centlein A2AM05 0 3 0 GAP and centrosome-associated protein A2AWA9 0 3 0 Proteasome (Prosome, macropain) 26S subunit, B2RT97 0 3 0 non-ATPase, 13 (blank) E9Q8T7 0 3 0 Disintegrin and metalloproteinase domain- E9QLC5 0 3 0 containing protein 22 (blank) F8VPK8 0 3 0 (blank) F8VPT3 0 3 0 LCA-related phosphatase P18052 0 3 0

269

Spectral Uniprot Protein Name Counts ID N1 N2 N3 Ras-related protein R-Ras2 P62071 0 3 0 Dynactin 2 Q3TPZ5 0 3 0 Putative uncharacterized protein Q3UGN1 0 3 0 BR serine/threonine-protein kinase 1 Q5RJI5 0 3 0 Oculocerebrorenal syndrome of Lowe Q6NVF0 0 3 0 Exocyst complex 84 kDa subunit Q6PGF7 0 3 0 Protein EFR3 homolog B Q6ZQ18 0 3 0 Leucine carboxyl methyltransferase 2 Q8BYR1 0 3 0 Elongation factor G 1, mitochondrial Q8K0D5 0 3 0 CCR4-associated factor 3 Q8K0V4 0 3 0 Cytoplasmic aconitase Q8VDC3 0 3 0 Ataxin-1 ubiquitin-like-interacting protein A1U Q99NB8 0 3 0 Calcyclin-binding protein Q9CXW3 0 3 0 Ethylmalonic encephalopathy protein 1 homolog Q9DCM0 0 3 0 Ankycorbin Q9EP71 0 3 0 Chorein A1ILG8 0 0 2 Phosphate cytidylyltransferase 1, choline, beta A2A450 0 0 2 isoform Ubiquitin associated domain containing 1 A2AHB3 0 0 2 Protein phosphatase 2C, magnesium dependent, A2AJQ0 0 0 2 catalytic subunit Inversin A2AM57 0 0 2 GrinchGEF A2AWP8 0 0 2 F-box and leucine-rich repeat protein 16 A2RT62 0 0 2 FK506 binding protein 1b A9E3L2 0 0 2 Nuclear factor of kappa light polypeptide gene B0V2H3 0 0 2 enhancer in B-cells inhibitor-like 1 Cyclin-dependent kinase-like 5 B1AU62 0 0 2 Sodium channel voltage-gated type II alpha 1 B1AWN6 0 0 2 PHD finger protein 3 B2RQG2 0 0 2 RIKEN cDNA E430012M05, isoform CRA_b B7ZMP1 0 0 2 Putative uncharacterized protein Churc1 D3YWJ4 0 0 2 Putative uncharacterized protein D3YXJ5 0 0 2 ENSMUSP00000098200 DPY30 domain-containing protein 1 E0CXB0 0 0 2 Deleted in liver cancer 1 protein homolog E9PXD2 0 0 2 (blank) E9Q3P4 0 0 2 (blank) E9Q440 0 0 2 B-cell antigen receptor Ig beta-associated protein 1 E9Q5D6 0 0 2 (blank) E9Q6C7 0 0 2

270

Spectral Uniprot Protein Name Counts ID N1 N2 N3 (blank) E9QAP7 0 0 2 Conserved helix-loop-helix ubiquitous kinase E9QNL4 0 0 2 (blank) F7AIF3 0 0 2 Ran GTPase-activating protein 1 F8WGD1 0 0 2 (blank) G3X8T6 0 0 2 (blank) G3X982 0 0 2 (blank) G3X9X1 0 0 2 Coiled-coil domain-containing protein C1orf110 H3BLK0 0 0 2 homolog 60 kDa SS-A/Ro ribonucleoprotein O08848 0 0 2 Neural cell adhesion molecule 2 O35136 0 0 2 Hrs-binding protein O88811 0 0 2 3T3-L1 lipid-binding protein P04117 0 0 2 47 kDa heat shock protein P19324 0 0 2 DNA primase 49 kDa subunit P20664 0 0 2 (blank) P34960 0 0 2 Calcium signal-modulating cyclophilin ligand P49070 0 0 2 DnaJ homolog subfamily C member 2 P54103 0 0 2 Augmenter of liver regeneration P56213 0 0 2 26S protease regulatory subunit 10B P62334 0 0 2 60S ribosomal protein L10-like P86048 0 0 2 Carnitine O-palmitoyltransferase 1, liver isoform P97742 0 0 2 Ccbl1 protein Q05CI8 0 0 2 Usher syndrome type IIa protein homolog Q2QI47 0 0 2 Putative uncharacterized protein Q3TJL3 0 0 2 Trichoplein keratin filament-binding protein Q3TVW5 0 0 2 Putative uncharacterized protein Q3U1V3 0 0 2 EV and lactate/malate dehydrogenase domain- Q3U1V6 0 0 2 containing protein Valine--tRNA ligase Q3U2A8 0 0 2 Protein zyg-11 homolog B Q3UFS0 0 0 2 Putative uncharacterized protein Q3UH70 0 0 2 (blank) Q3UH99 0 0 2 Neuron-associated developmentally-regulated Q3UIA2 0 0 2 protein GRAM domain-containing protein 2 Q3V3G7 0 0 2 Putative uncharacterized protein Q58E59 0 0 2 Reeler protein Q60841 0 0 2 Adenomatous polyposis coli protein Q61315 0 0 2 3-dehydrosphinganine reductase Q6GV12 0 0 2

271

Spectral Uniprot Protein Name Counts ID N1 N2 N3 Ninein-like protein Q6ZQ12 0 0 2 Cardiomyopathy-associated protein 5 Q70KF4 0 0 2 Corneodesmosin Q7TPC1 0 0 2 Taste receptor type 2 member 136 Q7TQA8 0 0 2 Slit homolog 1 protein Q80TR4 0 0 2 Hypertension-related protein 1 Q80U63 0 0 2 Zinc finger protein 618 Q80YY7 0 0 2 Lipid phosphate phosphatase-related protein type 1 Q8BFZ2 0 0 2 Amyloid-beta protein intracellular domain- Q8BIZ1 0 0 2 associated protein 1 Protein TMEM103 Q8BK75 0 0 2 Protein FAM53C Q8BXQ8 0 0 2 Putative uncharacterized protein Q8C0V2 0 0 2 Putative uncharacterized protein Q8C3P2 0 0 2 Putative uncharacterized protein Q8C725 0 0 2 3-5 RNA exonuclease OLD35 Q8K1R3 0 0 2 Disintegrin and metalloproteinase domain- Q8K410 0 0 2 containing protein 32 ESCRT-I complex subunit VPS37B Q8R0J7 0 0 2 1-phosphatidylinositol-4,5-bisphosphate Q8R3B1 0 0 2 phosphodiesterase delta-1 Ether-a-go-go potassium channel 2 Q920E3 0 0 2 PP2A subunit B isoform B55-delta Q925E7 0 0 2 Uncharacterized protein C6orf203 homolog Q9CQF4 0 0 2 Uncharacterized protein C12orf45 homolog Q9CX66 0 0 2 Putative uncharacterized protein Q9CZ86 0 0 2 Chemokine-like factor superfamily member 5 Q9D6G9 0 0 2 Autophagy-related protein 101 Q9D8Z6 0 0 2 Alcohol dehydrogenase PAN2 Q9ERI6 0 0 2 SH3 domain-binding glutamic acid-rich-like Q9JJU8 0 0 2 protein Neutral sphingomyelinase 2 Q9JJY3 0 0 2 Thioredoxin reductase 1, cytoplasmic Q9JMH6 0 0 2 (blank) Q9QUG2 0 0 2 Chromobox homolog 1 (Drosophila HP1 beta) A2A6C8 0 1 0 Oxysterol-binding protein-related protein 9 A2A8Z1 0 1 0 Ribose-phosphate pyrophosphokinase A2AHI4 0 1 0 Interleukin 6 A2RTD1 0 1 0 Lysophospholipase 2 B1AV56 0 1 0 Phosphodiesterase 4B, cAMP specific B1AWC9 0 1 0

272

Spectral Uniprot Protein Name Counts ID N1 N2 N3 Nebulette B7ZCI2 0 1 0 Putative uncharacterized protein 4631416L12Rik D3YYY8 0 1 0 (blank) E9PWP3 0 1 0 R3hdm1 protein E9Q9Q2 0 1 0 Rab GTPase-binding effector protein 1 E9QPP9 0 1 0 (blank) F7A0B0 0 1 0 Otopetrin-3 F8VQ32 0 1 0 (blank) F8WI30 0 1 0 Small ubiquitin-related modifier 3 G3UZA7 0 1 0 (blank) H3BKZ0 0 1 0 Alpha-synuclein O55042 0 1 0 MK3 P16390 0 1 0 Arylsulfatase G Q3TYD4 0 1 0 (blank) Q3UHC7 0 1 0 F-box only protein 2 Q3USR5 0 1 0 Keratin associated protein 1-4 Q3V2D6 0 1 0 ATPase, H+ transporting, lysosomal V1 subunit Q5HZY7 0 1 0 G1 E3 ubiquitin-protein ligase TRIM41 Q5NCC3 0 1 0 DBF4-type zinc finger-containing protein 2 Q5SS00 0 1 0 homolog Breast cancer anti-estrogen resistance protein 1 Q61140 0 1 0 (blank) Q61754 0 1 0 ESCRT-I complex subunit MVB12B Q6KAU4 0 1 0 THAP domain-containing protein 4 Q6P3Z3 0 1 0 Inositol monophosphatase 3 Q80V26 0 1 0 (blank) Q80YD6 0 1 0 PH domain leucine-rich repeat-containing protein Q8BXA7 0 1 0 phosphatase 2 PIAS-like protein Zimp7 Q8CIE2 0 1 0 2,5-oligoadenylate synthetase-like 10 Q8VI93 0 1 0 Endothelial differentiation inhibitory protein D10 Q91WC0 0 1 0 (blank) Q91YK2 0 1 0 Lens epithelium-derived growth factor Q99JF8 0 1 0 Uncharacterized aarF domain-containing protein Q9D0L4 0 1 0 kinase 1 Coiled-coil domain-containing protein 54 Q9DAL3 0 1 0 4-trimethylaminobutyraldehyde dehydrogenase Q9JLJ2 0 1 0 (blank) Q9Z2U2 0 1 0

273

Table A.2: Proteins Identified Only in Control Samples

Uniprot Spectral Counts Protein Name ID C1 C2 C3 67 kDa cytokeratin P04104 347 0 0 Ankyrin-2 Q8C8R3 66 52 76 MCG133649, isoform CRA_a B2RQQ1 41 0 8 Myosin, heavy polypeptide 7, cardiac B2RXX9 41 0 0 muscle, beta Myosin heavy chain 1 Q5SX40 41 0 0 Myosin heavy chain 8 P13542 39 0 0 Myosin, heavy polypeptide 2, skeletal G3UW82 38 0 0 muscle, adult Calcium pump 1 Q8R429 29 0 0 Plasma membrane Ca++ transporting ATPase E9Q828 27 0 0 4 splice variant b Afadin E9Q316 22 13 16 U1 small nuclear ribonucleoprotein A Q62189 15 0 33 Receptor mediated endocytosis-8 D4AFX7 13 11 4 Calcium/calmodulin-dependent protein Q923T9 11 0 14 kinase type II subunit gamma Kinesin heavy chain isoform 5A P33175 10 9 0 SM22-beta Q9WVA4 10 0 0 Copine IX Q1RLL3 8 0 0 Tropomyosin 3, gamma E9Q5J9 7 0 0 Alpha-actinin skeletal muscle isoform 3 O88990 7 0 0 ATP-dependent RNA helicase eIF4A-1 P60843 7 0 0 ABP-280-like protein Q80X90 7 0 0 Actin-related protein 1B Q8R5C5 7 0 0 High density lipoprotein (HDL) binding Q3U4Z7 6 3 4 protein, isoform CRA_d Alpha-PAK O88643 6 5 0 Trafficking protein particle complex subunit F8VQF9 6 3 0 10 Alpha-actinin skeletal muscle isoform 2 G3UW84 6 0 0 CRK1 P47811 6 0 0 Maspin P70124 6 0 0 Ikaros family zinc finger protein 2 P81183 6 0 0 Putative uncharacterized protein Q3TYK4 6 0 0 60S ribosomal protein L10 Q6ZWV3 6 0 0

274

Uniprot Spectral Counts Protein Name ID C1 C2 C3 JNK3 alpha1 protein kinase Q80W82 6 0 0 Protein phosphatase 1 glycogen-associated Q99MR9 6 0 0 regulatory subunit APG7-like Q9D906 6 0 0 Mitogen activated protein kinase kinase Q543B5 4 5 10 kinase 7 Talin-1 F8WGT0 4 5 2 Putative uncharacterized protein Bcl7c D3Z3W8 4 0 12 Calmodulin P62204 4 6 0 Heterogeneous nuclear ribonucleoprotein M B8JK30 4 0 4 Microtubule-associated protein 4 F7CK47 4 0 4 MBK1 P16388 4 0 4 Casein kinase II subunit alpha Q60737 4 0 4 Disks large homolog 4 Q62108 4 0 4 Drosophila retinal degeneration B homolog 2 Q6ZPQ6 4 0 4 PAB-dependent poly(A)-specific H3BKF3 4 3 0 ribonuclease subunit 3 60S ribosomal protein L8 P62918 4 3 0 Proteasome subunit alpha type Q542H2 4 3 0 Membrane-organizing extension spike P26041 4 2 0 protein Exocyst complex component 1 Q6P1Y9 4 2 0 Cell cycle and apoptosis regulatory protein 1 Q8CH18 4 2 0 Coactivator independent of AF-2 Q91W39 4 2 0 Zinc finger CCHC domain-containing protein Q9CYA6 4 0 2 8 Connectin A2ASS6 4 0 0 Putative uncharacterized protein E9Q2W5 4 0 0 (blank) E9Q912 4 0 0 Channel-associated protein of synapse-110 F6T8G5 4 0 0 Laminin A chain P19137 4 0 0 Cytovillin P26040 4 0 0 (blank) P35761 4 0 0 Phosphorylase b kinase regulatory subunit Q7TSH2 4 0 0 beta Distinct subgroup of the Ras family member Q91Z61 4 0 0 1 Diadenosine tetraphosphate synthetase Q9CZD3 4 0 0 Insulin protease Q9JHR7 4 0 0 Activin receptor-interacting protein 1 Q9WVQ1 3 6 4 Novel protein containing six WD40 domains A2ACM0 3 3 6 at C-terminus

275

Uniprot Spectral Counts Protein Name ID C1 C2 C3 Rho GTPase-activating protein 14 F8VPQ4 3 3 6 Nudix (Nucleoside diphosphate linked B1B0E7 3 3 4 moiety X)-type motif 10 40S ribosomal protein S15 P62843 3 39 0 Janus kinase 2 G5E852 3 0 4 Protein transport protein Sec24A Q3U2P1 3 0 4 MKIAA4058 protein Q571A6 3 0 4 Putative uncharacterized protein Q3TTV6 3 3 0 Ras-related GTP binding D B1AWT3 3 2 0 Putative uncharacterized protein Gm7263 D3YXU0 3 0 2 Chromodomain helicase DNA binding E9PYL1 3 0 2 protein 5 EH and SH3 domains protein 1 E9Q0N0 3 2 0 PIAS-like protein Zimp10 Q6P1E1 3 0 2 (blank) A2A9B9 3 0 0 Neuronal pentraxin 1 A2ACL9 3 0 0 Putative uncharacterized protein Dip2a D3Z7D3 3 0 0 MCG7378 E9PW43 3 0 0 Brain protein 44-like protein E9Q0V4 3 0 0 (blank) F6UCX4 3 0 0 80 kDa M-calpain subunit O08529 3 0 0 Beta-dystrobrevin O70585 3 0 0 Alpha-1 type I collagen P11087 3 0 0 Phospholipase A-2-activating protein P27612 3 0 0 Cyclin-A2 P51943 3 0 0 Gamma-tubulin complex component 3 P58854 3 0 0 Inositol polyphosphate 5-phosphatase J P59644 3 0 0 Dual specificity mitogen-activated protein P70236 3 0 0 kinase kinase 6 Putative uncharacterized protein Q3U111 3 0 0 Zinc finger and SCAN domain-containing Q3URR7 3 0 0 protein 10 Phosphate cytidylyltransferase 2, Q3USD5 3 0 0 ethanolamine MCG19050, isoform CRA_d Q5BKQ9 3 0 0 Akt phosphorylation enhancer Q5SNZ0 3 0 0 17 kDa myosin light chain Q60605 3 0 0 Guanine nucleotide exchange factor H1 Q60875 3 0 0 Arginase-1 Q61176 3 0 0 Chondroitin sulfate proteoglycan core protein Q62059 3 0 0 2

276

Uniprot Spectral Counts Protein Name ID C1 C2 C3 Diacylglycerol kinase theta Q6P5E8 3 0 0 Rho guanine nucleotide exchange factor 18 Q6P9R4 3 0 0 Trinucleotide repeat-containing gene 18 Q80WC3 3 0 0 protein Protein FAM49A Q8BHZ0 3 0 0 Melanoma inhibitory activity protein 3 Q8BI84 3 0 0 Hsc20 Q8K3A0 3 0 0 (blank) Q99MZ3 3 0 0 Prefoldin 1 Q9CQF7 3 0 0 CYPIIC55 Q9D816 3 0 0 2-methyl branched chain acyl-CoA Q9DBL1 3 0 0 dehydrogenase NIF3-like protein 1 Q9EQ80 3 0 0 RanBP-type and C3HC4-type zinc finger- Q9WUB0 3 0 0 containing protein 1 Fox-1 homolog C Q8BIF2 1 8 8 Eukaryotic translation initiation factor 4 Q6NZJ6 1 6 8 gamma 1 Mediator complex subunit 12 A2AGH6 1 3 8 A18 hnRNP P60824 1 2 4 Cystatin C A2APX2 1 2 2 Signal transducing adapter molecule 1 P70297 1 0 8 Zinc finger protein 326 O88291 1 0 6 Solute carrier family 1 (High affinity B2RQX4 1 0 4 aspartate/glutamate transporter), member 6 Deubiquitinating enzyme 10 P52479 1 0 4 EH-domain containing 4 Q3TM70 1 0 4 Protein phosphatase 1H Q3UYC0 1 0 4 Adapter-related protein complex 3 sigma-2 Q8BSZ2 1 0 4 subunit 50 kDa nucleoporin Q9JIH2 1 0 4 Protein numb homolog Q9QZS3 1 0 4 Metastasis-associated 1-like 1 Q9R190 1 0 4 Sidekick homolog 2 (Chicken) A2A6P3 1 3 0 Diacylglycerol kinase zeta A2AHK0 1 3 0 Gamma-tubulin complex component 2 G5E859 1 3 0 Dedicator of cytokinesis protein 7 Q8R1A4 1 3 0 Putative uncharacterized protein Q9CZ46 1 3 0 Neuroligin 4 B0F2B4 1 2 0 PDZ domain containing 8 B9EJ80 1 2 0 Putative uncharacterized protein Safb D3YXK2 1 0 2

277

Uniprot Spectral Counts Protein Name ID C1 C2 C3 Putative uncharacterized protein D3YZD8 1 2 0 1810020D17Rik (blank) E0CXG6 1 2 0 Glycogen synthase kinase 3 beta E9QAQ5 1 2 0 (blank) F2Z408 1 2 0 (blank) F6THF3 1 2 0 MCG121875 F6TIC7 1 0 2 CAZ-associated structural protein 2 F8VPM7 1 2 0 Apolipoprotein J Q06890 1 2 0 Putative uncharacterized protein Q3UNV7 1 2 0 Putative uncharacterized protein Q3UZG4 1 0 2 Heterogeneous nuclear ribonucleoprotein Q6PAK3 1 2 0 methyltransferase-like protein 4 3D3/LYRIC Q80WJ7 1 0 2 RNA-binding motif protein 12B-A Q80YR9 1 2 0 WD repeat-containing protein 26 Q8C6G8 1 2 0 (blank) Q8CE13 1 2 0 NmrA-like family domain-containing protein Q8K2T1 1 2 0 1 Ribonuclease inhibitor Q91VI7 1 2 0 26S proteasome non-ATPase regulatory Q99JI4 1 0 2 subunit 6 Diadenosine 5,5-P1,P6-hexaphosphate Q9JI46 1 2 0 hydrolase 1 Alpha glucosidase 2 alpha neutral subunit A1A4T2 1 0 0 Troponin C2, fast A2A4Z2 1 0 0 Proteasome subunit beta type A2A882 1 0 0 Histamine N-methyltransferase A2AQK4 1 0 0 Ral GEF with PH domain and SH3-binding A2AR50 1 0 0 motif 1 ATPase class I type 8B member 2-like A3FIN4 1 0 0 protein Forkhead-associated domain-containing A6PWD2 1 0 0 protein 1 MCG3895, isoform CRA_a A8Y5G1 1 0 0 Putative transmembrane protein A9DA50 1 0 0 mV/BamHI#3 Proteasome (Prosome, macropain) 26S B7ZCF1 1 0 0 subunit ATPase 3 G protein-coupled receptor 98 B8JJE0 1 0 0 Putative uncharacterized protein Gm5481 D3YU72 1 0 0 Putative uncharacterized protein Mtap7 D3YWN7 1 0 0

278

Uniprot Spectral Counts Protein Name ID C1 C2 C3 MCG11021 D3Z4S3 1 0 0 Signal-regulatory protein alpha E0CYM8 1 0 0 (blank) E9PV03 1 0 0 Putative uncharacterized protein E9PWL0 1 0 0 (blank) E9PYB0 1 0 0 Novel protein E9Q1W1 1 0 0 (blank) E9Q2C0 1 0 0 Major vault protein E9Q3X0 1 0 0 (blank) E9Q4X4 1 0 0 (blank) E9QKF1 1 0 0 Putative uncharacterized protein Bat2l2 E9QKG5 1 0 0 (blank) F6SAA7 1 0 0 40S ribosomal protein S17 F7BBI3 1 0 0 Adam3 protein F8VQ03 1 0 0 Protein quaking F8WHX7 1 0 0 Hornerin F8WJ23 1 0 0 (blank) G3UWG0 1 0 0 SEC23-interacting protein G3X928 1 0 0 Bromodomain and WD repeat-containing G5E8J3 1 0 0 protein 2 Phosphatase and actin regulator 1 G5E8P7 1 0 0 Scaffold protein Pbp1 O08992 1 0 0 RalBP1-associated Eps domain-containing O54916 1 0 0 protein 1 B4 integrin interactor O55135 1 0 0 DRB sensitivity-inducing factor large subunit O55201 1 0 0 67 kDa neutrophil oxidase factor O70145 1 0 0 Putative RNA-binding protein 3 O89086 1 0 0 Proto-oncogene A-Raf P04627 1 0 0 Neuronal proto-oncogene tyrosine-protein P05480 1 0 0 kinase Src Adenine phosphoribosyltransferase P08030 1 0 0 Activated RNA polymerase II transcriptional P11031 1 0 0 coactivator p15 Plasminogen activator inhibitor 2, P12388 1 0 0 macrophage (blank) P29121 1 0 0 65 kDa glutamic acid decarboxylase P48320 1 0 0 IRES-specific cellular trans-acting factor 45 P50580 1 0 0 kDa (blank) P52843 1 0 0 Carbonic anhydrase-related protein 10 P61215 1 0 0 279

Uniprot Spectral Counts Protein Name ID C1 C2 C3 Adapter-related protein complex 1 sigma-1A P61967 1 0 0 subunit Protein Wnt-10a P70701 1 0 0 Cell division protein kinase 2 P97377 1 0 0 Annexin A4 P97429 1 0 0 Alpha-2 type I collagen Q01149 1 0 0 (blank) Q07139 1 0 0 Nucleolar protein 8 Q3UHX0 1 0 0 Putative uncharacterized protein Q4VAE6 1 0 0 Chorea-acanthocytosis protein homolog Q5H8C4 1 0 0 Exocyst complex component 2 Q5SUZ6 1 0 0 Metavinculin Q64727 1 0 0 Aminopeptidase-like 1 Q6NSR8 1 0 0 Cysteine-S-conjugate beta-lyase 2 Q71RI9 1 0 0 (blank) Q7M766 1 0 0 Disheveled-associated activator of Q80U19 1 0 0 morphogenesis 2 Protein SDA1 homolog Q80UZ2 1 0 0 Activator of 90 kDa heat shock protein Q8BK64 1 0 0 ATPase homolog 1 RNA-binding Raly-like protein Q8BTF8 1 0 0 Ankyrin repeat domain-containing protein 46 Q8BTZ5 1 0 0 Myc-induced nuclear antigen Q8CD15 1 0 0 Aldehyde dehydrogenase family 4 member Q8CHT0 1 0 0 A1 F-box only protein 45 Q8K3B1 1 0 0 Ankyrin repeat and SAM domain-containing Q8K3X6 1 0 0 protein 4B Eukaryotic peptide chain release factor GTP- Q8R050 1 0 0 binding subunit ERF3A Tripartite motif-containing protein 29 Q8R2Q0 1 0 0 (blank) Q8R2Q4 1 0 0 Hcp beta-lactamase-like protein C1orf163 Q921H9 1 0 0 homolog Loss of heterozygosity 11 chromosomal Q99KC8 1 0 0 region 2 gene A protein homolog Neurexin I-alpha Q9CS84 1 0 0 Ubiquinone biosynthesis methyltransferase Q9CXI0 1 0 0 COQ5, mitochondrial Mediator complex subunit 10 Q9CXU0 1 0 0 Elongation factor p18 Q9D1M4 1 0 0

280

Uniprot Spectral Counts Protein Name ID C1 C2 C3 CDKN2A-interacting protein N-terminal-like Q9D211 1 0 0 protein 70 kDa WD repeat tumor rejection antigen Q9D2V7 1 0 0 homolog (blank) Q9D7N9 1 0 0 Docking protein alpha Q9DBG7 1 0 0 Bifunctional coenzyme A synthase Q9DBL7 1 0 0 UPF0498 protein KIAA1191 Q9DBN4 1 0 0 Nuclear distribution protein nudE-like 1 Q9ERR1 1 0 0 15 kDa selenoprotein Q9ERR7 1 0 0 Leucine-rich glioma-inactivated protein 1 Q9JIA1 1 0 0 Trafficking protein particle complex subunit Q9JME7 1 0 0 2-like protein Protein FAM50A Q9WV03 1 0 0 3(2),5-bisphosphate nucleotidase 1 Q9Z0S1 1 0 0 MCG49525 D3Z1I3 0 3 6 Valyl-tRNA-synthetase G7a/Bat6 G3UY93 0 3 6 Sm protein F P62307 0 5 4 ATP-dependent DNA helicase VIII P97855 0 3 6 Coiled-coil domain-containing protein 128 Q3TDD9 0 5 2 RAP2C, member of RAS oncogene family A2AD86 0 2 4 Drosophila retinal degeneration B homolog 1 O35954 0 2 4 (blank) B8XCJ6 0 3 2 Leucyl-tRNA synthetase F8WH45 0 3 2 Rab-12 P35282 0 3 2 Adenosine monophosphate deaminase 2 A2AE27 0 2 2 (Isoform L) Putative uncharacterized protein Eif4e2 D3YUV9 0 2 2 Serine/threonine-protein phosphatase 4 E9QKU6 0 2 2 regulatory subunit 4 (blank) F8VPZ3 0 2 2 Protein lingerer homolog 1 Q91VX2 0 2 2 Ran-binding protein 3 Q9CT10 0 2 2 ATP-dependent helicase SMARCA2 F2Z4A9 0 0 623 Putative uncharacterized protein Q3UDA4 0 0 143 Glyceraldehyde-3-phosphate dehydrogenase D2KHZ9 0 124 0 MCG9102 D3Z351 0 123 0 (blank) E9PX99 0 64 0 ADP-ribosylation factor 2 A2A6T7 0 55 0 Protein mago nashi homolog P61327 0 46 0 Putative uncharacterized protein Q3U0T9 0 28 0

281

Uniprot Spectral Counts Protein Name ID C1 C2 C3 Alpha-CP3 P57722 0 0 18 MCG49049 D3Z426 0 16 0 Kinesin-like protein KIF21A Q9QXL2 0 16 0 ERT2 Q63844 0 0 14 Cell division control-related protein 1 Q9Z2Q6 0 0 14 (blank) F6T4M4 0 0 12 MCG2650 D3YTT7 0 11 0 (blank) E9Q4Z2 0 0 10 Ataxin-2-binding protein 1 Q9JJ43 0 0 10 (blank) G3UX98 0 0 8 (blank) Q5Y5T5 0 0 8 Syntrophin acidic 1 A2AKD8 0 0 6 Myosin, light polypeptide kinase B1B1A8 0 0 6 Brain calcium channel II E9QK20 0 0 6 cAMP-dependent protein kinase catalytic P05132 0 0 6 subunit alpha MCG15287 Q3UEN8 0 0 6 Laminin subunit alpha-5 Q61001 0 6 0 Bone sialoprotein 2 Q61711 0 6 0 E3 ubiquitin-protein ligase HECW2 Q6I6G8 0 6 0 (blank) Q8CJ27 0 6 0 Glutamate/H(+) symporter 2 Q9DB41 0 6 0 Coiled-coil-forming protein 1 Q9ESK9 0 0 6 Gelsolin A2AL35 0 5 0 Kinesin family member 2B A2RSC8 0 5 0 Ca2+-dependent activator protein for A4PI81 0 5 0 secretion 2 isoform b LIM domain only protein 3 E9PWI9 0 5 0 Splicing factor, arginine/serine-rich 2 (SC- F7CZ20 0 5 0 35) Postmeiotic segregation increased 2 (S. F8VQD1 0 5 0 cerevisiae) Cell division protein kinase 14 O35495 0 5 0 Putative uncharacterized protein Q8BVQ9 0 5 0 GTP-binding protein 9 Q9CZ30 0 5 0 Maternal antigen that embryos require Q9R1M5 0 5 0 Adenylyltransferase and sulfurtransferase A2BDX3 0 0 4 MOCS3 Aldehyde dehydrogenase family 1, subfamily B2RTL5 0 0 4 A7 Rho GTPase activating protein 12 B2RUJ8 0 0 4 Putative uncharacterized protein Kcnt2 D3YXJ8 0 0 4 282

Uniprot Spectral Counts Protein Name ID C1 C2 C3 (blank) E9QMC4 0 0 4 Elongation factor 1-delta F6ZFU0 0 0 4 Putative uncharacterized protein Clip1 F8WIA1 0 0 4 Branched-chain-amino-acid aminotransferase F8WIH8 0 0 4 Numb-like protein O08919 0 0 4 40S ribosomal protein S5 P97461 0 0 4 Zinc finger protein 250 Q7TNU6 0 0 4 ELKL motif serine/threonine-protein kinase Q8VHJ5 0 0 4 3 3-ketoacyl-CoA thiolase A, peroxisomal Q921H8 0 0 4 (blank) Q9D451 0 0 4 Oxysterol-binding protein A2A716 0 3 0 Nik related kinase B1B0C9 0 3 0 Diaphanous-related formin-1 E9PV41 0 3 0 (blank) E9PVP1 0 3 0 MKIAA1300 protein E9Q3T3 0 3 0 UPF0684 protein C5orf30 homolog F6R5A0 0 3 0 Putative uncharacterized protein Nfix F6VBN9 0 3 0 (blank) F8VPT7 0 3 0 Protein Odd Oz/ten-m homolog 3 G3X907 0 3 0 Misshapen-like kinase 1 (Zebrafish) G3X9G2 0 3 0 (blank) O09053 0 3 0 Processed zona pellucida sperm-binding P20239 0 3 0 protein 2 Inositol 1,4,5-trisphosphate receptor type 3 P70227 0 3 0 Alpha T-catenin Q65CL1 0 3 0 (blank) Q69ZI1 0 3 0 A disintegrin and metalloproteinase with Q769J6 0 3 0 thrombospondin motifs 13 Coiled-coil domain containing 21 A2A9K2 0 0 2 ATPase family, AAA domain containing 3A A2AD89 0 0 2 Protein DDI1 homolog 2 A2ADY9 0 2 0 SH3-domain kinase binding protein 1 A2AG61 0 2 0 Retrotransposon gag domain containing 1 A2AH48 0 2 0 E3 ubiquitin-protein ligase UBR4 A2AN08 0 2 0 ATPase, class II, type 9A A2AQC3 0 2 0 Myosin cardiac muscle beta chain A2AQP0 0 0 2 Rab GTPase-activating protein 1-like A6H6A9 0 0 2 Mediator complex subunit 24 A6PW47 0 0 2 Acyl-Coenzyme A dehydrogenase, very long B1AR27 0 2 0 chain

283

Uniprot Spectral Counts Protein Name ID C1 C2 C3 MCG6846, isoform CRA_c B1AT82 0 0 2 Gamma-aminobutyric acid (GABA-A) B1AVY2 0 2 0 receptor, subunit alpha 3 Brain-specific angiogenesis inhibitor 1- B1AZ47 0 0 2 associated protein 2 Dynein light chain 2 B2KGQ2 0 0 2 Tnik protein B2RQ80 0 0 2 Bromodomain containing 2 B2RS09 0 0 2 Kelch-like 17 (Drosophila) B2RTE7 0 0 2 Histidine ammonia-lyase B2RXW1 0 0 2 RNA-binding motif protein 25 B2RY56 0 2 0 (blank) B7ZCC7 0 0 2 (blank) D3YTR0 0 0 2 Putative uncharacterized protein Gm14446 D3Z6F0 0 0 2 MCG3543, isoform CRA_a D3Z7W0 0 2 0 Salt-inducible kinase 3 E9PU87 0 0 2 Deubiquitinating enzyme 24 E9PV45 0 2 0 (blank) E9PWK8 0 0 2 (blank) E9PX59 0 2 0 MKIAA0696 protein E9Q0X1 0 0 2 (blank) E9Q9P5 0 0 2 (blank) E9QLA4 0 2 0 (blank) F6QI24 0 0 2 Deubiquitinating protein VCIP135 F6ZDG3 0 0 2 Cyclin C F7AZW6 0 2 0 (blank) F8VPZ9 0 2 0 (blank) F8WGV3 0 0 2 Androgen-induced proliferation inhibitor F8WHU5 0 2 0 SF3a66 G3UVU2 0 0 2 Polyadenylate-binding protein-interacting G3UYE5 0 2 0 protein 1 Poly [ADP-ribose] polymerase 6 G5E856 0 0 2 (blank) G5E874 0 2 0 (blank) H3BKF6 0 0 2 GTP-binding protein 1 O08582 0 0 2 AH receptor-interacting protein O08915 0 0 2 Flotillin-1 O08917 0 2 0 Neuromedin-B receptor O54799 0 2 0 MTG8-like protein O70374 0 2 0 Macropain subunit C8 O70435 0 2 0 A-kinase anchor protein 10, mitochondrial O88845 0 0 2

284

Uniprot Spectral Counts Protein Name ID C1 C2 C3 2-amino-3-ketobutyrate coenzyme A ligase, O88986 0 0 2 mitochondrial Ig heavy chain V-III region J606 P01801 0 0 2 Ig gamma-2B chain C region P01867 0 0 2 Ferritin heavy chain P09528 0 0 2 CYPIIA4 P15392 0 0 2 Antigen peptide transporter 2 P36371 0 2 0 ATP-dependent helicase IGHMBP2 P40694 0 0 2 (blank) P52785 0 2 0 4-alpha-hydroxy-tetrahydropterin P61458 0 2 0 dehydratase Deafness dystonia protein 2 homolog P62077 0 2 0 26S protease regulatory subunit 4 P62192 0 0 2 Heparin-binding brain mitogen P63089 0 0 2 Sialate O-acetylesterase P70665 0 2 0 Putative G-protein coupled receptor P83854 0 2 0 Dystrophia myotonica WD repeat-containing Q08274 0 0 2 protein BEN domain containing 7 Q0VE44 0 0 2 NOL1/NOP2/Sun domain family member 7 Q14AW5 0 0 2 Acyl-coenzyme A thioesterase 10, Q32MW3 0 0 2 mitochondrial Musashi homolog 2 (Drosophila) Q3TE41 0 0 2 Heterochromatin protein 1-binding protein 3 Q3TEA8 0 0 2 DnaJ (Hsp40) homolog, subfamily B, Q3TU79 0 0 2 member 1 (blank) Q3TWW 0 2 0 0 Inositol (Myo)-1(Or 4)-monophosphatase 2, Q3U3B7 0 2 0 isoform CRA_b Never in mitosis A-related kinase 10 Q3UGM2 0 2 0 BRCA1-A complex subunit MERIT40 Q3UI43 0 0 2 FLI-LRR-associated protein 1 Q3UZ39 0 2 0 Immunoglobulin superfamily member 10 Q3V1M1 0 2 0 N-myc downstream regulated gene 3 Q544I1 0 2 0 Transcription factor IIIB 150 Q571C7 0 0 2 Mitochondrial ribosomal protein L43 Q5RL20 0 0 2 (blank) Q5STE3 0 2 0 Olfactory receptor 464 Q5SW48 0 2 0 DARPP-32 Q60829 0 0 2 ATP-binding cassette sub-family B member Q61102 0 0 2 7, mitochondrial

285

Uniprot Spectral Counts Protein Name ID C1 C2 C3 Myocyte-specific enhancer factor 2D Q63943 0 0 2 Beta-glucocerebrosidase 2 Q69ZF3 0 0 2 Protein FAM167A Q6P1G6 0 0 2 Nestin Q6P5H2 0 0 2 ATP-dependent RNA helicase Dhx29 Q6PGC1 0 0 2 Syntaphilin Q80U23 0 0 2 Kinetochore-associated protein KNL-2 Q80WQ8 0 2 0 homolog Uncharacterized protein C12orf60 homolog Q810N5 0 0 2 Parkinson disease 7 domain-containing Q8BFQ8 0 0 2 protein 1 Golgi-associated PDZ and coiled-coil motif- Q8BH60 0 0 2 containing protein Regulator of differentiation 1 Q8BHD7 0 2 0 (blank) Q8BHX0 0 2 0 Protein FAM164A Q8BJH1 0 2 0 Importin subunit beta-3 Q8BKC5 0 2 0 Rho GTPase-activating protein 28 Q8BN58 0 2 0 Phosphodiesterase 1A, calmodulin-dependent Q8BRR9 0 0 2 Kelch-like protein 7 Q8BUL5 0 2 0 Putative mitochondrial carrier protein Q8BW66 0 0 2 FLJ44862 homolog GATE-binding factor 1 Q8BWM 0 2 0 0 Coiled-coil domain-containing protein 10 Q8BYN5 0 0 2 Potassium voltage-gated channel subfamily Q8BZN2 0 0 2 V member 1 Carbamoyl-phosphate synthase [ammonia], Q8C196 0 2 0 mitochondrial Vacuolar protein sorting-associated protein Q8CCB4 0 0 2 53 homolog Deubiquitinating enzyme 27 Q8CEG8 0 0 2 UPF0539 protein C7orf59 homolog Q8CF66 0 0 2 Pyroglutamyl-peptidase II Q8K093 0 2 0 Pitrilysin metalloproteinase 1 Q8K411 0 2 0 SF3a120 Q8K4Z5 0 2 0 Otoancorin Q8K561 0 2 0 Hepatocellular carcinoma-associated protein Q8R1L8 0 0 2 TD26 homolog COP9 homolog Q8VBV7 0 0 2 Angiomotin Q8VHG2 0 0 2

286

Uniprot Spectral Counts Protein Name ID C1 C2 C3 Phospholipase C beta 4 Q91UZ1 0 2 0 Probable protein BRICK1 Q91VR8 0 0 2 Dpy-30-like protein Q99LT0 0 2 0 DnaJ homolog subfamily A member 3, Q99M87 0 0 2 mitochondrial COX assembly mitochondrial protein Q9CPZ8 0 2 0 homolog Glutaredoxin-3 Q9CQM9 0 0 2 (blank) Q9CR73 0 0 2 Suppressor of G2 allele of SKP1 homolog Q9CX34 0 2 0 Putative uncharacterized protein Q9D5C1 0 2 33 N-acetyltransferase 5 (ARD1 homolog, S. Q9DB82 0 0 2 cerevisiae) Plasma membrane proteolipid Q9DCU2 0 2 0 Never in mitosis A-related kinase 7 Q9ES74 0 0 2 Enhancer of yellow 2 transcription factor Q9JIX0 0 0 2 homolog Uncharacterized protein C4orf14 homolog Q9JJG9 0 0 2 B-cell adapter containing a SH2 domain Q9QUN3 0 2 0 protein (blank) Q9QX15 0 0 2 Diacylglycerol kinase epsilon Q9R1C6 0 0 2 SUMO-activating enzyme subunit 1 Q9R1T2 0 2 0 Alpha-(1,6)-fucosyltransferase Q9WTS2 0 0 2 Hephaestin Q9Z0Z4 0 2 0 Regulator of G-protein signaling 6 Q9Z2H2 0 0 2

287

Table A.3: Proteins Identified in Both the Nicotine-Exposed and Control Samples

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed

AT-rich interactive domain-containing protein E9QAQ7 2607 1746 1A

Putative uncharacterized protein Q3UID0 1877 1509

Elongation factor 1-alpha 1 P10126 1791 309

Elongation factor 1-alpha B7ZBW3 1771 290

(blank) E9Q6R4 1488 701

ATP-dependent helicase SMARCA4 Q3TKT4 1388 1148

ATP-dependent helicase SMARCA2 H3BLH0 1086 1432

Putative uncharacterized protein Q9Z1A1 1077 973

Trinucleotide repeat-containing gene 6B protein Q8BKI2 1014 868

Eukaryotic translation initiation factor 2C 2 Q8CJG0 901 818

Actin, alpha cardiac muscle 1 P68033 886 238

Spectrin alpha 2 E9Q447 832 752

Heat shock 70 kDa protein 8 P63017 776 783

MCG1034428 D3YWP3 713 8

Heat shock 84 kDa P11499 700 317

Beta-II spectrin Q62261 694 623 Heterogeneous nuclear ribonucleoproteins O88569 678 470 A2/B1

Perlecan (Heparan sulfate proteoglycan 2) E9PZ16 663 464

Microtubule-associated protein 6 Q7TSJ2 658 575

288

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Actin, cytoplasmic 2 P63260 638 328

Heterogeneous nuclear ribonucleoprotein U Q8VEK3 582 423

Protein pigpen P56959 580 397

BRG1-associated factor 155 P97496 578 581

MCG15366, isoform CRA_c A2A4L8 563 495

60 kDa BRG-1/Brm-associated factor subunit A Q61466 520 573

Collagen alpha-1(XII) chain Q60847 499 479

Dihydropyrimidinase-related protein 2 O08553 484 520

Polyadenylate-binding protein 1 P29341 483 450

Tubulin beta-2C chain P68372 477 736

Tubulin beta-2A chain Q7TMM9 469 714

60 kDa BRG-1/Brm-associated factor subunit C Q6P9Z1 466 452

Tubulin beta-5 chain P99024 447 713

Heat shock 86 kDa P07901 431 280

Eukaryotic translation initiation factor 2C 1 E9Q5W9 427 403

17-beta-hydroxysteroid dehydrogenase 4 P51660 426 394

Alpha-tubulin 4 P68368 408 373

Microtubule-associated protein 1 A A2ARP8 403 308

Clathrin, heavy polypeptide (Hc) Q5SXR6 396 424

Tubulin beta-4 chain Q9D6F9 394 580

Myelin A1 protein F6RT34 388 665

Aconitate hydratase, mitochondrial Q99KI0 386 460

Heat shock-related 70 kDa protein 2 P17156 384 390

289

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Eukaryotic translation initiation factor 2C 3 Q8CJF9 381 364

53 kDa BRG1-associated factor B Q99MR0 376 377

Tubulin beta-3 chain Q9ERD7 373 543

Heterogeneous nuclear ribonucleoprotein A3 Q5FB19 372 247

MCG142115, isoform CRA_c Q3UHK5 369 979

Putative uncharacterized protein Q6ZWP4 368 356

Heterogeneous nuclear ribonucleoprotein A3 Q0VG47 362 264

DNA-binding p52/p100 complex, 100 kDa Q8VIJ6 357 252 subunit

Cytokeratin-28 A6BLY7 345 160

Actin-like 6A, isoform CRA_a Q505L1 339 343

Heterogeneous nuclear ribonucleoprotein H1 Q8C2Q7 332 314

2,3-cyclic-nucleotide 3-phosphodiesterase P16330 327 573

Sodium pump subunit alpha-1 Q8VDN2 325 808

Heterogeneous nuclear ribonucleoprotein A1 Q5EBP8 319 198

Dynamin-1 P39053 315 352

Cytoplasmic dynein 1 heavy chain 1 Q9JHU4 315 219

60 kDa chaperonin P63038 311 195

N-ethylmaleimide-sensitive fusion protein P46460 308 397

Catenin delta-2 O35927 304 225

[Acyl-carrier-protein] S-acetyltransferase P19096 303 173

Beta-actin-like protein 2 Q8BFZ3 301 316

290

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Acidic-type mitochondrial creatine kinase P30275 284 213

ATP-dependent RNA helicase DDX1 Q91VR5 284 202

Microtubule-associated protein 2 P20357 284 199

(blank) E9Q557 281 310

Putative uncharacterized protein Dpf2 D3Z5N6 281 289

UPF0027 protein C22orf28 homolog Q99LF4 275 257

(blank) F8VPN4 274 249

Trinucleotide repeat containing 6C B1ATC3 272 263

Plectin-1 E9QN87 268 83

Protein bassoon O88737 264 214 Heat shock 70kD protein 5 (Glucose-regulated A2AUF6 263 276 protein)

SEC16 homolog A (S. cerevisiae) A2AIX1 261 189

Heterogeneous nuclear ribonucleoprotein H2 A2BDV8 255 219

ATP-dependent RNA helicase A E9QNN1 251 169

Protein transport protein Sec23A Q01405 247 201

Heat shock protein 9 Q7TSZ0 243 271

Junction plakoglobin A2A4H7 232 309

Nono protein F6XLC7 232 207

Glycogen phosphorylase, muscle form Q9WUB3 228 194

60 kDa BRG-1/Brm-associated factor subunit B Q99JR8 223 247

Hnrpk protein H7BXB7 222 162

Keratin, type II cytoskeletal 2 oral Q3UV17 221 289

Glutamate decarboxylase P15105 218 210

291

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Ankyrin-2 E9PW46 215 160

Dynamin-like 120 kDa protein, form S1 P58281 213 101

Phosphorylase A2AWQ5 212 179

Crmp1 protein Q6P1J1 210 231 ATPase, Na+/K+ transporting, alpha 3 Q8VCE0 203 199 polypeptide

Serine/threonine-protein phosphatase B2RRX2 198 201

(blank) E9Q1Z0 198 372

Triosephosphate isomerase P17751 190 254

Calcium pump 2 F8WI94 189 153 IP3R-binding protein released with inositol Q80SW1 187 242 1,4,5-trisphosphate

Putative uncharacterized protein Q8BQ46 185 178 DEAD/H (Asp-Glu-Ala-Asp/His) box Q3TQX5 183 167 polypeptide 3, X-linked

Far upstream element-binding protein 2 Q3U0V1 181 155

Glutamate dehydrogenase 1, mitochondrial P26443 179 218

DEAD (Asp-Glu-Ala-Asp) box polypeptide 17, Q3U741 179 139 isoform CRA_a

BRG1-associated factor 45C P58269 177 181

200 kDa neurofilament protein P19246 172 222

Serum albumin P07724 172 54

DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 Q8BTS0 167 138

ATP citrate lyase Q3V117 167 129

Sbf1 protein B2RXX4 167 127

292

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Plectin-1 E9QK43 166 335

Histone H2A.x P27661 164 52

Aspartate aminotransferase, mitochondrial P05202 163 221

MCG19655 B1AXW7 163 188

14-3-3 protein zeta/delta P63101 161 296

ELAV-like protein 4 Q61701 161 135

ELAV (Embryonic lethal, abnormal vision, B1AXZ5 161 123 Drosophila)-like 2 (Hu antigen B)

Chromosome segregation 1-like (S. cerevisiae) A2A5S1 161 34

Spectrin beta 3 Q68FG2 159 87

Protein unc-18 homolog 1 O08599 157 220 ATPase, Ca++ transporting, plasma membrane F8WHB1 157 202 2, isoform CRA_a

Putative uncharacterized protein Dpf1 D3YY44 156 134

Putative adenosylhomocysteinase 3 E9PV16 153 175

Na(+)/K(+) ATPase alpha(III) subunit Q6PIC6 151 754

K(+) channel subunit beta-2 E0CXZ9 148 161

Cytoplasmic FMR1-interacting protein 2 Q5SQX6 148 150

Heterogeneous nuclear ribonucleoprotein U-like F8VPM4 147 126 protein 2

(blank) G5E829 146 180

Pyruvate carboxylase, mitochondrial E9QPD7 146 121

14-3-3 protein gamma subtype A8IP69 145 252 Malate dehydrogenase, mitochondrial P08249 145 247

293

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed 94 kDa glucose-regulated protein P08113 145 140

Protein transport protein Sec31A Q3UPL0 143 118

Putative uncharacterized protein Synj1 D3Z656 143 117

Mll2 protein Q6PDK2 139 97 Putative uncharacterized protein D3YWG1 139 146 ENSMUSP00000095927

2-phospho-D-glycerate hydro-lyase P17182 138 179

AU-rich element RNA-binding protein 1 Q60668 136 123

Guanine nucleotide-binding protein G(o) P18872 135 279 subunit alpha Constitutive coactivator of PPAR-gamma-like Q6A0A9 135 75 protein 1 B-cell CLL/lymphoma 7 protein family member Q9CXE2 134 154 A

NCK-associated protein 1 A2AS98 134 140

ATP synthase subunit alpha, mitochondrial Q03265 130 305

Lipophilin P60202 130 196

ATP-dependent helicase RENT1 Q9EPU0 130 95 Sec24 related gene family, member B (S. Q80ZX0 129 129 cerevisiae)

Anastellin G5E8B8 129 73

MAP1 light chain LC1 P14873 128 90

Deubiquitinating enzyme FAF-X P70398 127 77

Heat shock 70 kDa protein 1 P17879 127 207 ATPase, Ca++ transporting, plasma membrane Q0VF55 126 168 3

294

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Fibrinogen, alpha polypeptide E9PV24 125 152

Fructose-bisphosphate aldolase A6ZI44 124 226

MCG5546, isoform CRA_c Q6PHQ9 124 111

DnaJ homolog subfamily C member 29 E9QL60 122 120

Cytoplasmic FMR1-interacting protein 1 Q7TMB8 122 118

Glycogen [starch] synthase, muscle Q9Z1E4 121 121

Interleukin enhancer-binding factor 3 Q9Z1X4 121 96

Heat shock protein 1-like A1L347 121 77

LDH heart subunit P16125 120 200

Beta-globin A8DUK4 119 217

Dmx-like 2 B0V2P5 119 89

Dihydropyrimidinase-related protein 3 E9PWE8 118 159

Src substrate cortactin Q60598 118 105

MCG11326, isoform CRA_a D3Z3N4 118 61

Isocitrate dehydrogenase 3 (NAD+) beta Q91VA7 115 95

ATP synthase subunit beta, mitochondrial P56480 114 378

Ataxin-2 E9QM77 113 91

Pyruvate kinase isozymes M1/M2 P52480 112 189

Alpha-adducin Q9QYC0 111 127

Putative uncharacterized protein Q91VM5 110 33

66 kDa neurofilament protein P46660 109 296

Dynamin-3 E9QLL2 108 127

295

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed 100 kDa coated vesicle protein A P17426 107 120

Ewing sarcoma breakpoint region 1 Q5SUS9 107 55

14-3-3 protein epsilon P62259 106 183

Putative uncharacterized protein Q3TML0 106 105

Putative uncharacterized protein Q3TGG2 106 48

Dynamin-like 120 kDa protein, form S1 H7BX01 105 175

MCG1048875, isoform CRA_c D3YVN7 104 108

Actin-like protein 2 P61161 104 101

Caprin-1 Q60865 104 93 Tyrosine 3-monooxygenase/tryptophan 5- A2A5N2 102 165 monooxygenase activation protein, beta Heterogeneous nuclear ribonucleoprotein U-like Q8VDM6 102 76 protein 1

Laminin subunit beta-2 Q61292 102 49

LDH muscle subunit G5E8N5 101 165

Ubiquitin-associated protein 2-like Q80X50 101 94

Gphn protein A0JNY3 100 96

Alpha-globin P01942 99 200

10-formyltetrahydrofolate dehydrogenase Q8R0Y6 99 79

Synapsin I O88935 98 210

68 kDa neurofilament protein P08551 97 197

B-CK Q04447 97 150

Putative uncharacterized protein Carm1 D3YUP1 96 93

MCG126220 F6VW30 95 158

296

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Cleavage and polyadenylation specificity factor Q9EPU4 95 74 160 kDa subunit

5-oxo-L-prolinase Q8K010 94 106

Beta-catenin Q02248 94 78

Zinc finger RNA-binding protein O88532 94 48 Calmodulin-dependent calcineurin A subunit P48453 93 100 beta isoform

Putative uncharacterized protein Q50HX4 91 146

Trinucleotide repeat-containing gene 6A protein Q3UHK8 91 68

14-3-3 protein eta P68510 90 139

CDC10 protein homolog E9Q9F5 90 106

Heterogeneous nuclear ribonucleoprotein L G5E924 90 52

Septin-11 Q8C1B7 89 88

Actin cross-linking family 7 E9QA63 88 71

Complement C1q subcomponent subunit B P14106 88 69

Inositol 1,4,5-trisphosphate receptor type 1 P11881 88 39

Ras-related protein Rab-3A P63011 87 184

Ras-related protein Rab-3C P62823 87 150

Annexin A7 F8WGC2 87 69

Ubc protein P0CG50 86 154

Beta-2-globin P02089 86 119

O-GlcNAc transferase subunit p110 Q8CGY8 85 76

28S ribosomal protein S22, mitochondrial Q9CXW2 85 67

297

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Complement component 1, q subcomponent, C Q6DI63 84 88 chain

Synaptogyrin-3 Q8R191 83 93

15S Mg(2+)-ATPase p97 subunit Q01853 83 92 Adaptor-related protein complex 2, beta 1 Q5SWR1 83 92 subunit

160 kDa neurofilament protein P08553 82 166

CCT-theta P42932 82 96

(blank) E9Q3L2 82 59

Alpha-2-macroglobulin receptor Q91ZX7 82 51

Peroxiredoxin-2 Q61171 81 84

RING finger protein 22 Q9R1R2 81 78

Cadherin-associated Src substrate E9Q8Z5 81 68

Hexokinase type I P17710 80 137

Calcium-responsive transactivator Q8BW22 80 96

2-phospho-D-glycerate hydro-lyase P17183 80 87

Putative uncharacterized protein Q3UER8 80 73

40S ribosomal protein S9 Q6ZWN5 80 65 Glycerol phosphate dehydrogenase 2, A2AQR0 79 58 mitochondrial

Interleukin enhancer-binding factor 2 Q9CXY6 79 48 Purine-rich single-stranded DNA-binding P42669 77 75 protein alpha

EF-3 P97807 76 78

Protein FAM98B Q80VD1 76 74

MCG140066 Q4VA29 75 122

298

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed (blank) F6U775 75 73

MCG121979, isoform CRA_c Q6ZQ61 75 61

ADP-ribosylation factor 5 P84084 75 53

Complement C1q subcomponent subunit A P98086 75 52

Far upstream element-binding protein 1 Q91WJ8 75 48

GTPase Ran P62827 75 30

100 kDa coated vesicle protein C P17427 74 87

Annexin A2 P07356 74 75

CCT-gamma P80318 74 68

Actin-binding protein 280 Q8BTM8 74 58

Fructose-bisphosphate aldolase B1AQE0 73 134

Vacuolar proton pump subunit alpha P50516 73 120

Guanine deaminase Q548F2 73 99

28S ribosomal protein S27, mitochondrial Q8BK72 73 54

Heterogeneous nuclear ribonucleoprotein D-like Q9Z130 73 26

ATP-specific succinyl-CoA synthetase subunit Q9Z2I9 72 91 beta

Putative uncharacterized protein Hnrpdl D3YTQ3 72 70

A45 P80315 72 86

40S ribosomal protein S3 P62908 71 58

Elav-like generic protein P70372 71 58

40S ribosomal protein S18 F5H8M6 71 56

Discs, large homolog 3 (Drosophila) A2BEF1 70 70

299

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Ig mu chain C region membrane-bound form P01873 70 47

Heterogeneous nuclear ribonucleoprotein F Q9Z2X1 70 45

ADP-ribosylation factor 4 E9Q972 70 33

Citrate synthase, mitochondrial Q9CZU6 69 92

ELAV-like protein 3 Q60900 69 63

Cytosolic alpha-D-mannosidase Q3ZCX9 69 46

Putative uncharacterized protein Q3UGX2 69 28

Heterogeneous nuclear ribonucleoprotein G Q9WV02 69 34

Super protein P60879 68 112

Ras-related protein Rab-10 P61027 68 110

CCT-zeta-1 E9QPA6 68 84

Purine-rich element-binding protein B O35295 68 74

Glial fibrillary acidic protein P03995 67 101

Alpha-CP2 Q61990 67 59

Heterogeneous nuclear ribonucleoprotein R Q8VHM5 67 47

Tectonin beta-propeller repeat-containing Q80VP0 66 34 protein 1

MKIAA0968 protein F8WIS9 65 172 Isocitrate dehydrogenase [NAD] subunit alpha, Q9D6R2 65 82 mitochondrial

Aspartate aminotransferase, cytoplasmic P05201 64 131 von Willebrand factor A domain-containing Q8R2Z5 64 38 protein 1

MKIAA0079 protein G3X972 64 63

300

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Calcium regulator SV2A Q9JIS5 63 61

Protein kinase C gamma type P63318 63 46

22 kDa neuronal tissue-enriched acidic protein Q91XV3 63 275

Alpha-CP1 P60335 62 62

Putative uncharacterized protein Q3U6P5 62 16

AHD-M1 P47738 61 55

BCL2-associated athanogene 4 A6H6S8 61 51

RasGAP-activating-like protein 1 Q9Z268 60 84

Alpha N-catenin E0CXB9 60 62 Pentatricopeptide repeat-containing protein 3, Q14C51 60 55 mitochondrial

ACC-alpha E9PUB1 60 16

Neural cell adhesion molecule 1 P13595 59 76

(blank) E9Q7L0 59 66

TGF-beta resistance-associated protein TRAG Q920I9 59 43

BM88 antigen Q9JKC6 58 116

Synapsin II Q64332 58 96

CCT-alpha P11983 58 59

Putative uncharacterized protein Q4FJL2 58 57 Long-chain specific acyl-CoA dehydrogenase, P51174 58 48 mitochondrial

(blank) E9Q6P5 58 43

Endomembrane proton pump 58 kDa subunit P62814 57 120

Neuronal-specific septin-3 Q9Z1S5 57 67

301

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Putative uncharacterized protein Mlec D3Z1M3 57 66

ERT1 P63085 57 64 Arf-GAP with GTPase, ANK repeat and PH F8VQE9 57 56 domain-containing protein 3

R3H domain-containing protein 2 Q80TM6 57 55

Novel protein (2810405J04Rik) B8JJG1 57 50

82 kDa FMRP-interacting protein Q5F2E7 57 41 Acyl-CoA synthetase bubblegum family Q99PU5 57 36 member 1

Entactin-2 O88322 57 30

Histone H4 B2RTM0 56 230

Putative uncharacterized protein Q8C605 56 60

6-phosphofructokinase, muscle type P47857 56 56

40S ribosomal protein S2 E9PV46 56 50

Antioxidant enzyme AOE372 O08807 55 36

DnaJ (Hsp40) homolog, subfamily C, member 6 B1AYC5 54 46

Axonal membrane protein GAP-43 P06837 53 132

Ras-related protein Rab-6A P35279 53 86

Heterogeneous nuclear ribonucleoprotein A/B Q20BD0 53 62

C1qTNF5 Q4ZJN4 53 61

CCT-beta P80314 53 60

GAP SH3 domain-binding protein 2 P97379 53 40

Epiplakin Q8R0W0 53 20 Beta-1-globin P02088 53 147

302

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Small nuclear ribonucleoprotein Sm D2 E9Q2T1 53 37

Rab1B Q0PD66 53 26

Protein Ndr2 Q9QYG0 52 82

Adapter-related protein complex 2 mu subunit P84091 52 64

Heat shock 70 kDa protein 12A Q8K0U4 52 52

Actin-related protein 2/3 complex subunit 3 Q9JM76 52 42

MCG16669, isoform CRA_b Q544R5 52 35

Mitochondrial ribosomal protein S5 A2AHS8 51 65

Add97 Q9QYB8 51 65

Glutathione S-transferase GT8.7 P10649 51 51

Putative uncharacterized protein Q8CC13 51 51

Nucleolysin TIAR P70318 51 50

MKIAA0531 protein Q8CHF1 51 41

3-methylcrotonyl-CoA carboxylase 1 Q99MR8 51 36

Small nuclear ribonucleoprotein 70 (U1) A2RS68 50 75

Guanine nucleotide binding protein, alpha 13 A2AA49 50 56

Carboxyl-terminal LIM domain-binding protein P70662 50 39 2

MCG49644 D3Z4Z0 50 17

Adenine nucleotide translocator 1 P48962 50 121 Sodium/potassium-dependent ATPase subunit P14094 49 100 beta-1

MCG140784 Q792Z1 49 96

Fibrinogen, B beta polypeptide, isoform CRA_a Q3TGR2 49 56

303

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Reticulon-3 Q9ES97 49 55

Long chain 3-hydroxyacyl-CoA dehydrogenase Q8BMS1 49 42

Protein-glutamine gamma-glutamyltransferase 2 P21981 49 41

Eukaryotic translation elongation factor 1 Q4FZK2 49 23 gamma

Neurofascin Q810U3 49 35 Guanine nucleotide-binding protein P62874 48 83 G(I)/G(S)/G(T) subunit beta-1

CCT-epsilon P80316 48 57

Tripeptidyl aminopeptidase Q64514 48 47

Paraspeckle component 1 Q8R326 48 41

Putative uncharacterized protein Q9CPN9 48 38

Actin-RPV P61164 48 34

Caskin-1 Q6P9K8 48 31

Laminin B2 chain F8VQJ3 48 25

CapZ alpha-2 P47754 47 62 mRNA (guanine-N(7)-)-methyltransferase Q9D0L8 47 36

Collapsin response mediator protein 3 O35098 47 25

40S ribosomal protein S8 G3XA55 47 29 B-cell chronic lymphocytic leukemia/lymphoma O08664 46 51 7C protein

Acetoacetyl-CoA thiolase Q8QZT1 46 51

Single-stranded DNA binding protein 2 Q540I3 46 50

Adapter-related protein complex 3 subunit beta- Q9JME5 46 23 2

304

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Adenine nucleotide translocator 2 P51881 46 98

58 kDa glucose-regulated protein P27773 45 59

Septin-6 Q9R1T4 45 43

Protein transport protein Sec23B Q9D662 45 35

150 kDa dynein-associated polypeptide O08788 45 29

Protein TANC2 A2A690 45 25

Actin-related protein 2/3 complex subunit 4 P59999 44 47

2-oxoglutarate dehydrogenase complex Q60597 44 45 component E1

Putative uncharacterized protein Ylpm1 D3YWX2 44 32

Adenylate cyclase-inhibiting G alpha protein P08752 44 48

Histone H2A B2RWH3 44 25

Cytosolic malate dehydrogenase P14152 43 86 Guanine nucleotide binding protein (G protein), B2RSH2 43 68 alpha inhibiting 1

C-terminal-binding protein 1 O88712 43 48

Protein WAVE-1 Q8R5H6 43 47

Actr3 protein Q3ULF7 43 44

Doublecortin-like and CAM kinase-like 1 Q9JLM8 43 43

Galactocerebrosidase P54818 43 39

Lamin A B3RH23 43 15 Calcium/calmodulin-dependent protein kinase E9Q1T1 42 114 type II subunit delta

Vacuolar proton pump subunit E 1 P50518 42 77

305

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Mammalian lin-seven protein 1 Q8JZS0 42 73

Antioxidant enzyme B166 H3BJQ7 42 61

28S ribosomal protein S34, mitochondrial Q9JIK9 42 56

B-cell CLL/lymphoma 7 protein family member Q921K9 42 50 B

Amphiphysin Q7TQF7 42 49

Collapsin response mediator protein 5 Q9EQF6 42 39 Neural RAP guanine nucleotide exchange Q8CHG7 42 35 protein

Putative uncharacterized protein Pzp D3YW52 42 31

Septin 8 B1AQZ0 42 26

Putative uncharacterized protein Q3TLR3 42 19 Plasma membrane Ca++ transporting ATPase 4 Q6Q477 42 96 splice variant b

214 kDa nucleoporin Q80U93 42 13

Aspartyl aminopeptidase Q9Z2W0 41 36 Constitutive coactivator of PPAR-gamma-like Q8C3F2 41 33 protein 2

Actin-related protein 2/3 complex subunit 2 Q9CVB6 41 31

40S ribosomal protein S4, X isoform P62702 41 29

28S ribosomal protein S9, mitochondrial Q9D7N3 41 27

Protein SCAI Q8C8N2 41 40

4F2 cell-surface antigen heavy chain G3UWA6 40 71

Exophilin-1 P47708 40 47

Glycine- and tyrosine-rich RNA-binding protein Q7TMK9 40 39

306

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Conventional kinesin heavy chain Q61768 40 38

28S ribosomal protein S26, mitochondrial Q80ZS3 40 32

Hepatoma-derived growth factor-related protein Q3UMU9 40 28 2

Elongation factor 2 P58252 39 51

Antioxidant protein 1 P20108 39 46

Flavoprotein subunit of complex II Q8K2B3 39 39

Putative uncharacterized protein Fip1l1 D3Z4V2 39 33

Mitochondrial ribosomal protein S23 Q5SXC8 39 31

Bifunctional aminoacyl-tRNA synthetase Q8CGC7 39 29

Pumilio 1 (Drosophila) B1ASF2 39 26

Ig gamma-3 chain C region P03987 39 9

CaM kinase-like vesicle-associated protein Q3UHL1 38 47

Alpha-dystrobrevin G5E8S5 37 28

UDP--Glc:glycoprotein glucosyltransferase Q6P5E4 37 27

Disco-interacting protein 2 homolog B Q3UH60 37 26 Sodium- and chloride-dependent GABA P31650 36 94 transporter 3

Microtubule-associated protein A2A5Y6 36 91

Clathrin light chain B Q6IRU5 36 80

42 degrees C-HSP Q61699 36 52

Growth factor receptor bound protein 2 Q3U5I5 36 42

Myocilin O70624 36 40

307

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed DAZ associated protein 1, isoform CRA_b Q3UGB5 36 40

Downstream of CHOP4 Q3UHK6 36 28 Methylcrotonoyl-Coenzyme A carboxylase 2 B2RUK5 36 33 (Beta)

PP2A subunit A isoform PR65-alpha Q76MZ3 35 49

Profilin II Q9JJV2 35 41

MKIAA0885 protein Q80TP8 35 24

Myotubularin-related protein 1 Q9Z2C4 35 23

Disks large homolog 1 E9Q9H0 35 22

Heat shock protein 75 kDa, mitochondrial Q9CQN1 35 33

Vimentin P20152 35 26

Protein SEC13 homolog Q9D1M0 34 59

Small nuclear ribonucleoprotein Sm D3 P62320 34 46

Phenylalanine--tRNA ligase beta chain Q9WUA2 34 38 Serine/threonine kinase receptor associated B2RUC7 34 33 protein Aldhehyde dehydrogenase family 5, subfamily B2RS41 34 28 A1

Citrate synthase Q80X68 34 31

Putative uncharacterized protein Akap5 H3BIV5 34 8

Copine IV Q8BLR2 33 58

Enoyl-CoA hydratase 1 Q8BH95 33 52

Clathrin light polypeptide (Lca) B1AWD9 33 44

Amphiphysin II O08539 33 34

308

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed ATP-dependent RNA helicase p54 P54823 33 33

Actin-related protein 2/3 complex subunit 1A Q9R0Q6 33 29

40S ribosomal protein S20 P60867 33 27

M-Sema F-associating protein of 75 kDa Q9Z0E0 33 22

DEAH box protein 15 O35286 33 17

Histone H3 A2A852 33 95 Membrane protein palmitoylated 2 (MAGUK B1AQF9 33 50 p55 subfamily member 2)

Putative uncharacterized protein Q3URG1 32 60

91 kDa synaptosomal-associated protein Q61548 32 35

Protein FAM126B Q8C729 32 34

Putative uncharacterized protein Gyg D3Z5N4 32 30

Putative uncharacterized protein E9QKC6 32 24

Phosphoglycerate kinase 1 P09411 32 24 3-hydroxyisobutyrate dehydrogenase, Q99L13 32 23 mitochondrial

ATP-dependent helicase SKIV2L2 Q9CZU3 32 22

Isoleucine--tRNA ligase Q8BIJ6 32 15

CCT-eta P80313 32 42

H1 VAR.2 P43274 31 113

UDP-glucose pyrophosphorylase Q91ZJ5 31 47

Coronin-1A O89053 31 44

Single-stranded DNA binding protein 3 B1AS35 31 34

Superoxide dismutase [Mn], mitochondrial P09671 31 34

309

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed eIF-3 p48 P60229 31 34

Eukaryotic translation initiation factor 4B Q8BGD9 31 34

DnaJ homolog subfamily C member 10 Q9DC23 31 33

102 kDa cadherin-associated protein P26231 31 15

Cadherin-associated Src substrate E9Q8Z8 31 45

SH3-domain GRB2-like 2 A2ALV3 30 43

Inositol monophosphatase 1 O55023 30 43

CDKN2A-interacting protein Q8BI72 30 34

Adenylosuccinase P54822 30 28

Putative uncharacterized protein Gls D3Z7P3 30 25

Putative uncharacterized protein Gm10119 D3Z6C3 30 22

Heat shock 70 kDa protein 4L P48722 30 22

Sorcin Q6P069 30 22 hnRNP associated with lethal yellow protein Q64012 30 21

Putative uncharacterized protein Q3TRJ1 30 20

Collagen alpha-1(XIV) chain Q80X19 30 19

Putative uncharacterized protein Cit E9QPY8 30 15

Histone H1 P10922 30 83

1-Cys peroxiredoxin O08709 30 22

Janusin Q8BYI9 29 58

A10 Q61753 29 40

SH3-domain GRB2-like endophilin B2 A2AWI7 29 38

Adenosylhomocysteinase P50247 29 35

310

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed MCG21131, isoform CRA_d B2KF41 29 33

Glutathione S-transferase P 1 P19157 29 29

BET3 homolog O55013 29 28

Adaptor-associated kinase 1 Q3UHJ0 29 21

Putative uncharacterized protein Inpp4a D3Z230 29 20

Acyl-CoA synthetase long-chain family Q5F2D0 29 19 member 6

Centrosomin P23116 29 18

60S ribosomal protein L26 P61255 29 8

Guanine nucleotide-binding protein alpha-q P21279 28 49

6-phosphofructokinase, liver type P12382 28 31

Leucine-zipper-containing LZF Q3TDK6 28 31

140 kDa Ser/Arg-rich domain protein Q6NV83 28 30

Putative uncharacterized protein Gm5805 D3Z1P6 28 28

Ubiquitously transcribed tetratricopeptide repeat A2AL70 28 27

3-ketoacyl-CoA thiolase Q99JY0 28 24

Kinesin-related microtuble-based motor protein E9Q9G6 28 18

37 kDa laminin receptor precursor P14206 28 57

Lin-7 homolog C (C. elegans) A2ARI2 28 26

Putative uncharacterized protein Q4VAA1 27 39

Vacuolar proton pump subunit H Q8BVE3 27 34

Lasp1 protein Q543N3 27 32

Abi2 protein Q6AXD2 27 32

311

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Major prion protein P04925 27 31

IMPDH-II P24547 27 29

Glyoxalase II G5E8T9 27 27

MCG3853 B9EIC7 27 24

Desmoglein 1 beta B2RQH0 27 23

Glutaryl-CoA dehydrogenase, mitochondrial Q60759 27 19

Phosphoglycolate phosphatase Q8CHP8 27 14 Cullin-associated and neddylation-dissociated Q6ZQ38 27 13 protein 1

Carbamoyl-phosphate synthetase 2, aspartate B2RQC6 27 8 transcarbamylase, and dihydroorotase

(blank) F7D6E8 27 21

Dihydrolipoamide dehydrogenase O08749 27 21

Importin subunit beta-1 P70168 27 3

Excitatory amino acid transporter 2 P43006 27 87

RAB3B, member RAS oncogene family A2A7Z6 27 71

Glioblastoma-amplified sequence O55126 26 39

28S ribosomal protein S29, mitochondrial G3X9M0 26 33

MCG123443 A4FUS1 26 29

45 kDa-splicing factor Q8JZX4 26 28

WD repeat-containing protein mio Q8VE19 26 28 SH3-domain GRB2-like (Endophilin) F8WIR7 26 27 interacting protein 1

MCG141011, isoform CRA_e Q571J7 26 27

312

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed 28S ribosomal protein S6, mitochondrial P58064 26 25

28S ribosomal protein S31, mitochondrial Q61733 26 25

Pyridoxal kinase Q8K183 26 20

Deubiquitinating enzyme 5 P56399 26 16 1-phosphatidylinositol-5-phosphate 4-kinase 2- O70172 26 13 alpha

RNA-binding motif protein 14 Q8C2Q3 26 11

CCR4-associated factor 1 Q6ZQ08 26 7

Syntaxin-1B P61264 25 70

Mammalian ependymin-related protein 1 Q99M71 25 41

5-3 exoribonuclease 2 A2ATK1 25 34

Glycine cleavage system P protein Q91W43 25 32 Cleavage and polyadenylation specificity factor Q9QXK7 25 28 73 kDa subunit

Threonine synthase-like 1 (Bacterial) Q149D1 25 27

MCG22088 D3Z6N6 25 25

54 kDa VacA-interacting protein Q9ESJ4 25 25

Putative uncharacterized protein Gm4963 D3YXG3 25 24

UPF0465 protein C5orf33 homolog Q8C5H8 25 24

Peptidylarginine deiminase II Q08642 25 23

Interleukin 1 receptor accessory protein-like 1 B1ASU0 25 22

L-fucose kinase Q7TMC8 25 20

Branched chain ketoacid dehydrogenase E1, Q3U3J1 25 16 alpha polypeptide

313

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Isocitrate dehydrogenase [NAD] subunit P70404 25 15 gamma, mitochondrial AT-hook DNA-binding motif-containing Q6PAL7 25 14 protein 1

ADP-ribosylation factor 3 P61205 25 54

Membrane protein, palmitoylated 6 (MAGUK Q3UN60 25 31 p55 subfamily member 6), isoform CRA_b

GDI-1 Q99PT1 25 5

H1 VAR.4 P43277 24 85

(S)-3-amino-2-methylpropionate transaminase P61922 24 52

Plakophilin-1 P97350 24 38

Septin-9 Q80UG5 24 38

ICD-M P54071 24 31 p164 ROCK-2 E9PYM9 24 16

Protein kinase C beta type P68404 24 13 Calcium-dependent activator protein for Q80TJ1 24 11 secretion 1

Lysosomal-trafficking regulator 2 Q9EPN1 24 11 Carboxyl-terminal LIM domain-binding protein O55203 24 9 1

Vesicle-associated membrane 2 B0QZN5 24 35

28S ribosomal protein S35, mitochondrial Q8BJZ4 24 17

Alpha-actinin-4 P57780 24 15

Prothymosin alpha P26350 24 11

Cofilin, non-muscle isoform F8WGL3 23 43

314

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Actin-binding protein 1 Q62418 23 34

La ribonucleoprotein domain family member 1 Q6ZQ58 23 27

Ubiquitin-activating enzyme E1 B9EHN0 23 20

GRB10-interacting GYF protein 2 Q6Y7W8 23 18

40S ribosomal protein S13 P62301 23 12 DNA-directed RNA polymerase II 140 kDa Q8CFI7 23 12 polypeptide

Tcf20 protein B9EHJ7 23 6

Putative uncharacterized protein Mapk3 D3Z3G6 23 36

Armadillo-related protein Q68FH0 23 26

Alpha-actinin cytoskeletal isoform Q7TPR4 23 10

Chromosome condensation 1-like G1EE51 22 30 Breast carcinoma-amplified sequence 1 Q80YN3 22 30 homolog

Asparaginase-like protein 1 Q8C0M9 22 30 Glycerol-3-phosphate dehydrogenase [NAD+], P13707 22 26 cytoplasmic

Actin-like protein 3B Q641P0 22 21

Guanine monphosphate synthetase B2RRH9 22 20

NIK- and IKBKB-binding protein Q3U0M1 22 18

Retinoblastoma-binding protein 5 Q8BX09 22 18

Adducin-like protein 70 Q9QYB5 22 18

Aczonin E9QK94 22 17

Putative uncharacterized protein Q3UMA3 22 13 bNOS F8WGF2 22 12

315

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed La autoantigen homolog P32067 22 12

Fibrous sheath component 2 P48774 22 27

Laminin subunit alpha-4 P97927 22 13

Early endosome antigen 1 Q8BL66 22 7

DEAH box protein 30 Q99PU8 22 7

Putative uncharacterized protein Q9CX86 22 8

3-oxoacid-CoA transferase 1 Q9D0K2 21 39 Capping protein (Actin filament) muscle Z-line, A2AMV7 21 38 beta

Hippocalcin-like 4 B2RRY8 21 31 Succinyl-CoA ligase [GDP-forming] subunit Q9WUM5 21 25 alpha, mitochondrial

Puromycin-sensitive aminopeptidase Q11011 21 23

WD repeat domain 5 A2AKB1 21 21 2-oxoglutarate dehydrogenase complex Q9D2G2 21 21 component E2

Agrin A2ASQ1 21 17 Propionyl-Coenzyme A carboxylase, alpha Q3UGC8 21 17 polypeptide, isoform CRA_b

(blank) G3X9L7 21 16

Entactin P10493 21 13 Eukaryotic translation initiation factor 4 E9QNX3 21 11 gamma, 3

Neuronal protein NP25 Q9R1Q8 21 18

Putative uncharacterized protein Q3U4B1 21 15

Uncharacterized protein KIAA0564 homolog Q8CC88 21 7

316

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Chondroitin sulfate proteoglycan 3 P55066 21 4

DNA-directed RNA polymerase II subunit A P08775 21 2

Rab5b protein B2RPS1 20 35

Putative uncharacterized protein Acad8 D3YTT4 20 32 Phytanoyl-CoA hydroxylase-interacting 20 29 protein-like Q8BGT8

Calcineurin subunit B type 1 Q63810 20 28

Abl-interactor 1 B7ZCT9 20 21

Putative uncharacterized protein Q8BTL0 20 20

C-1-tetrahydrofolate synthase, cytoplasmic F6YW06 20 18 Thioredoxin domain containing 4 (Endoplasmic 20 17 reticulum) B1AY76

MCG13422, isoform CRA_a Q3V214 20 15

Destrin Q4FK36 20 13

WD repeat domain 68 B1AR85 20 12

Aspartate--tRNA ligase Q922B2 20 20

MCG123182 Q3UCW0 20 16

40S ribosomal protein S11 E9PXE7 20 7

MCG9909 A2APD4 20 24 Calcium/calmodulin-dependent protein kinase 20 68 II, beta Q5SVJ0 Guanine nucleotide-binding protein subunit 19 29 alpha-11 P21278

FAD pyrophosphorylase Q8R123 19 29

28S ribosomal protein S2, mitochondrial Q924T2 19 27

317

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Protein SSXT Q62280 19 26

Nitrogen fixation gene 1 (S. cerevisiae) Q8C6I5 19 26

Potassium voltage-gated channel, shaker-related 19 24 subfamily, beta member 3 Q8VD73 Hypoxanthine guanine phosphoribosyl 19 21 transferase 1 B1B0W8

COP9 signalosome complex subunit 3 O88543 19 20

Tyrosyl-tRNA synthetase A2A7S7 19 17

BAI1-associated protein 1 Q6RHR9 19 17

Putative uncharacterized protein Q8CD76 19 16 Kelch repeat and BTB domain-containing 19 14 protein 11 Q8BNW9 Cleavage and polyadenylation specificity factor 19 12 100 kDa subunit O35218

Trafficking protein particle complex subunit 5 Q9CQA1 19 9

CAP-Gly domain-containing linker protein 2 Q9Z0H8 19 7

Sept5 protein B7ZNM7 19 51 Succinate dehydrogenase complex, subunit B, 19 43 iron sulfur (Ip) A2AA77

Putative uncharacterized protein E9Q0S6 19 4 Dynamin family member proline-rich carboxyl- 19 27 terminal domain less E9PUD2

Catalase P24270 19 7

Ciliary rootlet coiled-coil protein Q8CJ40 19 1

19 6 Nucleolysin TIA-1 P52912

318

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed

3-ketoacyl-CoA thiolase, mitochondrial Q8BWT1 18 27

Kinesin-2 E0CZ72 18 26

Aldehyde dehydrogenase family 6 member A1 Q9EQ20 18 16

40 kDa erythrocyte membrane protein O89112 18 15

60 kDa poly(U)-binding-splicing factor Q3UEB3 18 15

Inter-alpha-trypsin inhibitor heavy chain H5 Q8BJD1 18 15

(blank) E9Q401 18 13

Splicing factor arginine/serine-rich 4 (SRp75) A2A837 18 12

Putative uncharacterized protein Rps19 D3Z722 18 12

MCG9666, isoform CRA_d Q14A95 18 12

Kinectin F8VQC7 18 8

Penta-EF hand domain containing 1 A3KFW0 18 38

DnaJ (Hsp40) homolog, subfamily C, member 6 B1B0B8 18 16

YTH domain family protein 3 Q8BYK6 18 10

Phosphoribosyl pyrophosphate synthase I G3UXL2 18 3

130 kDa leucine-rich protein Q6PB66 18 1

Calponin, acidic isoform Q9DAW9 17 25

AIR carboxylase Q9DCL9 17 24

Protein WAVE-3 Q8VHI6 17 22

Acyl-CoA thioesterase 7 E9PYH2 17 21

Putative uncharacterized protein F8WIV2 17 17

Choline phosphatase 3 O35405 17 15

319

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Nucleolin P09405 17 15 Dual specificity mitogen-activated protein 17 15 kinase kinase 1 P31938

Histone deacetylase 6 Q9Z2V5 17 14

N-myc downstream regulated gene 1 Q545R3 17 13

Neurite outgrowth inhibitor Q99P72 17 10

Phosphate carrier protein, mitochondrial G5E902 17 70 Single-stranded DNA-binding protein, 17 14 mitochondrial Q9CYR0 Phosphatidylinositol-4-phosphate 5-kinase type 17 13 I gamma F8WHW6

Putative uncharacterized protein Q3U367 17 10

Nebulette Q9DC07 17 6 Cofactor required for Sp1 transcriptional 17 2 activation subunit 2 A2ABV5

Glutamate/H(+) symporter 1 Q9D6M3 16 47 Protein kinase C and casein kinase substrate in 16 32 neurons 1 Q543Y7 Eukaryotic translation initiation factor 3, 16 26 subunit H Q5M9L0

Hydroxysteroid (17-beta) dehydrogenase 10 A2AFQ2 16 24

Malonyl-CoA decarboxylase, mitochondrial Q99J39 16 22

P68 P40142 16 21

Microtubule-associated protein Q2UZW7 16 20

Constitutive coactivator of peroxisome 16 20 proliferator-activated receptor gamma Q6RI63

320

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Apba1 protein B2RUJ5 16 18

A kinase (PRKA) anchor protein 8 Q059U9 16 17

Phosphofurin acidic cluster sorting protein 1 Q8K212 16 13 PAX transactivation activation domain- 16 12 interacting protein Q6NZQ4 Vacuolar protein sorting-associated protein 16 16 11 homolog G3X8X7

28S ribosomal protein S21, mitochondrial P58059 16 11

Liprin-alpha 3 B8QI35 16 9

NFX1-type zinc finger-containing protein 1 Q8R151 16 7

1,4-beta-N-acetylmuramidase C P17897 16 39 ADP-ribosylation factor 1 GTPase-activating 16 23 protein Q9EPJ9 Cleavage and polyadenylation specificity factor 16 20 subunit 5 Q9CQF3

Calcium-dependent tyrosine kinase G3X8V1 16 15

Programmed cell death 6 interacting protein B8JJL8 16 13

Putative uncharacterized protein Q3ULB0 16 7

Deubiquitinating enzyme 15 Q8R5H1 16 7

Laminin M chain F8VQ43 16 6

EH domain-containing protein 3 Q9QXY6 16 8

Dynactin light chain p24 Q9Z0Y1 16 6

Synaptophysin B1AVA7 15 41

GAP-associated tyrosine phosphoprotein p62 Q60749 15 22

Fascin Q61553 15 22

321

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed MCG20813 A2A9F5 15 20

Pdk3 protein Q4FJR4 15 15 Cytoplasmic polyadenylation element-binding 15 13 protein 2 E9Q969

GDP-mannose pyrophosphorylase A Q922H4 15 13 Adapter-related protein complex 3 subunit 15 12 delta-1 O54774

Ras-related protein Rab-18 P35293 15 12

Isoleucine--tRNA ligase Q8BU30 15 12

High temperature requirement protein A2 Q9JIY5 15 12

60S ribosomal protein L24 E9QNJ0 15 11

Annexin A11 P97384 15 13

Centaurin beta 2 D4AFX6 15 9 Pyruvate dehydrogenase phosphatase regulatory 15 8 subunit, mitochondrial Q7TSQ8

ATP-dependent RNA helicase DDX48 Q91VC3 15 6

Ank1 protein E9QLM8 15 3

Ras-related protein Rab-33B O35963 15 73

ASH2-like protein Q91X20 15 6

Threonine--tRNA ligase Q3UQ84 15 4

UPF0533 protein C5orf44 homolog Q3TIR1 15 2

182 kDa tankyrase-1-binding protein P58871 15 2

Plexin-A2 P70207 15 2

Neural visinin-like protein 1 P62761 14 26 Protease (Prosome, macropain) 26S subunit, 14 23 ATPase 5 B1ARK2

322

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed 60S ribosomal protein L19 P84099 14 21

Isovaleryl coenzyme A dehydrogenase A3KGG9 14 20 eIF3 p110 Q8R1B4 14 20

28S ribosomal protein S28, mitochondrial Q9CY16 14 19

Pyrroline-5-carboxylate reductase Q3TMZ1 14 18

Cyp-C-associated protein Q07797 14 17

LanC-like protein 2 Q9JJK2 14 17

BC032203 protein B2RX81 14 14

Gbeta5 P62881 14 13 Eukaryotic translation initiation factor 3, 14 12 subunit 1 A2AE04

G protein pathway suppressor 1 G3UXW9 14 12

Nuclear receptor coactivator 6 Q5XJV5 14 12

Actin-interacting protein 1 O88342 14 11

MKIAA0617 protein Q6ZQ84 14 11

WD repeat-containing protein 37 Q8CBE3 14 10 Cleavage and polyadenylation specificity factor 14 8 subunit 6 H3BJW3

Putative uncharacterized protein Q8C266 14 36

Cornifin-A Q62266 14 24

Cullin 5 G3X914 14 17

Parkinson disease protein 7 homolog Q99LX0 14 15

E3 ubiquitin-protein ligase NEDD4-like Q8CFI0 14 13

Serine/arginine-rich protein-specific kinase 2 O54781 14 11

323

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed

Small nuclear ribonucleoprotein Sm D1 P62315 14 11

Fructosamine 3 kinase B1ATT8 14 10

WW domain-binding protein 2 E9Q1S7 14 4

U1 small nuclear ribonucleoprotein A E9Q4T6 14 42 eIF3 p47 Q9DCH4 14 13

AFG3-like protein 2 Q8JZQ2 14 12 Cytoplasmic polyadenylation element-binding 14 7 protein 4 Q7TN98

ER-Golgi SNARE of 24 kDa O08547 14 3

Copine V Q8JZW4 14 12

Protein KIAA1045 Q80TL4 13 25

Focal adhesion kinase 1 E9QMQ8 13 20 Calcium/calmodulin-dependent serine protein 13 18 kinase F8WGK4

Putative uncharacterized protein F8WIJ5 13 14

Aflatoxin B1 aldehyde reductase member 2 Q8CG76 13 13

CMP-N-acetylneuraminic acid synthase Q99KK2 13 13 Serine/threonine-protein phosphatase PP1-beta 13 11 catalytic subunit P62141

CCAAT-binding transcription factor I subunit A P62960 13 11

Formin-binding protein 1 Q80TY0 13 11 Collagen/fibrinogen domain-containing protein 13 10 1 O70165

60S ribosomal protein L22 P67984 13 10

Methionine adenosyltransferase 2 Q3THS6 13 9

324

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed (blank) B3F3T1 13 34

Phenylalanine--tRNA ligase alpha chain Q8C0C7 13 18

Carbohydrate kinase domain-containing protein Q9CZ42 13 18 Phosphatidylinositol-4-phosphate 5-kinase, type 13 15 II, beta Q3UJ95

Protein midA homolog, mitochondrial Q9CWG8 13 15

17-beta-hydroxysteroid dehydrogenase 8 P50171 13 14

Mu-crystallin homolog O54983 13 12

Protein Odd Oz/ten-m homolog 2 Q9WTS5 13 10 [Pyruvate dehydrogenase [lipoamide]] kinase 13 8 isozyme 1, mitochondrial Q8BFP9

Serine--tRNA ligase Q9JJL8 13 7

Putative uncharacterized protein E9Q530 13 6 49 kDa TATA box-binding protein-interacting 13 6 protein P60122

Cytosolic NADP-isocitrate dehydrogenase F8WIY0 13 4

EPM2A-interacting protein 1 Q8VEH5 13 3

Sm protein E P62305 13 17

Ubiquitin-conjugating enzyme O88738 13 11

Putative uncharacterized protein Q3TIX6 13 6

Itchy F6YH70 13 4

(blank) E9Q616 13 6

Protein LSM12 homolog Q9D0R8 12 25

Leukocyte elastase inhibitor A Q9D154 12 24

325

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed MCG5487, isoform CRA_b A2BH07 12 20

Mitochondrial ribosomal protein S15 A2A8Y4 12 17

Cartilage-linking protein 1 Q9QUP5 12 16

Histidine triad nucleotide binding protein B1AZK3 12 12

Kinesin family member 3A B1AQZ2 12 10 DNA-directed RNA polymerase II 33 kDa 12 9 polypeptide E9Q898 Mitochondrial ribosomal protein S11, isoform 12 9 CRA_b Q3U8Y1

28S ribosomal protein S18-2, mitochondrial Q99N84 12 8

Uncharacterized protein C1orf77 homolog Q9CY57 12 6

Synaptogyrin-1 O55100 12 42 FtsJ methyltransferase domain-containing 12 14 protein 2 Q9DBC3

Breast carcinoma amplified sequence 3 B1AR74 12 11

AU-specific RNA-binding enoyl-CoA hydratase Q9JLZ3 12 10

Protein-tyrosine phosphatase SYP P35235 12 8

Adapter protein containing PH domain, PTB 12 4 domain and leucine zipper motif 1 Q8K3H0

Intracellular hyaluronan-binding protein 4 E9QKB2 12 2

60S ribosomal protein L31 P62900 12 17

CaMK-like CREB regulatory kinase 2 Q6PGN3 12 8 cAMP-dependent protein kinase type II-beta 12 6 regulatory subunit P31324

326

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Neuronal olfactomedin-related ER localized 12 4 protein O88998 Inactive ubiquitin carboxyl-terminal hydrolase 12 1 52 Q8BGF7

Brevican core protein Q61361 12 8

(blank) E9Q7G0 12 5

Protein arginine N-methyltransferase 1 Q9JIF0 12 3

Mitchondrial ribosomal protein S7 A2A9W4 11 19

Methylthioadenosine phosphorylase A2ANM4 11 16 (N-acetylneuraminyl)- 11 14 galactosylglucosylceramide Q09200 [Pyruvate dehydrogenase [lipoamide]] kinase 11 14 isozyme 2, mitochondrial Q9JK42

Tetratricopeptide repeat protein 15 Q8K2L8 11 13

Neuroligin-1 Q99K10 11 10

80 kDa nuclear cap-binding protein Q3UYV9 11 9

PAI1 RNA-binding protein 1 Q9CY58 11 9

Protein FAM195A Q9CQB2 11 8

3,2-trans-enoyl-CoA isomerase, mitochondrial P42125 11 5

Ras-related protein Rab-11B P46638 11 25 ATPase, H+ transporting, lysosomal V1 subunit 11 22 G2 Q54A87

Complex I-MLRQ Q62425 11 19

Protein-tyrosine phosphatase MEG2 O35239 11 11

MCG129789, isoform CRA_b Q3TKV1 11 11

Rho GTPase-activating protein 12 Q8C0D4 11 11

327

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed

Protein phosphatase 1 regulatory subunit 3F Q9JIG4 11 11 Electron transferring flavoprotein, alpha 11 10 polypeptide B1B1B4

COP9 signalosome complex subunit 4 O88544 11 9 La ribonucleoprotein domain family member 11 9 4B Q6A0A2

Protein phosphatase methylesterase 1 Q8BVQ5 11 6

Bullous pemphigoid antigen 1 E9PXE5 11 4

MCG1040650 A2AG40 11 3

STAT5a variant delta E18 B2C3G8 11 3

ATP synthase, H+ transporting, mitochondrial 11 18 F1 complex, delta subunit, isoform CRA_c Q4FK74

Importin alpha-S1 Q60960 11 9

Aldehyde dehydrogenase 1 family, member B1 B1AWX7 11 6

TANK-binding kinase 1 A1L361 11 5 Mitochondrial import inner membrane 11 3 translocase subunit TIM44 O35857

Putative WDC146 Q8K4P0 11 3

Adenylyl cyclase-associated protein 2 Q9CYT6 11 3

Uncharacterized protein KIAA1107 Q80TK0 11 1

Adenylate cyclase-stimulating G alpha protein Q6R0H7 11 32

Putative uncharacterized protein D3Z2H9 11 8 ENSMUSP00000072197

Propanoyl-CoA:carbon dioxide ligase subunit Q99MN9 11 4 beta

328

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed

Putative uncharacterized protein Pebp1 D3Z1V4 10 38

Protein RUFY3 E9QL96 10 22

UPF0515 protein C19orf66 homolog Q8CAK3 10 22

Glycerol-3-phosphate dehydrogenase 1-like B2RSR7 10 16

UPF0424 protein C1orf128 homolog E9QK32 10 16

Vacuolar protein sorting-associated protein 26B Q8C0E2 10 16 Cleavage and polyadenylation specific factor 4 10 12 isoform 2 E0CXT7

Thymidylate kinase LPS-inducible member Q3U5Q7 10 12 Class A helix-loop-helix transcription factor 10 10 ME2 E9Q8G4 Apoptotic chromatin condensation inducer in 10 10 the nucleus Q9JIX8

Ena/vasodilator-stimulated phosphoprotein-like P70429 10 9

Putative uncharacterized protein Mpst D3YYT8 10 8 Adaptor-related protein complex 3, sigma 1 10 8 subunit G3XA56

28S ribosomal protein S14, mitochondrial Q9CR88 10 7

Mediator complex subunit 20 Q9R0X0 10 7

Putative uncharacterized protein Napg D3Z4B2 10 19

Microtubule-associated protein 4 E9PZ43 10 19

Carbonate dehydratase II P00920 10 19

60S ribosomal protein L35 Q6ZWV7 10 15

10 12 (blank) Q8BVC4

329

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed 40S ribosomal protein S26 P62855 10 9

(blank) Q8VG87 10 9

(blank) E9PWG2 10 7 Dynamin family member proline-rich carboxyl- 10 30 terminal domain less Q8K1M6

Leubrin Q8K406 10 18

Ras-related protein Rab-2A P53994 10 11

Cytoplasmic dynein 1 light intermediate chain 2 Q6PDL0 10 11 Dual-specificity tyrosine-(Y)-phosphorylation 10 10 regulated kinase 1a A9C475

Uncharacterized protein C6orf174 homolog Q6NZL0 10 5

Asparagine--tRNA ligase Q8BP47 10 2

Elongator complex protein 1 Q7TT37 10 1

Mammalian lin-seven protein 2 O88951 10 25 Ganglioside-induced differentiation-associated 10 4 protein 1-like 1 Q8VE33

Plexin-A4 Q80UG2 10 3

Heat-responsive protein 12 P52760 10 2

Calretinin Q08331 9 28

ATP synthase gamma chain Q3UD06 9 17

Aldehyde dehydrogenase family 18 member A1 Q9Z110 9 15

Vacuolar proton pump subunit C 1 Q9Z1G3 9 15

Methionine--tRNA ligase E9QB02 9 10

COP9 signalosome complex subunit 5 O35864 9 9

Formyltetrahydrofolate synthetase Q3V3R1 9 9

330

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Methionine adenosyltransferase 2 subunit beta Q99LB6 9 9

Arginine--tRNA ligase Q9D0I9 9 8

Heterogeneous nuclear ribonucleoprotein L-like Q921F4 9 7

Calbindin-28K A2AS59 9 28

Neuron cytoplasmic protein 9.5 Q9R0P9 9 25

ARF GTPase-activating protein GIT1 Q68FF6 9 14

Phosphatidylinositide phosphatase SAC1 Q9EP69 9 13 Secretory carrier-associated membrane protein Q9JKD3 9 11 5 Dual specificity phosphatase 3 (Vaccinia virus B1AQF4 9 9 phosphatase VH1-related)

60S ribosomal protein L13 P47963 9 9

Adenylyl cyclase-associated protein 1 P40124 9 8

Argininosuccinate synthase P16460 9 7 Serine/threonine-protein phosphatase 2A P62715 9 5 catalytic subunit beta isoform

Glutathione peroxidase Q76LV0 9 3

3-hydroxy-3-methylglutarate-CoA lyase P38060 9 2

Histone H2B Q921L4 9 24 Cleavage and polyadenylation specificity factor Q8BTV2 9 15 subunit 7

Dynein cytoplasmic 1 light intermediate chain 1 Q3TWG5 9 8

ASF/SF2 H7BX95 9 7

Splicing factor, arginine/serine-rich 7 Q8BL97 9 7

PTPNU-3 B0V2N1 9 6

331

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed CW17 Q64213 9 5

Actin-binding protein anillin Q8K298 9 5

Papln protein B7ZN28 9 1

Dipeptidyl peptidase X E9QN98 9 5

Toll interacting protein A6PWS1 9 3

Glucose-6-phosphate 1-dehydrogenase X Q00612 9 2

Protein NipSnap homolog 1 E9Q0E4 8 24

66 kDa tyrosine-rich heat shock protein Q8QZY1 8 23 Glyoxylate reductase/hydroxypyruvate 8 12 reductase Q3T9Z2 Microtubule-associated protein, RP/EB family, 8 10 member 1 Q3U4H0

Importin alpha Q1 O35343 8 9 CDP-diacylglycerol--inositol 3- 8 9 phosphatidyltransferase Q8VDP6

Growth arrest-specific protein 7 Q60780 8 7

L-iditol 2-dehydrogenase Q64442 8 7

Cadherin-11 P55288 8 6

Mediator complex subunit 31 Q9CXU1 8 5

Alpha-1,3-mannosyltransferase ALG2 Q9DBE8 8 4

Cytochrome c, somatic G3UWG1 8 65

Glycoprotein m6b A2AEG6 8 22

Cell division protein kinase 5 P49615 8 19 cAMP-dependent protein kinase catalytic 8 18 subunit beta P68181

332

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed

Putative uncharacterized protein Gm5908 D3Z6J9 8 16

Kinesin-like protein KIF21A E9Q0J5 8 15

Erythrocyte protein band 4.1-like 1 A2AUK5 8 14

Protein FAM168A Q8BGZ2 8 13

5-nucleotidase domain-containing protein 3 Q3UHB1 8 12

Ig kappa chain C region P01837 8 9

Macropain iota chain Q9QUM9 8 9

Dynactin subunit 5 Q9QZB9 8 9

Clasp2 protein E9QB05 8 8 eIF3 p116 E9PX78 8 7 Ankyrin repeat and FYVE domain-containing 8 6 protein 1 Q810B6 Rab3 GTPase-activating protein 150 kDa 8 5 subunit E9QKE4

Argininosuccinate lyase Q91YI0 8 5

Casein kinase II subunit beta G3UZX4 8 4

MCG14259, isoform CRA_a Q14C24 8 4

Nucleoporin SEH1 Q8R2U0 8 4

NUDT16-like protein 1 Q8VHN8 8 4

Actin-related protein 2/3 complex subunit 5 A3KGQ6 8 16

Sorting nexin-9 Q91VH2 8 16 Adapter-related protein complex 2 sigma 8 12 subunit P62743 Deubiquitinating enzyme OTUB1 Q7TQI3 8 12

333

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed

Methionine-tRNA synthetase 2 (Mitochondrial) A2RT28 8 11

Deubiquitinating enzyme CYLD Q80TQ2 8 8

General vesicular transport factor p115 Q9Z1Z0 8 4

Cellular glutathione peroxidase P11352 8 3

Putative uncharacterized protein Q3TXT7 8 2

Mediator complex subunit 22 Q62276 8 2

NPC-derived proline-rich protein 1 E9QLZ9 8 1 Pre-mRNA-splicing factor SF3b 155 kDa 8 1 subunit G5E866

Annexin A1 E9QA30 8 27

Creatine kinase, muscle A2RTA0 8 3

ADP-ribosylation factor 6 P62331 8 3

28S ribosomal protein S16, mitochondrial Q9CPX7 8 3

Vacuolar protein sorting-associated protein 29 Q9QZ88 8 2 Cutaneous T-cell lymphoma-associated antigen 8 1 5 homolog H3BJS0

Vacuolar protein sorting-associated protein 33B P59016 8 1

Centrosomal protein of 164 kDa Q5DU05 8 1

Putative GTP-binding protein Parf Q5U3K5 8 1

Tubulointerstitial nephritis antigen-like 1 A3KFW2 7 10

Aldehyde dehydrogenase family 7 member A1 Q9DBF1 7 8

FIS1 homolog Q9CQ92 7 5

Electron transfer flavoprotein subunit beta Q9DCW4 7 19

334

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Neuronal pentraxin receptor F6T7F3 7 12

Cysteine and glycine-rich protein 1 P97315 7 12 Fumarylacetoacetate hydrolase domain- Q3TC72 7 12 containing protein 2A Calcium-independent alpha-latrotoxin receptor E9Q3V9 7 11 1 2-methyl branched chain acyl-CoA E9Q5L3 7 11 dehydrogenase

Rhodanese P52196 7 10

Adenylate kinase 3 Q9WTP7 7 10

Pde10a Q7TPG2 7 9

Chromatin assembly factor 1 subunit C E9PYH8 7 6

Ig kappa chain V-V region K2 P01635 7 5

110 kDa cell membrane glycoprotein Q9JKV1 7 4

Pyruvate dehydrogenase complex, component X A2AWH6 7 3

BNIP2 motif-containing molecule at the C- Q52KR3 7 3 terminal region 1

Putative uncharacterized protein Q3TRJ7 7 43

Guanosine diphosphate dissociation inhibitor 1 P50396 7 24

Brain protein H5 P28661 7 18 Epidermal growth factor receptor pathway Q60902 7 15 substrate 15-related sequence

Lon protease homolog, mitochondrial E9Q120 7 10

ADP-ribosylation factor-like protein 10C Q9CQW2 7 10

CDC42-binding protein kinase beta Q7TT50 7 7

335

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Mitogen-activated protein kinase kinase kinase Q8CF89 7 7 7-interacting protein 1

Neuroligin 3 A2AGI2 7 5

Homer protein homolog 3 Q99JP6 7 5 26S proteasome non-ATPase regulatory subunit Q9CR00 7 5 9

Mediator complex subunit 18 Q9CZ82 7 4

Ig heavy chain V region AC38 205.12 P06330 7 2

Casein kinase I isoform alpha E9PWB2 7 1

ATP synthase subunit O, mitochondrial Q9DB20 7 17

Heat shock protein 1 (Chaperonin 10) Q4KL76 7 15

Rgs7 protein Q80XD3 7 8

Ras-related protein Rap-2b P61226 7 6

1,4-alpha-glucan-branching enzyme Q9D6Y9 7 4

Nucleoporin 98 Q6PFD9 7 3 GTP-specific succinyl-CoA synthetase subunit Q9Z2I8 7 3 beta Branched-chain alpha-keto acid dehydrogenase P53395 7 2 complex component E2

Enaptin F8WIS7 7 1

Protein VAC14 homolog Q80WQ2 7 1

GDP-mannose pyrophosphorylase B Q8BTZ7 7 1

Tax1-binding protein 1 homolog Q3UKC1 6 8

Neuroplastin P97300 6 29

Inorganic pyrophosphatase 2, mitochondrial Q91VM9 6 24

Putative uncharacterized protein Q3UWU7 6 14

336

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Proteasome subunit beta type A2CFA7 6 13

MCG21883, isoform CRA_e Q3UKM0 6 11

WD repeat-containing protein 47 Q8CGF6 6 11

Tetratricopeptide repeat protein 9A Q3V038 6 9

28S ribosomal protein S10, mitochondrial G5E8U5 6 8 Acyl-CoA synthetase family member 2, 6 8 mitochondrial Q8VCW8

Putative uncharacterized protein Dync1i1 D3Z0M6 6 7

Granule cell differentiation protein P62774 6 7

CD40 receptor-associated factor 1 Q60803 6 7

Adapter-related protein complex 3 mu-2 subunit Q8R2R9 6 6

2-aminoethanethiol dioxygenase Q6PDY2 6 5

Complex III subunit 1 Q9CZ13 6 60

PDHE1-A type I P35486 6 30

ATP synthase, H+ transporting, mitochondrial 6 19 F0 complex, subunit b, isoform 1 Q5I0W0

Cbln1 protein Q7TNF5 6 18

Mitogen activated protein kinase 9 Q5NCK7 6 14

Sepiapterin reductase Q91XH5 6 12

BTB/POZ domain-containing protein KCTD12 Q6WVG3 6 7

Cannabinoid receptor interacting protein 1 F7C0H6 6 4

Adaptor protein complex AP-1 mu-1 subunit P35585 6 4

Malic enzyme 2 Q99KE1 6 4

337

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Noelin-3 P63056 6 3

Rho GTPase activating protein 21 B7ZCJ1 6 2 p65 H6RXZ1 6 33

Sodium-calcium exchanger Q8K596 6 15 Brain-specific Na(+)-dependent inorganic Q3TXX4 6 14 phosphate cotransporter Protein tyrosine phosphatase, receptor type Z, B9EKR1 6 11 polypeptide 1

28S ribosomal protein S25, mitochondrial Q9D125 6 11

Neural cell expressed, developmentally down- B2RSC8 6 9 regulated 4

Splicing factor, arginine/serine-rich 2 (SC-35) E9Q975 6 9

Ig kappa chain V-VI region XRPC 44 P01675 6 9 Protein kinase, cAMP dependent regulatory, E9Q276 6 8 type I, alpha

Ig kappa chain V-II region 26-10 P01631 6 8

Dihydropteridine reductase Q8BVI4 6 5

UPF0598 protein C8orf82 homolog Q8VE95 6 4

Putative uncharacterized protein Hmcn1 D3YXG0 6 2

Calcineurin-like phosphoesterase domain- Q8BFS6 6 2 containing protein 1

CUL4- and DDB1-associated WDR protein 12 Q8C0M0 6 2

Putative uncharacterized protein Mgll H7BXA7 6 1

Phosphoribosyl pyrophosphate synthetase- Q5SWZ0 6 1 associated protein 2

338

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Cofactor required for Sp1 transcriptional Q8VCD5 5 9 activation subunit 6

Beta-B1 crystallin E9Q789 5 7

MCG2706 A2AV97 5 6

P39 P51863 5 36 N-ethylmaleimide sensitive fusion protein A2APW8 5 16 attachment protein beta

(blank) E9Q474 5 15

Coronin, actin binding protein 1C Q499X7 5 14

3-oxoacyl-[acyl-carrier-protein] reductase Q91VT4 5 14

Novel protein (A930041I02Rik) B0R0E3 5 9

Endoplasmic reticulum resident protein 72 P08003 5 8

Rho-related GTP-binding protein RhoG P84096 5 8

Nucleoporin 88 Q5QNT9 5 8

Myotubularin-related protein 9 Q9Z2D0 5 8

Ferrochelatase Q544X6 5 7

Actin-related protein 2/3 complex subunit 5 Q9CPW4 5 7

Glutathione reductase, mitochondrial P47791 5 5 eIF3 p66 O70194 5 4

Glucosamine-6-phosphate deaminase 1 O88958 5 3

Dehydrogenase/reductase SDR family member Q99L04 5 3 1 Calcium-activated neutral proteinase small O88456 5 2 subunit

Delayed early response protein 6 P34884 5 2

339

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Contactin-1 P12960 5 32

ATP-dependent RNA helicase eIF4A-2 P10630 5 20

(blank) G5E8K5 5 18

Rho-related GTP-binding protein RhoB P62746 5 14

Secernin-1 Q9CZC8 5 14 cAMP-dependent protein kinase type II-alpha 5 13 regulatory chain Q8K1M3

Cytosol aminopeptidase Q9CPY7 5 12

BTB/POZ domain-containing protein KCTD3 Q8BFX3 5 8

Alanine--tRNA ligase Q8BGQ7 5 7

MCG4664, isoform CRA_b Q58DZ5 5 6 eIF-3 p25 Q9DBZ5 5 6

2-hydroxyacyl-CoA lyase 1 Q9QXE0 5 6 Eukaryotic translation initiation factor 3, 5 5 subunit 4 (Delta) Q544H0

Histidine triad nucleotide binding protein 2 Q5M9J2 5 5

Protein FAM177A1 Q8BR63 5 5

Pyrroline-5-carboxylate reductase 3 Q9DCC4 5 5

DNA-directed RNA polymerase II subunit H Q923G2 5 4

Putative uncharacterized protein Unc13a D3YTQ0 5 3

Leucine rich repeat containing 47 E9PV22 5 3

MCG68069 Q5SQB7 5 3

MCG3634, isoform CRA_b Q5SUR3 5 3

340

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Receptor-type tyrosine-protein phosphatase Q64487 5 3 delta Mitogen-activated protein kinase kinase kinase Q99K90 5 3 7-interacting protein 2

MCG6739, isoform CRA_a A2ABW8 5 1

(blank) E9Q7X7 5 1 Mitochondrial import inner membrane P62075 5 1 translocase subunit Tim13

Acylphosphatase Q5SPV7 5 1

Complex III subunit 2 Q9DB77 5 47

B allele F6TQW2 5 4 Apoptosis-inducing factor, mitochondrion- B1AU24 5 3 associated 1

(blank) Q8BI38 5 3

67 kDa glutamic acid decarboxylase P48318 5 2

Scaffold attachment factor B2 Q80YR5 5 2

Protein FAM103A1 Q9CQY2 5 2

Casein kinase 1, delta, isoform CRA_b Q3USK2 5 1

HESB-like domain-containing protein 2 Q9D924 5 1

Probable threonyl-tRNA synthetase 2, Q8BLY2 4 12 cytoplasmic

UPF0636 protein C4orf41 homolog B2RXC1 4 11 Mitochondrial import inner membrane Q9D880 4 11 translocase subunit TIM50

Cell differentiation protein RCD1 homolog Q9JKY0 4 10

Microsomal endopeptidase Q91YP2 4 6

341

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Polyadenylate-binding protein-interacting F6Y616 4 22 protein 1 Putative transferase C1orf69 homolog, Q8CAK1 4 12 mitochondrial

Enhancer of rudimentary homolog P84089 4 10

Mitotic checkpoint protein BUB3 Q9WVA3 4 9

DnaJ homolog subfamily A member 2 Q9QYJ0 4 5

Myelin-associated glycoprotein F8WJJ3 4 4

Ig lambda-1 chain C region P01843 4 4 Enoyl-CoA hydratase domain-containing Q9D9V3 4 4 protein 1

56 kDa U2AF65-associated protein Q9Z1N5 4 4

Calgizzarin F6SJ35 4 3

Pleckstrin homology domain containing, family F8WGQ4 4 3 C (With FERM domain) member 1

Protein FAM195B F8WHC3 4 3

Delta(3),delta(2)-enoyl-CoA isomerase Q9WUR2 4 3

26S protease subunit S5 basic Q8BJY1 4 9

Arsenite-resistance protein 2 Q99MR6 4 7

Ephrin type-A receptor 2 Q03145 4 6 c-H-ras F7BIB2 4 5

IMPDH-I F7DEU6 4 5

Cellular thyroid hormone-binding protein P09103 4 5

61 kDa Cam-PDE Q61481 4 5

342

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Serine/threonine-protein phosphatase PP1-alpha P62137 4 4 catalytic subunit

5-azacytidine-induced protein 2 Q9QYP6 4 4

[3-methyl-2-oxobutanoate dehydrogenase O55028 4 3 [lipoamide]] kinase, mitochondrial

Acyl-CoA thioesterase 11 Q543P6 4 3

Glutamate receptor-like protein 1A E9PZ54 4 2

Armadillo repeat-containing protein 8 G3X920 4 2

GTP-binding protein SAR1a P36536 4 2

RING-box protein 1 P62878 4 2

Protein prune homolog Q8BIW1 4 2

Dematin Q9WV69 3 11

Putative uncharacterized protein Q9CZI5 3 8

Gamma-1-tubulin P83887 3 6

Glutathione S-transferase kappa 1 Q9DCM2 3 6

AUT-like 1 cysteine endopeptidase Q8BGE6 3 4 Lamina-associated polypeptide 2, isoforms Q61033 3 12 alpha/zeta NADH dehydrogenase [ubiquinone] Q9D6J6 3 11 flavoprotein 2, mitochondrial

Peptidase (Mitochondrial processing) alpha A2AIW8 3 9

LIM domain only protein 3 Q8BZL8 3 9

Oxidation resistance protein 1 Q4KMM3 3 8

CD81 partner 3 Q8R366 3 8

Putative uncharacterized protein Q5EBP9 3 7

343

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed 3-hydroxybutyrate dehydrogenase Q80XN0 3 7 Methionine-R-sulfoxide reductase B2, 3 6 mitochondrial Q78J03

Novel protein B2KG93 3 5

40S ribosomal protein S12 P63323 3 5

Neuronal calcium-binding protein 2 Q91ZP9 3 5

Hsc70-interacting protein Q99L47 3 5

Alpha4 phosphoprotein E9Q2M7 3 4

Putative uncharacterized protein Q543P7 3 4

Four and a half LIM domains 1 A2AEY2 3 3

U1 small nuclear ribonucleoprotein C Q569X3 3 3

Transaldolase Q93092 3 3

Rho GTPase-activating protein RICH2 Q5SSM3 3 2

2-phospho-D-glycerate hydro-lyase P21550 3 39

Myelin oligodendrocyte glycoprotein Q3UY21 3 29 Eukaryotic translation initiation factor 4E 3 10 member 2 Q0P688

CNDP dipeptidase 2 Q9D1A2 3 10

Complex I-30kD Q9DCT2 3 10

Rap1 GTPase-activating protein A2ALS4 3 7

BTB/POZ domain-containing protein KCTD8 Q50H33 3 7

MKIAA0866 protein Q69ZX3 3 7 Medium-chain specific acyl-CoA 3 6 dehydrogenase, mitochondrial P45952

Anchorin CII P48036 3 5

344

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Bromodomain-containing protein 9 Q3UQU0 3 5

ADP-ribosylation factor-like protein 15 Q8BGR6 3 5

Double-stranded RNA-binding protein Staufen Q8CJ67 3 5 homolog 2

28S ribosomal protein S24, mitochondrial Q9CQV5 3 5

Deubiquitinating enzyme 7 E9PXY8 3 3

(blank) E9Q071 3 3 Calcium homeostasis endoplasmic reticulum G5E8I8 3 3 protein Membrane-associated progesterone receptor O55022 3 3 component 1

AMOG P14231 3 3

Putative uncharacterized protein Q3U125 3 3

FAST kinase domain-containing protein 3 Q8BSN9 3 3

Phosphoinositide 3-kinase regulatory subunit 4 Q8VD65 3 3

Putative uncharacterized protein Q3TLP8 2 31

Arginine and glutamate-rich protein 1 Q3UL36 2 18

Ribosomal protein L23 A2A6F9 2 7 Eukaryotic translation initiation factor 3 subunit Q99JX4 2 5 M

Haloacid dehalogenase-like hydrolase domain- Q9CYW4 2 5 containing protein 3

40S ribosomal protein S24 P62849 2 4

Thy-1 antigen P01831 2 54

345

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Pyruvate dehydrogenase E1 component subunit Q9D051 2 25 beta, mitochondrial

Complement component 2 (Within H-2S) B8JJN0 2 8

Serine hydroxymethyltransferase Q9CZN7 2 6 Nuclear pore complex-associated intranuclear E9PZZ3 2 5 coiled-coil protein TPR

Methylmalonyl-CoA isomerase P16332 2 5

56 kDa selenium-binding protein P17563 2 5

Protein FAM49B Q921M7 2 5

CDW13/WDR61 A0MNP4 2 4

Ubiquitin carrier protein A2RTT4 2 4

EG620155 protein F6VWT4 2 4 Cofactor required for Sp1 transcriptional Q9DB40 2 4 activation subunit 8

Neuronal membrane glycoprotein M6-a P35802 1 47

FK506 binding protein 1a A2AT05 1 22 Hsp90 chaperone protein kinase-targeting Q61081 1 9 subunit

Mediator complex subunit 6 Q921D4 1 7

UPF0565 protein C2orf69 homolog Q9D9H8 1 7

Formin-binding protein 1-like E9PUI5 1 6

Cytochrome c oxidase, subunit Vb Q9D881 1 5

Heat shock protein 4 Q3U2G2 1 4

Putative uncharacterized protein Q4VA32 1 7

Exocyst complex component 3 Q6KAR6 1 7

346

Additive Spectral Uniprot Counts Protein Name ID Nicotine Control Exposed Synapsin III Q8JZP2 1 7

Carbonyl reductase 1 B2RXY7 1 6

CCR4-associated factor 2 Q8C5L3 1 6 Stromal membrane-associated GTPase- B1AVZ1 1 5 activating protein 2

Plastin 3 (T-isoform) B1AX58 1 5

Importin alpha Q2 O35344 1 5

Imprinted and ancient gene protein O55091 1 5 5-AMP-activated protein kinase subunit Q91WG5 1 5 gamma-2

Beta-Pix Q9ES28 1 5

347