The Direct and Functional Interaction of Tubulin with Transient Receptor Potential Melastatin 2

The Direct and Functional Interaction of Tubulin With Transient Receptor Potential Melastatin 2

Colin Elliott Seepersad

A thesis submitted in conformity with the requirements for the degree of Masters of Science Cell & Systems Biology University of Toronto

The Direct and Functional Interaction of Tubulin With Transient Receptor Potential Melastatin 2

Colin Elliott Seepersad

Masters of Science

Cell & Systems Biology University of Toronto

2011 Abstract

Transient Receptor Potential Melastatin 2 (TRPM2) is a widely expressed, non-selective cationic channel with implicated roles in cell death, chemokine production and oxidative stress. This study characterizes a novel interactor of TRPM2. Using fusion proteins comprised of the

TRPM2 C-terminus we established that tubulin interacted directly with the predicted C-terminal coiled-coil domain of the channel. In vitro studies revealed increased interaction between tubulin and TRPM2 during LPS-induced macrophage activation and taxol-induced microtubule stabilization. We propose that the stabilization of microtubules in activated macrophages enhances the interaction of tubulin with TRPM2 resulting in the gating and/or localization of the channel resulting in a contribution to increased intracellular calcium and downstream production of chemokines.

Acknowledgments

I would like to thank my supervisor Dr. Michelle Aarts for providing me with the opportunity to pursuit a Master’s of Science Degree at the University of Toronto. Since my days as an undergraduate thesis student, Michelle has provided me with the knowledge, tools and environment to succeed. I am very grateful for the experience and will move forward a more rounded and mature student. Special thanks to my committee members, Dr. Mauricio Terebiznik, and Dr. Dinesh Christendat who provided the insight and direction for my progress. I would also like to acknowledge NSERC and the University of Toronto Fellowship for providing the funding necessary for my studies.

I extend my gratitude and thanks to the many friends I’ve made along the way. Past and present members of the Aarts Lab, you have provided the support, advice and humor needed to keep me going; Danny Chan, Kevin Sam, Russell Bent, Melanie Ratnam, Darren Gigliozzi, Naghmeh Lesani and Jonathan Chan – Thank you. I am most gracious for the external support and the endless amount of advice, expertise and RAW cells – thank you to all the members of the Harrison and Terebiznik Labs. Thanks to Chris Yong-Kee, Sherri Thiele, Ari Chow and Sam Khalouei. Extended thanks to Dr. Shelley Brunt and Dr. Mauricio Terebiznik for your invaluable and warm advice, support and expertise. Lastly I would like to thank my friends and family for the love, encouragement and motivation.

iii

Table of Contents

Acknowledgments...... iii

Table of Contents...... iv

List of Tables...... vii

List of Figures...... viii

List of Appendices ...... ix

List of Abbreviations...... x

Chapter 1...... 1

1 Introduction ...... 1 1.1 Transient Receptor Potential Channels...... 1 1.2 The TRPM Subfamily...... 5 1.2.1 TRPM1 ...... 5 1.2.2 TRPM3 ...... 6 1.2.3 TRPM4 & TRPM5 ...... 6 1.2.4 TRPM6 & TRPM7 ...... 7 1.2.5 TRPM8 ...... 7 1.3 TRPM2...... 7 1.4 TRPM2 Modes of Activation ...... 13 1.5 The Pharmacology of TRPM2...... 16 1.6 The Physiological and Pathological Roles of TRPM2...... 16 1.7 Hypothesis and Objectives ...... 18

Chapter 2...... 20

2 Materials and Methods...... 20 2.1 Cloning ...... 20 2.1.1 The Generation of GST-Fusion Constructs...... 20 2.2 Protein Pull-Down and Tubulin Interaction Assay...... 23 2.2.1 Protein purification...... 23

2.2.2 Mouse Brain Homogenization...... 24 2.2.3 Protein Pull-Down Assay...... 24 2.2.4 Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS)...... 25 2.2.5 Tubulin Binding Assay...... 25 2.3 SDS-Polyacrylamide Gel Electrophoresis ...... 25 2.4 Western Blotting...... 25 2.5 Cell Culture...... 26 2.6 Immunocytochemistry...... 26

Chapter 3...... 27

3 Results ...... 27 3.1 Identifying TRPM2 C-Terminal Protein Interactions ...... 27 3.1.1 Generation of Full-Length GST-TRPM2 C- and N-Terminus Fusion Constructs...... 27 3.1.2 Expression of GST-TRPM2 C- and N-Terminus Fusion Constructs in BL-21 DES E. coli 33 3.1.3 Identification of TRPM2 C-Terminus Protein Interactors Through Protein-Pull Down Assays and Mass Spectrometry...... 34 3.2 Investigating Tubulin’s Interaction With the Full-Length TRPM2 C-Terminus...... 34 3.2.1 Tubulin Binds Directly to the C-Terminus of TRPM2 ...... 34 3.3 Investigating the Binding Location of Tubulin Along the C-Terminus of TRPM2...... 41 3.3.1 Generation of Truncated GST-TRPM2 C-Terminus Fusion Constructs...... 41 3.3.2 Tubulin Interacts Directly With the C-Terminal Coiled-Coil Domain of TRPM2...... 53 3.3.3 The C-terminal Coiled-Coil of TRPM2 Contains a Putative Tubulin-Binding Motif Similar to that Found on TRPV1 ...... 53 3.4 In Vitro Analysis of The Interaction of Tubulin with TRPM2 in RAW 264.7 Macrophages... 59 3.4.1 TRPM2 Co-Localizes With Tubulin In RAW Cells...... 59

Chapter 4...... 65

4 Discussion ...... 65 4.1 TRPM2 And Its Interaction With Tubulin: The Potential Role At The Membrane...... 65 4.1.1 Localization of TRPM2 to the Membrane by Tubulin ...... 65 4.2 A Potential Role for TRPM2 in Activated Macrophages in Context With Tubulin ...... 66 4.3 Future Studies Characterizing TRPM2 Interactors and the Functional Interaction Between Tubulin and TRPM2...... 68

Chapter 5...... 72

5 Summary ...... 72

References ...... 73

Appendices...... 81

List of Tables

Table 1. Modes of TRPM2 Channel Activation ...... 15

Table 2. Physiological and Pathological Roles of TRPM2...... 17-18

vii

List of Figures

Figure 1. General TRP Channel Structure for Groups 1 and 2...... 4

Figure 2. The Channel Structure of TRPM2...... 12

Figure 3. Plasmid Map of pCI-NEO-TRPM2 and Excision of the C-Terminus ...... 29

Figure 4. Plasmid Map of pGEX-5X-1-TRPM2-C-Terminus ...... 32

Figure 5. GST-TRPM2-C-Terminus Fusion Protein...... 36

Figure 6. LC/MS/MS Results From the Protein Pull-Down Assay Utilizing Immobilized GST- TRPM2-C-Terminus Fusion Construct...... 38

Figure 7. Tubulin Binds Directly to the C-Terminus of TRPM2 ...... 40

Figure 8. Predicted Regions Within the C-Terminus of TRPM2 Containing Coiled Domains ...... 44

Figure 9. TRPM2 C-Terminus Truncated Clones...... 46

Figure 10. Analysis of Long-Coil and Coil-Only Constructs Following Cloning and Transformation...... 48

Figure 11. Analysis of Long-NUDT9-H and NUDT9-H-Only Constructs Following Cloning and Transformation ...... 50

Figure 12. Expression of the TRPM2 C-Terminus Fusion Constructs by Western Blot Analysis ...... 52

Figure 13. Direct Tubulin-Binding Assay with the GST-TRPM2-Long-Coil and GST-TRPM2- Coil-Only Fusion Constructs ...... 56

Figure 14. Helical-Wheel Model Depicting the Formation of the Proposed Tubulin-Binding Domain of TRPM2 ...... 58

Figure 15. TRPM2 and Tubulin Immunostaining in Resting Macrophages ...... 62

Figure 16. TRPM2 and Tubulin Immunostaining in LPS-Activated Macrophages ...... 64

Figure 17. Model: TRPM2 and Tubulin In Macrophages ...... 71 viii

List of Appendices

Appendix 1. Plasmid Map of the pGEX-5X-1-TRPM2-N-Terminus vector and Restriction Digest Analysis...... 79-80

Appendix 2. Expression of the pGEX-5X-1-TRPM2-N-Terminus Fusion Protein...... 81-82

List of Abbreviations

AD Alzheimer’s Disease ADP Adenosine Diphosphate ADPR Adenosine Diphosphate Ribose ALS Western Pacific Amyotrophic Lateral Sclerosis AMP Adenosine Monophosphate BD Bipolar Disorder CaM Calmodulin CLIP-170 Cytoplasmic Linker Protein-170 cNAD Cyclic Nicotinamide Adenine Dinucleotide EDTA Ethylenediaminetetraacetic acid FBS Fetal Bovine Serum GST Glutathione S-Transferase HRP Horseradish Peroxidase IPTG Isopropyl β-D-1-thiogalactopyranoside Kv Voltage Gated Potassium Channel LC/MS/MS Liquid Chromatography Mass Spectrometry LPS Lipopolysaccharide NAADP Nicotinic Acid Adenine Dinucleotide Phosphate NAD Nicotinamide Adenine Dinucleotide NUDT9-H Human Nucleoside Diphosphate-Linked Moiety X-type Motif 9 OAADPR O-Acetyl-ADPR PARP Poly(ADPR) Polymerase PARG Poly(ADPR) Glycohydrolase PBS Phosphate Buffered Saline PCR Polymerase Chain Reaction PD Parkinson’s Disease SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis SIR2 Silent Information Regulator 2 TBI Traumatic Brain Injury TBS Tris Buffered Saline TRP Transient Receptor Potential YT 2X Yeast Extract-Tryptone

x 1

Chapter 1

1 Introduction

Ion channels play a central role in a variety of fundamental cellular processes such as muscle contraction, cell proliferation, gene transcription, signaling, and cell death (Pedersen, Owsianik and Nilius 2005). The transient receptor potential (TRP) channel superfamily consists of over 50 voltage-independent channels that are found in a wide array of eukaryotes, including worms, fruit flies, zebrafish, mice and humans. There are currently 28 identified mammalian TRP channels that exhibit diversity in cation selectivities and specific methods of activation. TRPM2 is a widely expressed cation channel containing a unique ADPR domain. TRPM2 has been suggested to play a key role in such processes as neuronal cell death following ischemic injury (Aarts and Tymianski 2005), chemokine production in immune cells (Yamamoto, Shimizu, et al. 2008), and insulin secretion in β-cells. TRPM2 is a recently identified cationic channel and there is still little known in regards to its protein-protein interactions. Consequently the goal of this thesis was to outline and characterize a novel protein interactor of TRPM2. By determining the intracellular interactions of TRPM2, it may be possible to understand its role in many areas of physiology. Furthermore, interactors may exist to gate or inhibit TRPM2 providing new targets for therapeutic intervention in various disease models. The following thesis describes tubulin as a novel interactor with the C-terminal coiled-coil domain of TRPM2 and suggests a functional role for the interaction between tubulin and TRPM2 in macrophages.

1.1 Transient Receptor Potential Channels

TRP channels function as physiological sensors in response to perturbations to the external environment, such as with temperature, chemicals, sound and light. The ion flux through these channels are initiated by an array of stimuli where constituent subunits allosterically interact with the channel thereby causing an activation and contribution to sensory physiology, namely vision, taste, hearing, touch, olfaction, thermo- and osmosensation (Venkatachalam and Montell 2007, Ramsey, Delling and Clapham 2006; Nelson, Beck and Cheng 2011). The characterization of these interactions, in both structure and location, is still largely under investigation leaving much to be resolved in terms of TRP channel activation.

TRP channels are composed of six transmembrane domains, with a pore-forming loop between the 5th and 6th transmembranous segments (Nelson, Beck and Cheng 2011). The C- and N-terminal domains lie intracellularly and vary in length and characteristics. It is common to find ankyrin repeats and coiled-coil domains on the N-terminus, which may take part in protein- protein interactions and ligand binding sites. The C-terminus share conserved regions among families such as the TRP box and coiled-coil domains, while also presenting individually unique and characteristic motifs such as enzymatic or kinase regions (Nelson, Beck and Cheng 2011). TRP channels are proposed to form a tetrameric quaternary structure where each subunit, similar to potassium channels, contributes to a shared selectivity filter and ion-conducting pore (Ramsey, Delling and Clapham 2006).

Mammalian TRP channels are divided into group 1 (Figure 1a) and group 2 TRPs (Figure 1b) and then further divided into seven subfamilies based upon their sequence homology and topological differences (Venkatachalam and Montell 2007). Group 1 TRP channels are comprised of five families displaying strong sequence homology to the original Drosophila TRP channel, which functions as a light sensing protein to regulate phospholipase C-dependent visual transduction in the photoreceptor cells of Drosophila (Montell and Rubin 1989; Venkatachalam and Montell 2007; Nilius, et al. 2007). The TRPC (classical) channels, share the closest sequence homology to Drosophila TRP. Seven different TRPC proteins (TRPC1-7) have been identified thus far. The other subfamilies of group 1 include TRPV (vanilloid; TRPV1 - 6 ), TRPM (melastatin; TRPM1 - 8), TRPA (ankyrin: TRPA1), and TRPN (no mechanopotential) (Venkatachalam and Montell 2007; Pedersen, Owsianik and Nilius 2005; Nilius 2008). Group 2 TRP channels include the subfamilies TRPP (polycystin) and TRPML (mucolipin), both of which contain three poorly described members thought to localize primarily to organelle membranes (Nilius, et al. 2007).

Figure 1. General TRP Channel Structure for Groups 1 and 2.

TRP channels have 6 transmembrane domains with a pore forming loop between segments 5 and 6 (indicated with an arrow), and two long intracellular tails. The general structure for (A) group 1 and (B) group 2 TRP channels are shown. These two groups differ based on sequence homology.

A B

N N

C C

1.2 The TRPM Subfamily

The mammalian TRPM channel subfamily consists of eight genetically and functionally diverse members that share roughly 20% amino acid identity with TRPC channels in the transmembrane region (Venkatachalam and Montell 2007, Ramsey, Delling and Clapham 2006). The TRPM subfamily is structurally similar to voltage-gated channels with six transmembrane domains and two long intracellular tails. The N-terminal region of TRPM channels has four stretches of amino acids that share sequence similarity. These four regions do not resemble any known structural motif and have not been defined functionally. The C-terminal region of the TRPM subfamily is variable in length and structure, however they contain a proposed coiled-coil domain thought to participate in the assembly of TRPM proteins into tetramers (Andrea Fleig 2004). TRPM members are organized into subsets based upon their amino acid sequence similarities and resultantly link TRPM1/3, TRPM4/5 and TRPM6/7 together; TRPM2 and TRPM8 are not grouped into any of the latter due to low sequence homology, but share between themselves 42% homology (Venkatachalam and Montell 2007, Ramsey, Delling and Clapham 2006, Eisfeld and Luckhoff 2007).

1.2.1 TRPM1

TRPM1 was the first mammalian TRPM channel identified and was defined according to the discovery of the channel in melanoma cells but has since been negatively correlated with melanoma development (Vassort & Alvarez 2009). The expression level of TRPM1 correlates inversely with the metastatic potential in some melanomic cell lines allowing it to be used as a prognostic marker for metastasis (Venkatachalam and Montell 2007, Ramsey, Delling and Clapham 2006). Recently TRPM1 was discovered to have an essential role in mediating visual transduction in retinal ON bipolar cells in both mouse and humans. In response to light in retinal ON bipolar cells, a decrease in glutamate results in the inactivation of metabotropic glutamate receptor 6 (mGluR6) leading to the open state of TRPM1. In contrast, the absence of light increases glutamate resulting in mGluR6 activation, which results in the downstream inactivation of TRPM1 (Koike, et al. 2010). There exist two known protein variants, TRPM1-S and TRPM1- L, a short variant without transmembrane domains, and a full-length clone respectively.

TRPM1-S interacts with the full-length variant to inhibit translocation to the plasma membrane (Ramsey, Delling and Clapham 2006, Venkatachalam and Montell 2007).

1.2.2 TRPM3

TRPM3 is a constitutively active Ca2+, Mn2+ and Zn2+ permeable channel and its gene encodes a number of variants that differ within the pore-forming region (Oberwinkler J 2007; Venkatachalam and Montell 2007; Ramsey, Delling and Clapham 2006; Islam 2011). Calcium entry through TRPM3 is enhanced by hypotonicity and cell swelling and may also be affected by depletion of intracellular Ca2+ stores and D-erythro-sphingosine (Ramsey, Delling and Clapham 2006; Venkatachalam and Montell 2007). TRPM3 is directly activated by supraphysiological concentrations of the steroid pregnenolone sulphate (PS) and although not physiologically relevant, PS has been used experimentally to show that TRPM3 activation in mouse islet cells results in increases in intracellular Ca2+ and the augmentation of glucose-stimulated insulin secretion (Islam 2011).

1.2.3 TRPM4 & TRPM5

TRPM4 and TRPM5 are unique among the TRP super family in that they contain a short acidic stretch of amino acids in their pore loops that renders them voltage-modulated and Ca2+- activated monovalent cation channels (VCAMs) (Venkatachalam and Montell 2007). TRPM4 and TRPM5 are activated by changes in membrane potential and are stimulated further by heat and by PIP2 (Nilius, et al. 2007, Venkatachalam and Montell 2007, Ramsey, Delling and Clapham 2006). TRPM4 was the first identified Ca2+-activated channel of the TRPM subfamily and conducts mainly Na+ and K+ with only minimal permeation to Ca2+. Although TRPM4 lacks permeability to Ca2+, it significantly impacts Ca2+ signals by providing a mechanism by which cells are able to depolarize in a Ca2+-dependent manner (Marigo, V. 2009). TRPM4 also exists as two splice variants, a functional VCAM, TRPM4b, and the non-functional TRPM4a that displays little activity (Venkatachalam and Montell 2007). TRPM4 has been implicated to regulate a number of things including: myogenic constriction of cerebral arteries, modulation of insulin secretion in response to glucose uptake by pancreatic β-cells, and modulation of the firing in brainstem neurons responsible for breathing rhythm (Guinamard, Sallé and Simard 2011).

1.2.4 TRPM6 & TRPM7

Unlike most other non-selective cation channels TRPM6 and TRPM7 show a higher permeability to Mg2+ than Ca2+ or Na+ and as such are proposed to play key roles in cellular and physiological Mg2+ homeostasis. Both TRPM6 and TRPM7 possess C-terminal atypical protein α-kinase domains, which categorize them as “chanzymes” (Venkatachalam and Montell 2007). It is under debate whether the binding of ATP to the kinase domain plays a physiological role in channel function as kinase inactivating mutations reportedly have no effect on channel activation however can change the sensitivity of the channel to Mg2+ inhibition (Venkatachalam and Montell 2007). TRPM6 is regulated by intracellular Mg2+, pH and ATP and has been implicated as an indirect player involved in Mg2+ homeostasis (van der Wijst, G J Hoenderop and Bindels 2009). TRPM7 is inhibited by Mg2+ and potentiated by low intracellular pH, ATP, lipids and potentially reactive oxygen species (ROS). A large amount of research has implicated TRPM7 in fundamental cell processes including cell death, survival and proliferation, as well as cell cycle progression and oxidative stress (Venkatachalam and Montell 2007, Ramsey, Delling and Clapham 2006; Bates-Withers, Sah and Clapham 2011).

1.2.5 TRPM8

TRPM8 is a voltage dependent and non-selective cation channel that is thermally regulated and stimulated by compounds that evoke the sensation of coolness (e.g. menthol, eucalyptol), and

PIP2 (Venkatachalam and Montell 2007). TRPM8 has a well-established role as a major sensor for ambient cool and cold temperatures, and also appears to play part an important role in chronic pain resulting in cold hypersensitivity or analgesia under conditions of nerve injury and inflammation (Yi Liu 2011).

1.3 TRPM2

TRPM2 is a non-selective chanzyme permeable to Ca2+, that is widely expressed throughout the body with its highest levels localized to pancreatic β-cells, immune cells (e.g., macrophages, monocytes, and lymphocytes), intestines, and in neurons and microglia found in various brain regions, namely the cerebellum, cortex, medulla and hippocampus (Eisfeld and Luckhoff 2007). Much like other TRP channels, TRPM2 has six transmembrane domains with a pore between the 5th and 6th segments and two long, unique, intracellular tails (Figure 2). Both the N- and C-Terminal tails have predicted coiled-coil domains while the C-terminal tail boasts

8 an adenosine diphosphate ribose (ADPR) hydrolase (Nudix) domain (Pedersen, Owasianik and Nilius 2005). TRPM2 channels can be activated by the binding of intracellular ADPR to the Nudix enzymatic domain found at the end of C-terminal tail (A L Perraud 2001). Other oxidative stress activates TRPM2 indirectly via the activation of glycohydrolases and ectoenzymes resulting in the downstream production of ADPR. Intracellular Ca2+ and calmodulin also modulate TRPM2 by interacting with calmodulin IQ-like motifs on the N- terminus. TRPM2 has been implicated in various pathological and physiological conditions including oxidative stress and cell death, chemotaxis, and bipolar disorders.

The N-Terminus

The long N-terminal cytoplasmic tail of TRPM2 includes four TRPM subfamily homology domains (MHD I-IV) and a number of characteristic domains (Figure 2a), many of which still require a great deal of understanding and characterization (Jiang, Yang, et al. 2010). The N-terminal domain is considered indispensable as the deletion of a stretch of 20 amino acid residues in TRPM2-ΔN (Δ538–557) abolishes TRPM2 channel function in neutrophils (Wehage, et al. 2002). This stretch of amino acids includes two IQ-like motifs similar to calmodulin (CaM) binding domains, a coiled-coil domain and two PxxP motifs. The CaM binding domains are proposed to play part in TRPM2 activation following an increase in intracellular Ca2+ and the subsequent Ca2+-saturation of CaM (Takahashi, et al. 2011). TRPM2 is rich in PxxP motifs, which are characteristic for interactions with other proteins. PxxP motifs have the ability to participate in binding with Src homology 3 (SH3) domain-containing proteins, which are globular protein interaction modules that regulate cell behaviour (lli Aitioa 2010; Li 2005). SH3- containing proteins have been found in proteins associated with actin cytoskeleton organization (G Mirey 2005), and adaptor proteins such as Crk, Grb2 and Nck (R B Birge 1996). In regards to TRP channels, this motif has only been studied in the TRPC family, but to date there has been no study of this motif in TRPM2 and it’s interactions with other proteins (Kühn, et al. 2009). Domain deletion studies have demonstrated that the N-terminal coiled-coil domain is required for channel expression and function, but not for subunit interaction. Upon deletion of the N- terminal coiled-coil, there was an observed reduction in TRPM2 expression and ADPR-evoked currents. However, subunit interaction was only affected when both the N- and C-terminal coiled-coils were deleted suggesting the N-terminal coiled-coil is not critical for subunit interaction (Jiang and Mei 2009).

The C-Terminus

The categorization of TRPM2 as a “chanzyme”, meaning both a channel and an enzyme, comes from the unique C-terminus, which provides TRPM2 with its enzymatic abilities (Figure 2a). TRPM2 boasts a coiled-coil domain and a 22 amino acid Nudix box motif, which is defined as a catalytic site found within a larger domain of roughly 300 amino acids (Figure 2b). This motif shares a high degree of homology with the human nucleoside diphosphate-linked moiety X-type motif 9 (NUDT9-H), a mitochondrial ADPR hydrolase that degrades ADPR to adenosine monophosphate and ribose-5-phostphate. ADPR binding can subsequently lead to the activation of TRPM2 (Eisfeld and Lückhoff 2007; Ramsey, Delling and Clapham 2006; Frank J P Kühn 2004). Crystal structure and biochemical analysis of the NUDT9-H domain has divided it into a 105 amino acid N-terminal portion which binds ADPR, and a 179 amino acid C-terminal portion which acts as the catalytic active site, or Nudix domain. In neutrophils, an ADPR insensitive splice variant of TRPM2 still develops cation currents in the presence of H2O2. However this splice-variant is missing 34 amino acids, known as the ΔC-stretch, from the N-terminal ADPR binding site of the NUDT9-H domain. It has been demonstrated by Heiner et al. (2005) that a single asparagine residue immediately down stream of the ΔC-stretch is essential for ADPR gating of TRPM2. (Heiner, et al. 2005).

Downstream of the pore region is a TRP box domain, a short hydrophobic and highly conserved region of amino acids found in TRPCs, TRPNs and TRPMs. The TRP box domain is located just C-terminal to the putative sixth transmembrane helix. It is predicted that the helix would extend past the membrane bi-layer cytosolically and include the TRP box, suggesting a role for the TRP box in serving as a coiled-coil zipper to hold the channel in a closed conformation. The functional role of the TRP box has yet to be confirmed, however TRP box regions in TRPV5 and TRPM5/8 have been implicated in the sensing of PIP2 levels (Gaudet 2007).

The predicted C-terminal coiled-coil motif present in TRPM2 lies C-terminally to the TRP box domain. Coiled-coil domains are one of the most common amino acid motifs. Coiled- coil domains have been described to play an important role in the assembly of homomeric and heteromeric protein complexes as well as mediating protein-protein interactions between various types of proteins (e.g. filamentous, motor, membrane and cytoskeletal proteins) (Lupas and

Gruber 2005; Apostolovic, Danial and Klok 2010; Li, Yu and Yang 2011). Coiled-coils are bundles of α-helices that are wound into a super-helical structure. These structures are most commonly composed of two, three or four helices that run in parallel or antiparallel directions. The defining characteristic of coiled-coils is the distinctive packing of the amino acid side chains in the core of the bundle known as knob-into-holes where the residue from one helix (knob) packs into the space surrounded by four side chains of the facing helix (hole). Residues that engage in this knob-into-holes interaction are generally hydrophobic, leaving the hydrophilic residues to the outside (Lupas and Gruber 2005).

Coiled-coil domains are predicted to exist in TRPC, TRPM, TRPP, and TRPV channels, with roles rooted in channel assembly and function. In the cold-sensing TRPM8 channel, it has been demonstrated by Tsuruda et al. (2006) that the C-terminal coiled-coil is necessary for channel assembly, tetramer formation and trafficking. The coiled-coil of TRPM2 has also been demonstrated to be critically involved in TRPM2 subunit interaction, which is required for the assembly of functional TRPM2 channels. Deletion or mutation of the hydrophobic residues in the coiled-coil resulted in a severe disruption of subunit interactions and significant loss of channel currents (Zhu-Zhong, et al. 2006). Aside from this, a great deal still exists in further characterizing the role of the C-terminal coiled- coil in TRPM2.

Figure 2. The Channel Structure of TRPM2.

(A) TRPM2 is a unique structure by reason that it acts as both an ion channel and an enzyme, thus denoting it a “chanzyme”. TRPM2 has 6 transmembrane segments with a pore-forming loop between the 5th and 6th transmembrane segment, and two long intracellular tails. The C- terminal tail boasts a predicted coiled-coil domain and an ADPR hydrolase (NUDT9-H) enzymatic domain. A TRP box also exists C-terminally to the 6th transmembrane domain, which is a short region of highly, conserved amino acids found in TRPC’s, TRPN’s and TRPM’s. The N-terminal tail has predicted IQ-like motifs similar to calmodulin binding domains. (B) The NUDT9-H domain is divided into an ADPR binding site and the catalytic domain. The Nudix box motif is the small region within the catalytic domain (highlighted with the bracket) between amino acids 1381-1405. Figure not to scale.

N C

1.4 TRPM2 Modes of Activation

TRPM2, a proposed endogenous redox sensor, has numerous modes of activation that orbit around the suggested idea that TRPM2 activation is triggered via oxidative stress. The first discovered and most efficient activator of TRPM2 is ADPR, which is believed to arise from the mitochondria and nucleus (McNulty and Fonfria 2005; Yamamoto, Takahashi and Mori 2010; Hecquet and Malik 2009). ADPR binds to a critical region N-terminal to the Nudix box, which acts as the catalytic site for NUDT9-H domain (Figure 2b). The binding of ADPR to this region not only confers a unique mode of activation for TRPM2, but also results in the degradation of ADPR to adenosine monophosphate (AMP) and ribose-5-phosphate (Eisfeld and Lückhoff 2007; Jiang, Yang, et al. 2010). DNA damage from oxidative stress is an indirect source of ADPR production through a pathway involving the poly(ADPR) polymerase (PARP), PARP-1. In response to DNA damage PARP-1 binds to damaged single- and double-stranded DNA breaks and catalyzes the cleavage of nicotinamide adenine dinucleotide (NAD+) into nicotinamide and ADPR. From here, ADPR is polymerized onto various nuclear proteins subsequently activating mechanisms for DNA repair and stimulating nuclear factor-mediated transcription. In addition to this mechanism, free ADPR is also generated through the hydrolysis of both NAD+ and cyclic NAD (cNAD) through the activity of glycohydrolases, mitochondrial NADase, and the ectoenzymes CD38 and CD157. Aside from PARP activity following DNA damage, the hyperactivity of poly(ADPR) glycohydrolases (PARG), and ADP-ribosyl protein lyase can result in the production of free ADPR from the formation and hydrolysis of poly-ADPR (Sumoza-Toledo and Penner 2010; Yamamoto, Takahashi and Mori 2010). Nucleotides such as cADPR, nicotinic acid adenine dinucleotide phosphate (NAADP) and NAD+ can activate TRPM2 directly, but may do so indirectly through their breakdown into ADPR. Although it has been demonstrated that high concentrations of NAD+ activates TRPM2, it is difficult to confirm that this was not the indirect result of its breakdown to ADPR. The concentration necessary for cADPR and NAADP to act as TRPM2 agonists is higher than what exists physiologically, however at lower concentrations they can act synergistically with ADPR to increase the sensitivity of TRPM2 (Sumoza-Toledo and Penner 2010). The silent information regulator 2 (SIR2) family of NAD+-dependent enzymes catalyzes the reaction in which the acetyl group from the substrate is transferred to the ADPR portion of NAD+ resulting in the metabolite O-

14 acetyl-ADPR (OAADPR). OAADPR has been shown to bind directly to the Nudix domain, suggesting it also has a role as a physiological regulator of TRPM2 (Grubisha, et al. 2006).

Hydrogen peroxide (H2O2) is the only identified extracellular stimulus of TRPM2. H2O2 has acted as an experimental paradigm to demonstrate that oxidative stress is capable of activating TRPM2 and inducing TRPM2 currents and increases in intracellular Ca2+ concentrations (Eisfeld and Luckhoff 2007). The mechanism by which hydrogen peroxide activates TRPM2 is still under investigation however, it has been postulated that oxidative stress potentiates the formation of ADPR polymers through the degradation of poly-ADPR by PARPs and PARGs (Eisfeld and Luckhoff 2007). It has been demonstrated that overexpression of 2+ ADPR pyrophosphatase is able to suppress H2O2-induced Ca responses, which suggests a role for ADPR binding in H2O2-induced TRPM2 activation (Yamamoto, Takahashi and Mori 2010).

Intracellular Ca2+ on its own does not activate TRPM2 however, it acts as a mandatory and dose-dependent modulator and cofactor of TRPM2 gating by shifting the concentration- response curve to ADPR leftwards (Hecquet and Malik 2009). Calmodulin (CaM), a small conserved protein, is known to bind to a stretch of the N-terminus of TRPM2. CaM has four Ca2+ binding domains known as EF hands that bind a single calcium with high affinity, and exhibits both a low affinity N- and high affinity C-terminal Ca2+-binding lobe (Martin Bähler 2002). A stretch of the TRPM2 N-terminus contains an IQ-like motif, which has a sequence similar to the IQ-like motif IQxxRGxxR, that represents a CaM binding domain (Tong, et al. 2006). Through the use of mutant TRPM2 channels, the direct binding of CaM to the N- terminus CaM IQ-like motifs was identified as the Ca2+ sensor responsible for the Ca2+- activation of TRPM2. It has been demonstrated that oxidant-induced Ca2+ entry through TRPM2 enhances the CaM interaction with the channel resulting in positive-feedback for channel activation (Hecquet and Malik 2009). CaM has also been shown to be the Ca2+ sensor for the activation or inactivation of other channels, such as the TRPC subfamily. TRPC also display a number of C-terminal binding sites for CaM that exists to allow Ca2+/CaM to either inhibit or facilitate the channel. The gating of TRPV6 is also positively regulated by CaM binding to the transmembrane domain of TRPV6 in a Ca2+-dependent manner.

A summary of the modes of TRPM2 channel activation are listed below:

Table 1. Modes of TRPM2 Channel Activation

As progress continues to elucidate further mechanisms by which TRPM2 is gated, there exists a general understanding of what triggers channel activation. Most modes of TRPM2 activation are rooted in the products of oxidative stress, and although some have been directly shown to activate the channel, others are still under investigation whether it be direct or indirect.

Activator Mechanism Reference ADPR ADPR binds to a critical region N-terminally of the Nudix box, which (A L Perraud acts as the catalytic site for NUDT9-H domain. This direct binding 2001) gates the channel and also results in the degradation of ADPR to AMP and ribose-5-phosphate Oxidative Stress Oxidative stress results in damaged DNA, which is repaired by PARP- (Fonfria, et al. + 1. PARP-1 catalyzes cleavage of NAD to ADPR 2004) NAD+ and cNAD NAD+ and cyclic NAD (cNAD) are hydrolyzed by glycohydrolases, (Fabio Malavasi mitochondrial NADase, and the ectoenzymes CD38 and CD157, 2006) resulting in the production of ADPR poly(ADPR) Hyperactivity of poly(ADPR) glycohydrolases (PARG), and ADP- (Caiafa, glycohydrolases ribosyl protein lyase can result in the production of free ADPR from Guastafierro and (PARG), and ADP- the formation and hydrolysis of poly-ADPR Zampie 2009) ribosyl protein (Esposito and lyase Cuzzocrea 2009) cADPR, NAADP Although they do not directly activate TRPM2, they are broken down (Beck, et al. into ADPR which will then gate the channel. At low concentrations 2006) they can act synergistically with ADPR to increase the sensitivity of TRPM2 OAADPR The silent information regulator 2 (SIR2) family of NAD+-dependent (Grubisha, et al. enzymes catalyzes the reaction whereby NAD+ is acetylated resulting 2006) in the metabolite OAADPR. OAADPR has been shown to bind directly to the Nudix domain, suggesting its role as a physiological regulator of TRPM2 H2O2 The mechanism by which this paradigmatic extracellular activator (Eisfeld and gates TRPM2 is still under question, however it is believed that Luckhoff 2007) oxidative stress potentiates the formation of ADPR polymers through the degradation of poly-ADPR by PARPs and PARGs Intracellular Ca2+ Intracellular Ca2+ acts as a mandatory and dose-dependent (Hecquet and modulator and cofactor of TRPM2 gating by shifting the Malik 2009) concentration-response curve to ADPR leftwards CaM CaM acts as a Ca2+ sensor and binds directly to the N-terminus CaM (Hecquet and IQ-like motifs. It has been identified as the sensor responsible for Malik 2009; the Ca2+-activation of TRPM2. Ca2+ entry through TRPM2 enhances Tong, et al. the CaM interaction with the channel resulting in positive-feedback 2006) for channel activation

1.5 The Pharmacology of TRPM2

A common theme amongst the TRPM subfamily is the lack of proper pharmalogical inhibitors. The pharmacology of TRPM2 is satisfactory and although inhibition can be achieved, it comes at the cost of specificity. While TRPM2 itself may be blocked, it is not done so alone, suggesting the need for further investigation into unique and specific blockers of TRPM2. Currently, fenamates such as flufenamic acid (FFA) act to inhibit TRPM2-mediated currents in HEK cells (Hill, et al. 2004). Fenamates are non-steroidal anti-inflammatory agents that produce anti-inflammatory effects in the nervous system likely through the inhibition of a wide spectrum of cation influx pathways (Eisfeld and Lückhoff 2007). Another directly acting non-specific TRPM2 blocker is the anti-fungal agents clotrimazole and econazole. These anti-fungal agents provide an irreversible TRPM2 block (Hill, McNulty and Randall 2004).

Drugs that indirectly interfere with TRPM2s activation include anti-oxidants such as catalase and mannitol, which are used as intracellular radical scavengers (Wehage, et al. 2002). PARP inhibitors are thought to prevent the binding of NAD to PARP reducing the amount of ADPR produced which can subsequently activate TRPM2 (Miller 2004).

1.6 The Physiological and Pathological Roles of TRPM2

The ongoing studies in regards to the biological importance of TRPM2 in various tissues and cells continually bring to light a greater understanding of its physiological roles. Although progress is made, it is hindered a great deal due to the lack of pharmalogical tools required to properly study the channel. What is obvious however, is the understanding that TRPM2 acts in response to oxidative stress which, not only results in an influx of Ca2+ but a sizeable permeability for Na+ which can evoke depolarization that may pose as an important regulatory factor in various cells and conditions (Eisfeld and Lückhoff 2007).

See Table 2 below for a summary of the physiological and pathological roles of TRPM2:

Table 2. Physiological and Pathological Roles of TRPM2. TRPM2 has been implicated in a number of neurodegenerative diseases brought upon by oxidant-induced cell death, as well as roles in other tissues in relation to both homeostasis and disease. The disruption of Ca2+ homeostasis and Ca2+ overload following TRPM2 activation is considered crucial. Although some of the pathological roles for TRPM2 have been elucidated, much has been proposed leaving many resulting downstream signaling cascades to be established.

Disease Affectors Reference CNS ischemia; oxidative • Ischemia highly increases the activation of (Fonfria, et al. 2004) stress and cell death PARP-1, which can activate TRPM2 during (Moran, et al. 2011) stroke through ADPR (Olah, et al. 2009) • Activation of TRPM2 induces the death of striatal neurons via oxidative stress • The increase in microglia activation following focal ischemia middle cerebral artery occlusion (MCAO) is paralleled with an upregulation of TRPM2 mRNA suggesting a role for the channel in CNS responses to oxidative stress and ischemia • Oxidative stress induces an increase in TRPM2 channels in astrocytes • TRPM2 -/- mice protect against focal ischemia of stroke Alzheimer’s Disease (AD) • TRPM2 is expressed in striatal and (Hill, et al. 2006) and Parkinson Disease (PD) hippocampal neurons and is responsible for neuronal death in response to amyloid-β42, H2O2 and TNF-α – this suggests a role for pathogenesis in AD and PD. Western Pacific • A variant of TRPM2 producing a missense (Hermosura, et al. Amyotrophic Lateral change in the channel protein where 2008) Sclerosis (ALS) proline1018 is replace by leucine1018 resulting in an inactive channel. This suggests the inability of the channel to sustain ion influx and may be the disruption contributing to ALS. Bipolar Disorders (BD) • Genetic analysis within BD suggests a location (Chun Xu 2009) of TRPM2 gene with a BD susceptibility locus • TRPM2 mRNA expression in B lymphoblast cells from BD type-I showing higher basal intracellular Ca2+ is significantly lower when compared to healthy and BD-1 patients with normal intracellular Ca2+ • The overtransmission in the G allele of rs1556314 at exon 11 of TRPM2 in BD-1 suggests that genetic variance in may confer pathogenesis of BD Traumatic Brain Injury • Following TBI there were significant increases (Naomi L Cook 2010) (TBI) in TRPM2 mRNA and protein expression in the cerebral cortex and hippocampus of injured animals. This suggests that TRPM2 may contribute to TBI injury processes such as oxidative stress, inflammation and neuronal death

Table 2. Physiological and Pathological Roles of TRPM2 (continued).

Diabetes • In pancreatic β-cells TRPM2 is suggested to (Nelson, Beck and play part in temperature-induced insulin Cheng 2011) secretion via cADPR • TRPM2 reported to be localized to the lysosomal compartments where it functions as a Ca2+-release channel to regulate insulin secretion Chemotaxis • Inflammation results in ROS production which (Yamamoto, Shimizu, can induce Ca2+ influx through TRPM2 and et al. 2008)(Yamamoto, activate redox-sensitive transcription factors Takahashi and Mori, such as NF-κB, resulting in chemokine Chemical physiology of production oxidative stress- activated TRPM2 and TRPC5 channels 2010)

1.7 Hypothesis and Objectives

Few studies address the molecular interactions of TRPM2. TRPM2 is widespread in both its expression and involvement in pathological processes and as such the characterization of its protein-protein interaction will help to elucidate its role in many areas of the body and potentially reveal new targets for therapeutic intervention in a number of related diseases. With this understanding, the objective of this study was rooted in characterizing a novel intracellular activator of TRPM2. Considering the architecture of TRP channels is similar to voltage-gated potassium channels (Kv) (Voets, et al. 2005), it is logical to believe that the intracellular tails of TRPM2 can, like Kv intracellular tails, interact with proteins. The Kv intracellular tails are usually involved in various aspects of channel regulation such as the interaction of the PDZ- domain (found on the end of the Kv tail domain) with scaffold proteins like the PSD-95 protein (Magidovich, Fleishman and Yifrach 2006). The N- and C-terminal domain of TRPM2 collectively contain predicted coiled-coils, PxxP motifs, and a TRP box. These motifs and domains are regions known and predicted to participate in protein-protein interactions and thus stood as good candidates for the further characterization of protein-protein interactions.

The use of TRPM2 C-terminus fusion constructs for interaction studies by method of protein pull-down assays and mass spectrometry revealed tubulin as a novel interactor of TRPM2. In analyzing the interaction we were able to hypothesize that tubulin binds directly to the predicted C-terminal coiled coil domain of TRPM2 at a predicted tubulin-binding motif.

After identifying the direct interaction of tubulin with TRPM2, we learned from the literature that microtubules are stabilized by cytoplasmic linker protein-170 (CLIP-170) in LPS- activate macrophages. An in vitro analysis of the interaction between tubulin and TRPM2 was developed to identify any identifiable change in the tubulin-TRPM2 interaction in immunostained RAW 264.7 macrophages. By treating both resting and LPS-activated macrophages with taxol and nocodazole, we were able to analyze whether the tubulin-TRPM2 interaction was enhanced, diminished, or if there was a change in TRPM2 localization from the membrane to the cytosol (or vice-versa). Combined with what was found in the literature, we were able to further our study by suggesting that the stabilization of microtubules by CLIP-170 in LPS-activated macrophages results in the increased interaction of tubulin with TRPM2. This increased interaction may result in the gating or localization of TRPM2 subsequently resulting in calcium-dependent downstream effects.

The primary objective was to define full-length and truncated TRPM2 N- and C-terminal fusion constructs for use in interaction studies involving protein pull-down assays and mass spectrometry. Following the identification of a novel interactor, the second objective was to investigate the interaction biochemically using direct tubulin-binding assays to identify binding location. This was supplemented with genetic analysis to identify a potential tubulin-binding motif. Lastly, after understanding the tubulin-TRPM2 interaction, an investigation into its functional involvement in macrophages was considered. Macrophages in either the resting or LPS-activated states were treated with taxol or nocodazole to visualize the interaction between tubulin and TRPM2 using immunostaining and confocal microscopy. Taken together, this study was able to suggest a novel interaction of tubulin with the C-terminus of TRPM2.

Chapter 2

2 Materials and Methods 2.1 Cloning

Conventional cloning techniques were utilized to generate full-length and truncated TRPM2 C-terminus clones. Regions of interest were obtained using either PCR or enzymatic digests and subsequently ligated into the pGEX-5X-1 vector, which allowed for inducible production of a GST-tagged fusion protein. All constructs were confirmed by enzymatic digest and sequencing (The Center of Applied Genomics; The Hospital for Sick Kids).

2.1.1 The Generation of GST-Fusion Constructs

The generation of TRPM2 C-terminus fusion constructs were completed using the mouse cDNA pCI-NEO-TRPM2 template construct. Areas of interest were isolated using restriction digest and polymerase chain reaction (PCR). Insert regions from restriction digests were placed directly into the expression vector pGEX-5X-1 and PCR inserts were sub-cloned into a pCR®- TOPO® vector and subsequently placed into pGEX-5X-1. This process involved plasmid purification, restriction digest, vector dephosphorylation, agarose gel purification, ligation and transformation.

2.1.1.1 Plasmid Purification

Cultures were prepared in 2X yeast extract-tryptone (YT) broth (1.6% peptone, 1% Yeast Extract, 0.5% NaCl) and inoculated from frozen stocks. Cultures were grown overnight for 16 hours in a 37°C shaking incubator at 180rpm. Small-scale DNA isolations were carried out through use of the Qiagen QIAprep Miniprep kit (Cat. No. 27104) and suggested instruction.

2.1.1.2 Polymerase Chain Reaction (PCR)

The amplification of regions of interest from template DNA was completed using PCR. Four truncated clones were generated using PCR and the iProof™ High-Fidelity PCR Kit (Bio- Rad; Cat. No. 172-5331). For reactions, 5ng of template, 0.5µM of both the forward and reverse primer, 0.02U/µL of enzyme and a 1X final concentration of iProof ™ HF buffer were used in a

21 total volume of 50µL. The forward primer for the coil-only and long-coil clones were 5’- CGGGATCCCGCCTGAAGATCCCTGC-3’ and 5’- CGGGATCCGCGGGGTGCAGGAACACAC-3’ respectively, with both sharing the reverse primer 5’-CGGAATTCCGCTCAGCATCTGGCTCATCGAAGGC-3’. The forward primers for the NUDT9-H-only and the long-NUDT9-H clones were 5’- CGGAATTCCGGGTTACCACGTGAG-3’ and 5’-CGGAATTCCGAGGGCCTTCGATGAG- 3’ respectively, with both sharing the reverse primer 5’- CGGTCGACCGGTGAGCTCCAAACAGTGA-3’. All reactions were carried out using the following conditions: Step Stage Temp (°C) Time Cycles 1 Denaturation 98 35 seconds 1 2 Denaturation 98 15 seconds 30 3 Annealing 50-72 (gradient) 25 seconds 30 4 Extension 72 25 seconds 30 5 Final extension 72 12 minutes 1

The PCR product was subcloned using the Zero Blunt® TOPO® PCR Cloning Kit (Invitrogen; Cat. No. K2800-20) after thermal cycler incubation using 2µL of fresh PCR product mixed with 10ng of pCR®-blunt II-TOPO® and 1µL of salt solution in a 5µL final volume. The mix was incubated at room temperature for 5 minutes and immediately transformed into E. coli (see section 2.1.1.5).

2.1.1.3 Agarose Gel Electrophoresis

1% agarose gels containing 1 µg/ml ethidium bromide were prepared in 1X TAE (0.04M Tris – Acetate, 0.001M ethylenediaminetetraacetic acid (EDTA)) for the use of separating DNA based upon size. DNA samples were prepared for loading by addition of a 6X DNA loading dye (Fermentas; R0611) at a 1X final concentration. DNA samples were loaded alongside a DNA ladder (Fermentas 1kb Plus DNA Ladder; SM1331) and run at 80V until desired separation was achieved. DNA separation was visualized using a UV light box.

2.1.1.4 DNA Extraction From Agarose Gel

Bands of interest (inserts previously excised by restriction digest) were visualized by UV and were excised from the agarose gel using a razor. DNA was purified from the gel using the QIAquick Gel Extraction Kit (Qiagen; 28704). Following the manufacturer’s instruction, the

22 excised agarose segment was dissolved using the appropriate buffers, and added to a DNA- binding column. After washing, DNA was eluted using the elution buffer provided.

2.1.1.5 Restriction Digest

Plasmids of interest were digested with desired restriction enzyme in appropriate buffer (at a 1X concentration) following manufactures suggestion (usually 10U of enzyme per µg of DNA), with BSA supplementation where necessary. The restriction enzymes used are outlined below according to application and reaction temperature.

Plasmid Contents/Target Region Enzyme 1 Enzyme 2 Temp- Fragment erature Length (bp) pCI-NEO-TRPM2 C-terminus BclI SalI 37°C 1503 pCI-NEO-TRPM2 N-terminus EcoRI StuI 2168 pCR®-Blunt II- TRPM2 C-terminus Coiled- EcoRI SalI 37°C 378 TOPO®-Coil Only Coil only pCR®-Blunt II- TRPM2 C-terminus Long EcoRI SalI 37°C 510 TOPO®-Long Coil Coiled-Coil pCR®-Blunt II- TRPM2 C-terminus EcoRI SalI 37°C 815 TOPO®-NUDT9-H NUDT9-H enzymatic Only domain only pCR®-Blunt II- TRPM2 C-terminus Long EcoRI SalI 37°C 878 TOPO®-Long NUDT9-H enzymatic NUDT9-H domain pGEX-5X-1 Cloning vector – C-terminus EcoRI SalI 37°C See above + Truncated clones pGEX-5X-1 Cloning vector – N- EcoRI SmaI 37/30°C See above terminus

2.1.1.6 Vector Dephosphorylation and Ligation

After the vector was digested with restriction enzymes, cut ends were dephosphorylated using Antarctic phosphatase (New England Biolabs; M0289S) following manufacturer’s instruction. After vector dephosphorylation, both vector and insert were gel purified. 30 fmol of vector and 90 fmol of insert were combined and ligated together using ExpressLink™ T4 DNA ligase (Invitrogen; A13726) following manufacturer’s instruction.

2.1.1.7 Transformation and Selection

About 100ng of ligated plasmid was used to transform DH5-α high-efficiency (New England Biolabs; C2987H), BL21 DE3 (New England Biolabs; C2527H) or One Shot® TOP10 chemically competent (Invitrogen; K2800-20) E. coli. Plasmid DNA was subsequently incubated with cells on ice for 30 minutes. When BL21 DE3 E. coli was employed, cells were

incubated on ice with 50mM CaCl2 to make cells competent prior to the incubation with DNA. Cells were heat shocked from ice to 42°C for 30-45 seconds and immediately placed back on ice for another 2 minutes. Cells then received pre-warmed media and were placed to incubate in a shaking 37°C incubator at 225rpm for 1 hour. Following the incubation, cells were plated in varying volumes on agar-ampicillin plates. Plates were incubated overnight in a 37°C incubator. The following day single colonies were selected and grown in selective media overnight. DNA isolations were carried out on overnight cultures and subjected to restriction enzyme digest to excise inserts. Digested DNA was run on a 1% agarose gel to analyze the cloning success. Positive clones were frozen in 25% glycerol and placed at -80°C for storage.

2.1.1.8 DNA Sequencing

DNA samples were submitted to The Centre for Applied Genomics (The Hospital for Sick Children) for sequencing. DNA samples were analyzed using Next-Generation Sequencing technologies, which offer high-throughput sequencing using the Solexa/Illumina Genome Analyzer II, Applied Biosystems SOLiD 3.0 and Roche 454 FLX Titanium and FLX Standard technologies. Sequencing sample reports were returned and analyzed through DNA alignments using CLC Genomics Workbench 4.

2.2 Protein Pull-Down and Tubulin Interaction Assay

2.2.1 Protein purification

Various cultures of BL21 DE3 bacteria containing GST-TRPM2 truncated fusion constructs were grown overnight in 2X YT medium supplemented with 50µg/mL ampicillin in a 37°C shaking incubator at 200 rpm. Overnight cultures were used to inoculate fresh 2X YT media supplemented with 50µg/mL ampicillin. Fresh culture was grown in a 37°C shaking incubator at 200 rpm until mid-log phase (O.D.600 0.6-0.8). Bacteria were induced with 3mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and allowed to incubate at 25°C in a shaking incubator at 200 rpm for 2-3 hours. Bacteria cultures were centrifuged at 5000rpm at 4°C for 10 minutes. The supernatant was removed and the pellet was resuspended and washed in ice-cold 1X TBS. Culture was spun again at 5000rpm at 4°C for 10 minutes. The supernatant was removed and the pellet was resuspended in a volume of ice-cold GST Lysis buffer (50mM Tris, pH 7.5, 300mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM DTT, 1% Triton X-100 and protease inhibitors). Culture was lysed using ultra-sonication at 5 increasing levels for 10

24 seconds with 2-minute incubations on ice between sonication. Insoluble materials were pelleted by centrifugation at 11,000xg. The supernatant was added to a fresh tube containing Glutathione–Sepharose 4B beads (GE Health-care) and incubated for 3 hours at 4°C. The beads were washed 3 times in ice-cold GST-lysis buffer. The immobilized fusion protein on Glutathione–Sepharose 4B beads was subsequently used in protein pull-down and tubulin- binding assays.

2.2.2 Mouse Brain Homogenization

Each gram of frozen stripped mouse brain (Pel-Freeze® Biologicals; 55005-1) was homogenized in 3mL of ice-cold homogenization solution A (0.32M Sucrose, 1mM NaHCO3,

1mM MgCl2, 0.5mM CaCl2, 0.01% Triton X-100, with protease inhibitor) using a motor driven (Jacobs Multi Craft & SDS Adaptor) homogenizer. Brain tissue was homogenized using up and down strokes until tissue was homogenous and then centrifuged at 1400xg at 4°C for 10 minutes. The supernatant was kept on ice while the pellet was resuspended in solution A at 10% of the original volume using the same homogenization technique. Resuspended fraction was centrifuged at 1400xg at 4°C for 10 minutes. The second supernatant was pooled with the first supernatant fraction and centrifuged at 710xg at 4°C for 10 minutes. The final supernatant was flash frozen in a dry ice-methanol bath and stored at -80°C.

2.2.3 Protein Pull-Down Assay

Homogenized brain tissue (see 2.2.2.) was thawed on ice and then spun at 14,000rpm at 4°C for 10 minutes. The supernatant was added to the purified GST-Fusion protein immobilized on Glutathione–Sepharose 4B beads (see 2.2.1.) and incubated at 4°C with vertical rotation for 2 hours. The beads were subsequently washed 4 times in 1X PBS (137mM NaCl, 2.7mM KCl,

4.3mM Na2HPO4, 1.47mM KH2PO4). Beads were packed into micro-spin columns and both GST-fusion protein and interacting proteins were eluted from the beads using GST elution buffer (50mM Tris-HCl pH 8.0, 10mM reduced glutathione). Eluted fraction was analyzed using liquid chromatography-mass spectrometry (LC-MS/MS).

2.2.4 Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS)

Protein samples from the protein pull-down assay (see 2.2.3) were submitted, uncleaned to the Advanced Protein Technology Centre (The Hospital for Sick Children). Protein samples were subjected to a tryptic digest before analysis. Mass spectrometry was carried out using the Applied Biosystems/MDS Sciex API QSTAR XL Pulsar MALDI QTOF. Sample analysis report was returned and analyzed for positive interactors based upon the confidence of identification and the absence of the proposed interacting protein from the control samples (see Figure 7).

2.2.5 Tubulin Binding Assay

Tubulin (Cytoskeleton Inc.; BK029) was prepared and polymerized following manufacturer’s instruction. Polymerized tubulin was incubated with immobilized GST-fusion protein on Glutathione–Sepharose 4B beads (see 2.2.1.) at room temperature with vertical rotation for 45 minutes. Following incubation beads were washed 3 times using 1X PBS and then packed in a micro-spin column. A final wash was completed in the micro-spin column and then beads were removed and boiled in Laemmli sample buffer (50mM Tris, 2% SDS, 1% β- mercaptoethanol, 10% glycerol, 0.02% bromphenol blue, 12.5mM EDTA). Eluted fraction was analyzed using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (see section 2.3.).

2.3 SDS-Polyacrylamide Gel Electrophoresis

Protein samples denatured in Laemmli buffer were separated by weight on a 4% stacking and 10% resolving SDS-polyacrylamide gel. Proteins were separated at 110V using the general Laemmli buffer system (Laemmli, 1970).

2.4 Western Blotting

Following protein separation using SDS-PAGE, proteins were transferred to solid nitrocellulose membrane in 1X transfer buffer (25mM Tris, 192mM glycine, 20% methanol) for 1 hour at 90V. Following the transfer, the membrane was blocked in a 5% non-fat milk solution in 1X TBS at room temperature for 1 hour. The membrane was probed with either rabbit anti- TRPM2 (1:300), Mouse anti-α-Tubulin (1:5000) or rabbit anti-GST (1:10,000; horseradish peroxidase [HRP]-conjugated) in a 1% non-fat milk solution in 1X TBS either for 1 hour (if conjugated with HRP), or placed at 4°C overnight otherwise. Following the incubation the

26 membrane was washed 3 times with 1X TBS and subsequently incubated with 2° antibody in a 1% non-fat milk solution in 1X TBS for 1 hour at room temperature. The blot was washed 3 times with 1X TBS and incubated with chemiluminescent substrate for 2 minutes. The membrane was immediately imaged using film or a Bio-Rad ChemiDoc™ XRS+ System with Image Lab software.

2.5 Cell Culture

The RAW 264.7 murine macrophage cell line (a generous gift from Rene Harrison) was cultured in heat inactivated and 0.45µM-pore filtered 10% Fetal Bovine Serum (FBS) supplemented Dulbecco's Modification of Eagle's Medium (DMEM) in a 37°C incubator with

5% CO2. Adherent cells were cultured in T75 flasks with passages every 3 days by scraping.

2.6 Immunocytochemistry

RAW 264.7 cells were passed onto acid-washed coverslips in a 6-well plate at a density of 3.0 x 105 cells/mL and allowed to adhere and equilibrate overnight. Cells were activated using 0.1µg/mL of ultra-pure LPS and left for 16 hours before fixing. Regardless of initial treatment, cells were treated with either 10µM nocodazole or taxol for 30 minutes before fixing. Cells were washed with warm PBS and fixed in freshly prepared 4% paraformaldehyde in PBS at room temperature for 20 minutes. Following fixation, wells were washed with PBS and permeabilized using permeabilization buffer (0.1% Triton X-100 in PBS with 100mM glycine) for 20 minutes at room temperature. Following permeabilization, wells were washed and cells blocked using 5% albumin in PBS for 1 hour. Coverslips were inverted onto a small volume of primary antibody solution containing anti-TRPM2 antibody (1:50; [abcam, Ab11168]) and anti- α-tubulin antibody (1:500; [sigma, T9026]) and left to incubate overnight at 4°C. Coverslips were washed the following day and incubated with secondary antibody solution containing Alexa Fluor 488 donkey anti-rabbit IgG (1:1000; [Invitrogen; A21206) and Alexa Fluor 546 goat anti- mouse IgG (1:2000; [Invitrogen; A11003) for 1 hour at room temperature away from light. Coverslips were washed and mounted on slides using Dako fluorescent mounting media. Slides were imaged using a using a Zeiss confocal laser-scanning microscope (Axioplan 2 imaging microscope with attached Laser Scanning Microscope-5 Lasermodul, 63X objectives).

Chapter 3

3 Results 3.1 Identifying TRPM2 C-Terminal Protein Interactions

Few studies exist defining protein-protein interactions with the intracellular tails of TRPM2 and with that arises a curiosity to investigate. The potential for protein-protein interactions exists when the characteristic motifs of TRPM2 are considered: predicted N- and C- terminal coiled-coil domains (Zhu-Zhong, et al. 2006), PxxP domains (Kühn, et al. 2009) and NUTD9-H enzymatic domain (Eisfeld and Lückhoff 2007); each a region allowing for potential subunit and protein interactions (A. Lupas 1996). Therefore, full-length and truncated clones of the TRPM2 intracellular domains were generated for use in pull-down assays. In revealing a potential C-terminal interactor, the use of truncated clones provided insight into its binding location. Furthering the understanding of these interactions may provide the knowledge needed to discern how these channels behave and activate in various disease models.

3.1.1 Generation of Full-Length GST-TRPM2 C- and N-Terminus Fusion Constructs

C-Terminus

The purified untagged full-length TRPM2 sequence was obtained from the pCI-NEO- TRPM2 plasmid (Figure 3a) and was transformed into dcm-/dam- host bacteria for future methylation sensitive enzymatic digests. A previously constructed map of this vector (generous gift from John MacDonald) provided the restriction sites required to excise the C-terminus from the full sequence. The purified pCI-NEO-TRPM2 vector was subjected to an restriction digest with BclI and SalI to excise the C-terminus, which was subsequently gel-purified (Figure 3b) for insertion into the prokaryotic vector pGEX-5X-1. The pGEX-5X-1 vector was selected as the backbone for the generation of clones as it provided an N-terminal GST tag to the fusion protein, allowing fusion protein purification and ease of detection in western blotting. After ligation of the excised C-terminus into pGEX-5X-1, the plasmid was transformed into DH5-α E. coli. The plasmid was amplified and the proper clones confirmed through restriction digest (Figure 4) and

28 sequencing. Selected clones were transformed into BL-21 DE3 bacteria, a T7 expression strain of E. coli deficient in the proteases Lon and OmpT suitable for expression of recombinant proteins.

N-Terminus

The purified pCI-NEO-TRPM2 vector was subjected to a restriction digest with EcoRI and StuI to remove the N-terminus and subsequently gel-purified for insertion into the prokaryotic vector pGEX-5X-1. After the ligation of the excised N-terminus into pGEX-5X-1, the plasmid was transformed into DH5-α E. coli. The plasmid was amplified and the proper clone confirmed through restriction digest (Appendix 1) and sequencing. Selected clones were transformed into BL-21 DE3 bacteria.

Figure 3. Plasmid Map of pCI-NEO-TRPM2 and Excision of the C-Terminus

(A) The 10kb pCI-NEO-TRPM2 plasmid contains the full murine TRPM2 sequence and acted as the template for the generation of clones. The C-terminus was removed from the plasmid by a double restriction digest using SalI and BclI. (B) The full-length plasmid linearized by a SalI digest is depicted in lane. Lane 2 shows the resulting C-terminus excised from the template DNA. The full C-terminus has a predicted weight of 1253bp and is indicated with an arrow.

Figure 4. Plasmid Map of pGEX-5X-1-TRPM2-C-Terminus.

(A) The C-terminus of TRPM2 was removed from pCI-NEO-TRPM2 and ligated into pGEX- 5X-1 yielding the 6.2kb plasmid shown. pGEX-5X-1 places an N-terminal GST tag onto the inserted protein, thus generating a GST-TRPM2-C-terminus fusion protein. (B) The C-terminus construct was linearized using SalI and is shown for sizing purposes in both lanes 1 and 2 of.

3.1.2 Expression of GST-TRPM2 C- and N-Terminus Fusion Constructs in BL-21 DES E. coli

C-Terminus

The expression of the full length C-terminus fusion construct was tested and optimized to ensure accurate expression at suitable concentrations needed for purification and interaction studies. After growing and inducing the culture with IPTG, cells were lysed and analyzed by western blotting. Probing with an anti-GST antibody (GE Healthcare) revealed a band at 75 kDa, which was consistent with the predicted molecular weight of the GST-TRPM2-C-terminus fusion construct (74.6 kDa) (Figure 5).

N-Terminus

The expression of the full length N-terminus fusion construct was tested and optimized to ensure accurate expression at suitable concentrations needed for purification and interaction studies. After growing and inducing the culture with IPTG, cells were lysed and analyzed by western blotting. Probing with an anti-GST antibody (GE Healthcare) revealed a faint band at 100 kDa and a stronger band at 75 kDa (Appendix 2). The band at 100 kDa, although weak, was consistent with the predicted molecular weight of the GST-TRPM2-N- terminus fusion construct. Following several trials of GST-TRPM2-N-terminus expression, it was determined that there existed a problem with the clone. Informal communications with Rachelle Gaudet (Associate Professor of Molecular and Cellular Biology, Harvard University, 2009) confirmed that the full-length TRPM2 N-terminus is difficult to express. This suggests that the fusion protein is either toxic to the host BL-21 E. coli, or there is aggregation or degradation of the protein. As a result, the following study was completed focusing only on the C-terminus of TRPM2.

3.1.3 Identification of TRPM2 C-Terminus Protein Interactors Through Protein-Pull Down Assays and Mass Spectrometry

The GST-TRPM2-C-Terminus fusion protein was purified and immobilized on GST beads and subsequently incubated with mouse brain lysate in order to identify potential interactors. Eluting the fusion protein from the beads allowed any interacting proteins bound to the GST-TRPM2-C-Terminus protein to be eluted as well. The eluted fraction was submitted for analysis by LC/MS/MS. Upon completion a report was returned listing the proteins analyzed along with the confidence in accuracy, the number of peptides screened, and the proposed protein (Figure 6). In order to be regarded as a confident interactor with the C-terminus of TRPM2, identified peptides had to initially be absent from control (pGEX) lanes. This was to ensure that proteins were interacting with TRPM2, not GST. Lanes 20B and 21B represent the immobilized GST-TRPM2-C-Terminus fusion protein that was incubated with mouse brain lysate. Interacting proteins found only in these lanes can be regarded with a high degree of confidence as potential TRPM2 C-terminus interactors. Furthermore, lanes labeled 20 and 21 are the same GST-TRPM2-C-Terminus fusion proteins, however, these samples were not incubated with mouse brain lysate. Similar to the GST control, potential interactors should not exist in these lanes in order to account for bacterial proteins that may have bound to the fusion constructs. Mass spec results of interest are highlighted in yellow. Results were able to confirm that the fusion protein utilized was indeed from TRPM2, and was detected to a high degree in both concentration and confidence. In addition to this, results also indicated a great deal of cytoskeletal interaction, more specifically tubulin alpha 1B, tubulin beta 2 and tubulin beta 3.

3.2 Investigating Tubulin’s Interaction With the Full-Length TRPM2 C-Terminus

3.2.1 Tubulin Binds Directly to the C-Terminus of TRPM2

GST-TRPM2-C-Terminus and GST were purified and immobilized on GST beads and incubated with purified tubulin that had been polymerized in vitro. After removing bead-bound protein by boiling, samples were analyzed by western blotting. Probing with an anti-GST and anti-α-tubulin antibody revealed that tubulin bound to the GST-TRPM2-C-Terminus construct (Figure 7) and did not interact with the GST control. This result suggests that tubulin is interacting directly along the C-terminus in at least one location.

Figure 5. GST-TRPM2-C-Terminus Fusion Protein.

BL-21 culture containing the pGEX-5X-1-TRPM2-C-Terminus plasmid was induced using 3mM IPTG. Cell lysates were analyzed by western blotting. This blot was probed with an anti-GST antibody revealing a band around 75 kDa, which is indicative of the GST-TRPM2-C-Terminus fusion protein. Single band at 25 kDa is that of GST.

Figure 6. LC/MS/MS Results From the Protein Pull-Down Assay Utilizing Immobilized GST-TRPM2-C-Terminus Fusion Construct

The identification of potential interactors of the TRPM2 C-terminus following a protein-pull down assay was carried out using LC/MS/MS. The tabled results display a list of 26 different proteins that have the potential to interact with the C-terminus. Proteins of interest were selected based upon the confidence of each result. The number of peptides scored is reported in each hit, and the colour (green) indicates over a 95% probability of being a true hit. Potential protein interactors should only exist in the lanes labeled 20B and 21B, as these were the only fusion constructs incubated with mouse brain lysate. With the exception of pgexB, the lanes labeled pgex, 20 and 21 only contained immobilized protein without any incubation with mouse brain lysate. These four controls ensured that peptides found in the 20B and 21B lanes were not non- specific proteins interactions from the bacteria, or with the GST tag. Proteins of interest are highlighted in yellow and include TRPM2, tubulin alpha 1B, tubulin beta 2 and tubulin beta 3.

Figure 7. Tubulin Binds Directly to the C-Terminus of TRPM2

Purified GST-TRPM2-C-Terminus fusion protein was immobilized on beads and incubated with polymerized tubulin. After washing, protein was eluted from the beads and analyzed by western blotting. Here we show the direct binding of tubulin (50kDa) with the C-terminus of TRPM2 (75 kDa). The GST control did not bind tubulin.

Anti-GST

Anti-α-Tubulin

Anti-GST

3.3 Investigating the Binding Location of Tubulin Along the C- Terminus of TRPM2

3.3.1 Generation of Truncated GST-TRPM2 C-Terminus Fusion Constructs

After the generation of the full-length TRPM2 C-terminus clone and the identification of tubulin as a direct binding partner, there existed a need to identify where along the C-terminus tubulin was binding. Truncated portions of the C-terminus were devised based upon the unique C-terminal characteristics. As the C-terminus boasts a predicted coiled-coil and NUDT9-H enzymatic domain, the C-terminus was broken into two portions based upon literature-suggested locations and sequence analysis using software and online tools for alignments and motif identification. The enzymatic domain defined by Scharenberg et al. (2003) in the human TRPM2 gene was aligned with the murine TRPM2 sequence to map the NUDT9-H domain. The predicted coiled-coil domain was also identified through another alignment using Scharenberg’s suggested region. To further this, an online coil analysis tool1 was employed to confirm that the coiled-coil did indeed lie within this predicted region (Figure 8). These two portions were further broken down into a short and long version so as to cover both the unique domain alone and an extended region including both the unique domain and the region lying just outside the domain (Figure 9). Primers were subsequently generated for each of the four unique truncated clones and PCR carried out. PCR products were inserted directly into a pCR®-TOPO® vector which utilizes covalently bound topoisomerase I allowing fast and direct introduction of the blunt PCR products into the vector. This plasmid was transformed into DH5-α E. coli, which was later used to amplify the PCR product. Inserts were removed from the pCR®-TOPO® vector using the artificially introduced EcoRI and SalI cleavage sites and directionally ligated into the pGEX- 5X-1 vector. Plasmids were transformed into DH5-α E. coli, amplified and checked for accuracy by restriction digest and sequencing (Figure 10 and 11). Once confirmed, plasmids were transformed into BL-21 DE3 E. coli and checked for accuracy by western blotting. The GST- TRPM2-long-coil expressed at a high concentration and was visible by western blot around 46

1 Coils Version 2.2: http://www.ch.embnet.org/software/COILS_form.html

42 kDa. However, the GST-TRPM2-coil-only did express, but appeared slightly smaller than the expected weight of 41 kDa when analyzed by western blot (Figure 12A). This may be due to cleavage within the coil as there exist smaller bands and smears below the stronger band below 35 kDa. The long-NUDT9-H and the NUDT9-H-only clones, although confirmed genetically by sequencing, did not express well (Figure 12B). In fact, only GST was produced in high amounts suggesting either a natural cleavage point between the tag and the recombinant protein or problems within the sequence. To solve this, sequences were rechecked and clones streaked on agar plates for re-isolation. Individual colonies were again re-selected and examined by restriction digest to ensure that the insert existed. After transforming the newly confirmed plasmids into fresh competent BL-21 DE3 E. coli the success of expression was re-examined. After inducing the cultures, bacteria were lysed and analyzed by western blot. Again, only GST was produced suggesting a larger problem that has yet to be determined.

Figure 8. Predicted Regions Within the C-Terminus of TRPM2 Containing Coiled Domains.

(A) The amino acid sequence of the region C-terminal to the sixth transmembrane segment was analyzed using the online prediction tool Coils Version 2.2. In brief, the program determines regions of high coil probability based upon known parallel two-stranded coiled-coils found in globular and coiled-coil proteins. Regions are analyzed either in segments of 14, 21, or 28 as indicated by green, blue and red respectively (Lupas and Gruber 2005). (B) The amino acid sequence of the region C-terminal to the sixth transmembrane; Highlighted regions are areas predicted to have a high probability for coiled-coil formation. Highlighted areas include the analyzed region to the left of the arrow from (A).

1045- FNYTF QEVQE HTDQI WKFQR HDLIE EYHGR PPAPP PLILL SHLQL LIKRI VLKIP AKRHK QLKNK LEKNE ETALL SWELY LKENY LQNQQ YQQKQ RPEQK IQDIS EKVDT MVDLL DMDQV KRSGS TEQRL ASLEE QVTQV TRALH WIVTT LKDSG FGSGA GALTL APQRA FDEPD AELSI RRKVE EPGDG YHVSA RHLLY PNARI MRFPV PNEKV PWAAE FLIYD PPFYT AEKDV ALTDP VGDTA EPLSK ISYNV VDGPT DRRSF HGVYV VEYGF PLNPM GRTGL RGRGS LSWFG PNHTL QPVVT RWKRN QGGAI CRKSV RKMLE VLVMK LPRSE HWALP GGSRE PGEML PRKLK RVLRQ EFWVA FETLL MQGTE VYKGY VDDPR NTDNA WIETV AVSIH FQDQN DMELK RLEEN LHTHD PKELT RDLKL STEWQ VVDRR IPLYA NHKTI LQKVA SLFGA HF -1506

Figure 9. TRPM2 C-Terminus Truncated Clones.

The C-terminus of TRPM2 was divided into 4 regions based upon characteristic domains. Both the coiled-coil (cc) and the enzymatic (NUDT9-H) domains were divided into “short” and “long” regions in order to determine the binding location of tubulin. The long-coil includes the region after the sixth transmembrane segment (TM6) to the end of the predicted coiled-coil. The coil- only region contains solely the predicted coiled-coil without any of the region N-terminal to the domain. The same exists for the enzymatic domain, with the long-NUDT9-H containing the remaining region C-terminal to the coiled-coil, and the NUDT9-H-only clone containing just the enzymatic domain.

Figure 10. Analysis of Long-Coil and Coil-Only Constructs Following Cloning and Transformation.

(A) After the generation and transformation of plasmids for the TRPM2 C-terminus long-coil and coil-only clones, single colonies were selected from agar plates and grown in selective media. Unique clones were analyzed by restriction digest in order to determine whether the insert was present in the pGEX-5X-1 backbone. The pGEX-5X-1 vector has a weight of 4.9kb. Combined with the Long-coil and coil-only inserts the plasmids appear at 5.49kb and 5.35kb respectively. Inserts removed by restriction digest are indicated with arrows. The long-coil clones LC1-3 and LC4 contain the 592bp insert, and the single coil-only clone, C1, contains the predicted 378bp insert. Bands present at 5kb are indicative of the linear pGEX-5X-1 backbone. Those bands running higher at and above 7kb are likely indicative of uncut plasmids due to incomplete restriction digest (by reason of excess plasmid DNA). Plasmids from successful clones were later transformed into BL21 DE3 E. coli. (B) Plasmid map of the 5.49kb pGEX-5X- 1-Long-Coil. (C) Plasmid map of the 5.35kb pGEX-5X-1-Coil-Only.

B C

Figure 11. Analysis of Long-NUDT9-H and NUDT9-H-Only Constructs Following Cloning and Transformation.

After the generation and transformation of plasmids for the TRPM2 C-terminus long-NUDT9-H and NUDT9-H-only clones, single colonies were selected from agar plates and grown in selective media. Unique clones were analyzed genetically by restriction digest in order to determine whether the insert was present in the pGEX-5X-1 backbone. The pGEX-5X-1 vector has a weight of 4.9kb. Inserts removed by enzymatic digest are indicated with an arrow. (A) The long-NUDT9-H clones LN2-7 contain the 878bp insert, and (B) the NUDT9-H-only clones N3- 5, contains the 815bp insert. Bands present at 4.9kb are indicative of the linear pGEX-5X-1 backbone. Plasmids from successful clones were later transformed into BL21 DE3 E. coli. (C) Plasmid map of the 5.79kb pGEX-5X-1-Long-NUDT9-H. (D) Plasmid map of the 5.85kb pGEX-5X-1-NUDT9-H-Only.

50 A

C D

Figure 12. Expression of the TRPM2 C-Terminus Fusion Constructs by Western Blot Analysis.

The four different truncated clones were cultured, induced and harvested in order to analyze the yield and success of fusion-protein production. (A) By western blot, it was quickly determined that the yield of both the GST-TRPM2-long-coil and GST-TRPM2-coil-only fusion constructs was suitable. The GST-TRPM2-long-coil existed at its appropriate weight of roughly 46 kDa, where as the GST-TRPM2-coil-only construct ran much lighter than the expected 41 kDa. The presence of smaller bands and smears (indicated with arrow) suggest that the GST-TRPM2-coil- only construct may be subject to degradation. (B) Western blot analysis of the GST-TRPM2- long-NUDT9-H and GST-TRPM2-NUDT9-H-only constructs revealed that the fusion-proteins did not express well. The only bands present were at 25 kDa, which is indicative of GST only. This suggests that the enzymatic domain is either being cleaved and degraded or not expressed due to a problem with the cloning. Genetic analysis has yet to reveal any problems with the sequence.

3.3.2 Tubulin Interacts Directly With the C-Terminal Coiled-Coil Domain of TRPM2

After determining the direct interaction of tubulin with the C-terminus, truncated portions of the C-terminus were used to determine where the binding was localized. By purifying and immobilizing GST-TRPM2-Long-Coil, GST-TRPM2-Coil, and GST fusion constructs to GST beads, it was possible to interact each segment separately with purified polymerized tubulin. After interaction, bound protein was removed from the beads by boiling in 1X sample buffer and analyzed by western blotting (Figure 13). After probing the blot with anti-GST and anti-α- tubulin antibodies, it was observed that tubulin bound directly to both the GST-TRPM2-Long- Coil and GST-TRPM2-Coil-Only constructs despite the earlier expression problems with the GST-TRPM2-Coil-Only construct. Tubulin did not bind to the GST control. Unfortunately, due to lack of recombinant protein expression, we were unable to investigate whether tubulin bound to solely to the coiled-coil region or to the ADPR hydrolase/Nudix domain as well. Tubulin binding may be confined solely to the coiled-coil domain however, additional binding could have been provided by the ADPR hydrolase/Nudix domain.

3.3.3 The C-terminal Coiled-Coil of TRPM2 Contains a Putative Tubulin- Binding Motif Similar to that Found on TRPV1

Coiled-coil domains consist of a bundle of α-helices that are wound into a superhelix. The number of helices and their orientation is dictated by the packing of amino acids and the polar and ionic interactions between residues that flank the hydrophobic core (A. Lupas 1996). When considering an α-helical conformation, there may be regions where basic amino acids are projected to one side, allowing for potential interaction with negatively charged residues (Goswami and Hucho 2008). Such is the case with TRPV1, which has been shown to interact with the cytoskeleton at a defined tubulin-binding motif on its C-terminus (Goswami et al. 2007). The coiled-coil region of TRPM2 was analyzed using a helical–wheel model2 similar to that used

2 http://cti.itc.virginia.edu/~cmg/Demo/wheel/wheelApp.html

54 for the characterization of the tubulin binging motif in TRPV1. By analyzing short 18 amino acid segments of the mouse TRPM2 sequence within the coiled-coil region (as was done with TRPV1), the model revealed that amino acids 1092-1109 align themselves in a fashion where a large group of basic amino acids project themselves to one side (Figure 14). This highly basic region within TRPM2’s C-terminal coiled-coil may provide a region for tubulin binding. Tubulin contains an unstructured region known as an E-hook, which is a C-terminal over-hang containing a large number of negatively charged glutamate residues. These E-hooks have been known to be essential for the interaction of tubulin with various microtubule-associated proteins.

Figure 13. Direct Tubulin-Binding Assay with the GST-TRPM2-Long-Coil and GST- TRPM2-Coil-Only Fusion Constructs.

GST-TRPM2-Long-Coil and GST-TRPM2-Coil-Only fusion constructs were immobilized on beads and incubated with polymerized tubulin. After washing, protein was eluted from the beads and analyzed by western blotting. Probing with anti-GST and anti-tubulin antibodies revealed that tubulin bound directly to both constructs and not to the GST control. The binding of tubulin to the GST-TRPM2-Coil-Only fusion construct suggests that degradation is likely to blame for its smaller weight as the GST-TRPM2-Long-Coil fusion construct, which also bound tubulin, contains and extended region of the GST-TRPM2-Coil-Only construct.

Figure 14. Helical-Wheel Model Depicting the Formation of the Proposed Tubulin-Binding Domain of TRPM2

The coiled-coil region of TRPM2 was analyzed using a helical–wheel model. In analyzing short 18 amino acid segments of the mouse TRPM2 sequence, the model revealed that amino acids 1092-1109 within the coiled-coil region align themselves in a fashion where a large group of basic amino acids (blue) project themselves to one side. This highly basic region within TRPM2’s C-terminal coiled-coil may provide a binding region for the unstructured region of tubulin known as an E-hook. An E-hook is the C-terminal over-hang that contains a large number of negatively charged glutamate residues known to be essential for the interaction of tubulin with various microtubule-associated proteins. Bold-faced amino acids within the 1092- 1109 stretch signify highly basic lysine (K) and arginine (R).

1092-1109: I K R I V L K I P A K R H K Q L K N

3.4 In Vitro Analysis of The Interaction of Tubulin with TRPM2 in RAW 264.7 Macrophages

3.4.1 TRPM2 Co-Localizes With Tubulin In RAW Cells

The interaction between tubulin and TRPM2 was investigated further using immunocytochemistry. RAW cells in both the resting and LPS-activated state were treated with taxol, a microtubule-stabilizing agent, and nocodazole, a microtubule-depolymerizing agent, to observe differences in the tubulin-TRPM2 interaction. Resting macrophages adopted a round morphology with little to no processes or extensions. Tubulin staining showed minimal amount of microtubules within the cytosol. TRPM2 existed in the cytosol and along the membrane in large amounts. Sparse amounts of co-localization existed between TRPM2 and tubulin in the cytosol close to the nucleus and around the cell membrane (Figure 15). When tubulin was depolymerized using nocodazole, macrophages lost the smooth round morphology and became rough and sharp in appearance. Tubulin staining was reduced to a minimum, and TRPM2 staining revealed a retraction into the cytosol from the membrane. Loss of co-localization between TRPM2 and tubulin was observed in the macrophages leaving small pockets of puncta in the cytosol. On the other hand, macrophages presented a more robust and round morphology with defined and fibrous microtubules when treated with taxol. TRPM2 staining increased towards the membrane, as did the amount of co-localization between TRPM2 and tubulin.

In the LPS-activated state macrophages adopted a broader and more elaborate morphology displaying projections from around the cell body. Also, untreated activated macrophage presented a more fibrous network of microtubules and a greater amount of membrane and cytosolically localized TRPM2. A great deal of co-localization between TRPM2 and tubulin was revealed in the cytosol and along projections of the untreated, activated macrophage (Figure 16). Increased amounts of co-localization between TRPM2 and tubulin appeared to exist as a result of a more stable and fibrous body of microtubules. This network of microtubules was hindered in the activated state when treated with nocodazole, however there still existed some interaction between TRPM2 and tubulin within the cytosol. TRPM2 staining in the activated and nocodazole treated macrophage did not appear to change much aside from being less prominent in the extensions. In contrast, activated macrophages subjected to taxol

60 treatment showed intense TRPM2 staining in the cytosol and along the processes all the way to the tips. A very strong and intense microtubule network was visualized when activated macrophages were treated with taxol. As a result, a considerable amount of co-localization between TRPM2 and tubulin was observed in the cytosol, around the membrane and along projections. It appears that macrophages in the activated state display a greater degree of co- localization between tubulin and TRPM2 suggesting a potential role for TRPM2 in activated macrophages.

Figure 15. TRPM2 and Tubulin Immunostaining in Resting Macrophages

Immunostaining of resting macrophages treated with either 10μM taxol or 10μM nocodazole to observe changes in the interaction between TRPM2 and tubulin. Immunostained using anti- TRPM2 (Abcam), anti-α-tubulin (Sigma), Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen) and Alexa Fluor 546 goat anti-mouse IgG (Invitrogen). Images taken using a Zeiss confocal laser-scanning microscope (Axioplan 2 imaging microscope with attached Laser Scanning Microscope-5 Lasermodul, 63X objectives).

Figure 16. TRPM2 and Tubulin Immunostaining in LPS-Activated Macrophages

Immunostaining of LPS-activated (0.1ng/mL LPS) macrophages treated with either 10µM taxol or 10µM nocodazole to observe changes in the interaction between TRPM2 and tubulin. Immunostained using anti-TRPM2 (Abcam), anti-α-tubulin (Sigma), Alexa Fluor 488 donkey anti-rabbit IgG (Invitrogen) and Alexa Fluor 546 goat anti-mouse IgG (Invitrogen). Images taken using a Zeiss confocal laser-scanning microscope (Axioplan 2 imaging microscope with attached Laser Scanning Microscope-5 Lasermodul, 63X objectives).

Chapter 4

4 Discussion

In order to identify a novel protein interactor of TRPM2, the use of GST-TRPM2 fusion proteins were employed. Purified full-length GST-TRPM2-C-terminus fusion construct was immobilized and incubated with mouse brain lysate in order to identify interacting partners using mass spectrometry. Pull-down studies revealed a number of interactors, tubulin being one of strong confidence. This interaction was confirmed using a direct tubulin-binding assay with the full GST-TRPM2-C-terminus fusion construct. After confirming that the C-terminus of TRPM2 was sufficient for a direct interaction, a potential binding location of tubulin was identified using truncated TRPM2 C-terminus clones. Additional direct tubulin-binding assays demonstrated that tubulin was binding to the coiled-coil region of the TRPM2 C-terminus. Genetic analysis revealed a tubulin-binding motif within the coiled-coil region hinting at a potential binding site. Immunostaining and co-localization studies in RAW 264.7 macrophages demonstrated increases in the association of tubulin with TRPM2 suggesting a role during inflammation.

4.1 TRPM2 And Its Interaction With Tubulin: The Potential Role At The Membrane

Modern advances in biochemical studies and imaging techniques have corrected the old notion that tubulin does not interact with the membrane. It is understood now more than ever that tubulin plays an immense role at the membrane in terms of scaffolding and protein interaction.

4.1.1 Localization of TRPM2 to the Membrane by Tubulin

Our results have indicated a significant interaction between TRPM2 and tubulin, which was observed in protein binding assays, and in vitro studies and immunostaining in RAW cells. Activated RAW cells had a more stabilized cytoskeleton when compared to resting cells. With this increased microtubule network came about a greater amount of co-localization between TRPM2 and tubulin in the cytosol, at the membrane and along projections. These interactions between TRPM2 and tubulin were disrupted upon treatment with nocodazole. This result

66 suggests that tubulin may be important for the localization and retention of TRPM2 in the membrane and along projections. This idea stems from the interaction of β-tubulin with TRPC1, which was shown in human adult retinal pigment epithelial (APRE) cells to be significant for the localization and retention of TRPC1 protein to the plasma membrane (Bollimuntha, Cornatzer and Singh 2005). Bollimuntha et al. (2005) employed Ca2+ imaging to demonstrate a decrease in Ca2+ influx following tubulin depolymerization in ARPE cells. Using thapsigargin to induce Ca2+-influx through TRPC1, the group was able support the importance of tubulin in localization and function.

4.2 A Potential Role for TRPM2 in Activated Macrophages in Context With Tubulin

The inflammatory response involves the activation of a number of cell types including leukocytes (neutrophils, monocytes and lymphocytes) and tissue fixed macrophages. Neutrophils and monocytes comprise most of the circulating inflammatory cells and normally exist in a non-activated state. These cells have the potential to rapidly activate into phagocytes under the stimulation of invading microbes, bacterial products, foreign material, endogenous mediators, trauma and hypoxia. In response to these stimulants, these highly active phagocytes possess the ability to secrete enzymes, ROS and mediators such as chemotactic cytokines known as chemokines (Bellingan 1999). Chemokines have a key role in the mediation and recruitment of inflammatory cells to sites of inflammation (Yamamoto, Shimizu, et al. 2008). The physiology behind the transition to the active state is a complex process with multiple pathways and effectors that differ depending on the cell type in question. Although complex, it is understood that the mechanisms above require both ROS and the influx of Ca2+, an important second messenger in cells that acts as a regulator of cell viability and the internal processes that occur within the cell (Petricevich, et al. 2008; Sun and Zemel 2008).

TRPM2 is expressed to a high degree in immune cells such as monocytes, neutrophils, macrophages, and T lymphocytes. The recent ambitions to define its role in immune cells are understandable, considering its well-known activation by ROS and the resulting influx of

67 calcium. Investigation into the role of TRPM2 in neutrophils and monocytes has revealed that its activation by H2O2 in the human monocytotic cell line, U937, amplifies downstream Ras and Erk signaling via Pyk2 which results in the nuclear translocation of NF-κB and the subsequent production of the chemokines interleukin-8 (CXCL8) and the macrophage inflammatory protein- 2 (CXCL2) (Yamamoto, Shimizu, et al. 2008). In addition to this Knowles et al. (2010) demonstrated that TRPM2 knockout mice displayed diminished levels of the cytokines interleukin-12 (IL-12) and interferon-γ (IFN-γ) following Listeria monocytogenes infection. A further inquiry into TRPM2s place in LPS-activated human monocytes, revealed its role in the production of IL-6, IL-8, IL-10 and TNF-α, which of course was accompanied by a time- dependent increase in intracellular Ca2+ (Wehrhahn, et al. 2010).

Aside from the production of chemokines, it has been demonstrated that the classical activation of macrophages by a combination of IFN-γ and LPS resulted in the increase of stabilized cytoplasmic microtubules by action of the cytoplasmic linker protein 170 (CLIP-170). CLIP-170 acts as a major regulator of microtubule stabilization during macrophage activation, which is necessary during cell spreading and phagocytosis (Patel, et al. 2009). Considering TRPM2s demonstrated role and its contribution to increases in intracellular Ca2+ and chemokine production, we propose a potential mechanism which may further the activation of TRPM2. Considering the direct interaction tubulin shares with the C-terminus of TRPM2, and the observed increase in co-localization of TRPM2 with untreated and taxol-stabilized tubulin in RAW cells, both in the resting and activated states, it can be proposed that macrophage activation increases the association of tubulin with TRPM2 potentially gating the channel resulting in increased calcium influx. This activation of TRPM2 may work as proposed in LPS- and H2O2-stimulated models resulting in the production of chemokines (Figure 17).

The interaction of tubulin with TRPM2 is not the first of its kind. In addition to the evidence from Bollimuntha et al. (2004) in regards to TRPC1’s interaction with tubulin, Goswami et al. (2004) have also previously characterized a direct interaction between TRPV1 and tubulin. This group was able to demonstrate a Ca2+-stabilizing effect of the TRPV1 C- terminal fragment on microtubules when depolymerization was cold induced; and the Ca2+- independent stabilization of microtubules when depolymerized by nocodazole (Goswami, et al. 2004). In addition to TRPV1, Goswami et al. (2010) have recently demonstrated that the C- terminus of TRPV4 interacts directly and functionally with microtubules, in a mutual manner to

68 reduce the Ca2+-influx through the channel. Using a number of cell types, they were able to show that the interaction of tubulin with TRPV4 acts to stabilize microtubules, even under depolymerizing conditions. Further, they demonstrated the presence of the interaction between TRPV4 and tubulin in enriched structures at submembranous regions, and that activation of TRPV4 resulted in morphological changes affecting lamellipodial, filopodial, growth cone and neurite structures (Goswami, Kuhn, et al. 2010). The number of cases of microtubule interaction with TRP channels is comforting, and supportive of the idea that tubulins interaction with TRPM2 may very well act to activate the channel, or provide a mutual interaction whereby TRPM2 is gated and microtubules stabilized.

4.3 Future Studies Characterizing TRPM2 Interactors and the Functional Interaction Between Tubulin and TRPM2

In a general sense, further investigation into additional interactors of TRPM2 would benefit the overall understanding of the cationic channel. By simply employing protein pull- down assays and mass spectrometry work, a number of other activators may be revealed. The use of specific tissues for the study could enhance this. For example, brain tissue from an ischemic model can be used to indentify players that may only exist in an ischemic setting.

Investigating the interaction from a functional point of view would greatly enhance the understanding of the role of the cytoskeletal interaction with the C-terminus of TRPM2. By using electrophysiology, the activity of TRPM2 could be measured upon stabilization and depolymerization of microtubules. Increases in channel activity upon microtubule stabilization may provide the evidence needed to support the idea that microtubules may act to gate the channel. In contrast, if channel activity increases with depolymerization, perhaps the interaction may exist to halt channel activity. This could occur by blocking important domains whereby interactors bind and subsequently gate TRPM2. As current work has only identified the coiled- coil region as a domain for tubulin interaction, it cannot be forgotten that there is still chance for interaction with the enzymatic domain of TRPM2. If tubulin should prove to have affinity for this area of TRPM2, it may very well act to block ADPR binding domain. The mutation of the proposed tubulin-binding domain may prove to attenuate the binding of tubulin. Perhaps a full- length TRPM2 construct containing this mutation may be useful if transfected into macrophages and observed for changes in activation profiles after stabilization or depolymerization of microtubules. Considering the CLIP-170 induced stabilization of microtubules that occurs in

69 macrophages, perhaps a mutated TRPM2 C-terminal coiled-coil domain would have an effect in activated macrophages whether it be in the downstream secretion of chemokines, or maybe even in terms of migration, phagocytosis, or overall calcium influx. When these future studies are considered, there is great potential to further understand the interaction between tubulin and the C-terminus of TRPM2 and elucidate a functional relationship that may prove to be useful in better understanding TRPM2s role in various diseases.

Figure 17. Model: TRPM2 and Tubulin In Macrophages

The activation of macrophages with LPS (1) results in the stabilization of the cytoskeleton (2), which results in the interaction of tubulin with C-terminal domain of TRPM2 at the membrane (3). This interaction may result in the gating of TRPM2 with subsequent influx of Ca2+ (4) activating the Ras/Erk pathway via Pyk2. Translocation of NF-κB into the nucleus results in the production of chemokines (5).

Chapter 5 5 Summary

Roughly a decade of research exists describing what we currently know about TRPM2 and although there has been a great deal of progress made, there is plenty left to unearth. With sound understanding of TRPM2s protein-interactors still lacking, the initial goal of this study was to characterize potential protein-protein interactions with the intracellular tails of the channel. The generation of the full-length intracellular C-terminus clone allowed the opportunity to carry out interaction studies. Combining protein-pull down assays and LC/MS/MS, it was revealed that tubulin interacted with the C-terminus of TRPM2. Deeper investigation into the interaction yielded the understanding that the association of tubulin with the C-terminus was direct, and not under the influence or support of adapter proteins. In order to identify whereabouts tubulin was binding along the C-terminus the lengthy task of constructing truncated clones was carried out. Two of the 4 clones were successfully used and were able to show that tubulin bound directly to the predicted coiled-coil domain of TRPM2s C-terminus. It was not possible to conclude whether the enzymatic domain directly bound tubulin, and as such, there is still much left to investigate. By carrying out in vitro studies with RAW 264.7 macrophages, it was shown that the interaction of tubulin with TRPM2 was enhanced in activated RAW cells and under taxol-induced stabilization of microtubules. With the understanding of TRPM2s proposed role in immune cells, it was proposed that the endogenous stabilization of microtubules by CLIP- 170 in macrophages resulted in increased amounts of interaction between tubulin and TRPM2. This increased interaction may result in the gating or localization of TRPM2 subsequently contributing to the increase in intracellular calcium required for the production of chemokines. It was difficult to propose the latter model without the completion of any functional studies; however, in future a series of proposed studies may provide the missing links to completely elucidate both the direct and functional interaction between tubulin and TRPM2.

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Appendices

Appendix 1. Plasmid Map of the pGEX-5X-1-TRPM2-N-Terminus vector and Restriction Digest Analysis

(A) Plasmid map of the 7.13kb pGEX-5X-1-TRPM2-N-Terminus. The N-terminus sequence (insert) of 2168bp was ligated into the 4.9kb pGEX-5X-1 backbone. (B) After the generation and transformation of the pGEX-5X-1-TRPM2-N-Terminus plasmid, single colonies were selected from agar plates and grown in selective media. Unique clones were analyzed by restriction digest in order to determine whether the insert was present. Here, the plasmid was digested with XmnI generating a predicted band of 3077bp (which contains the insert) and another band weighing 4056bp (containing the remainder of the pGEX-5X-1 vector). Plasmids from successful clones were later transformed into BL21 DE3 E. coli. (C)

Appendix 2. Expression of the pGEX-5X-1-TRPM2-N-Terminus Fusion Protein

Induced pGEX-5X-1-TRPM2-N-Terminus culture was lysed and analyzed by western blotting. Probing with an anti-GST antibody (GE Healthcare) revealed a faint band at 100 kDa and a stronger band at 75 kDa. The band at 100 kDa is consistent with the predicted weight of the fusion protein, however the expression is weak yielding a low concentration of fusion protein not suitable for protein work. There are no bands below, suggesting that degradation may not be an issue. A large band at 75 kDa has yet to be elucidated.