Investigating Amine Oxidase Domain Containing Genes-Amx-1 and Amx

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Investigating Amine Oxidase Domain Containing Genes - amx-1 and amx-2 - in

Caenorhabditis elegans

A thesis presented to

the faculty of

the College of Arts and Sciences of Ohio University

In partial fulfillment

of the requirements for the degree

Master of Science

Reetobrata Basu

December 2014

This thesis titled

Investigating Amine Oxidase Domain Containing Genes - amx-1 and amx-2 - in

Caenorhabditis elegans

REETOBRATA BASU

has been approved for

the Department of Biological Sciences

and the College of Arts and Sciences by

Janet S. Duerr

Associate Professor of Biological Sciences

Robert Frank

Dean, College of Arts and Sciences 3

ABSTRACT

BASU, REETOBRATA, M.S., December 2014, Biological Sciences

Investigating Amine Oxidase Domain Containing Genes - amx-1 and amx-2 - in

Caenorhabditis elegans

Director of Thesis: Janet S. Duerr

Monoamine (MA) neurotransmitters affect multiple behaviors in animals. MA homeostasis is achieved partly by monoamine-oxidase (MAO) enzymes - a drug target for many human neuropsychiatric disorders. In C. elegans the MA pathway is similar to that in humans and the worm shows MA dependent behaviors, affected by MAO inhibitor

(MAOI) treatments. We cloned, expressed and purified the C. elegans genes - amx-1 and amx-2 in heterologous systems. Absorption spectra indicated that AMX1 and AMX2 bind the redox cofactor flavin adenine dinucleotide (FAD). Biochemical assays with wild-type

(N2) or mutant worm lysates showed significant differences in MAO and histone demethylase (HDM) activities between them. Purified AMX1 and AMX2 had very low in vitro HDM activity independently, but both significantly increased the HDM activity of worm lysates. AMX1 had negligible in vitro MAO activity, whereas AMX2 had high in vitro MAO activity, with substrate and inhibitor specificities.

DEDICATION

To my mother Manisha Basu

ACKNOWLEDGMENTS

I would like to express my deepest appreciation for my advisor, Professor Janet

Duerr. Without her guidance and persistent help this dissertation would not have been possible.

I would also like to thank the members of my committee Professor Robert Colvin,

Professor Daewoo Lee and Professor Sarah Wyatt for their time, support and invaluable guidance in various areas in my thesis. For the help in several aspects of my research, I am greatly thankful to Professor Tomohiko Sugiyama and Professor Mark Berryman

(Ohio University, Athens, Ohio), Professor Ralf Baumeister and Ruth Jahne (Freiberg

University, Germany), and Professor David Katz and Professor Kelly Williams (Emory

University, Atlanta, Georgia).

I must thank Amrita Basu, Jian Li, and Nilesh Khade for their constant help and support in different parts of my thesis. I would like to thank my parents and my wife for their love and encouragement and for always being my strength.

TABLE OF CONTENTS

Page

Abstract ...... 3 Dedication ...... 4 Acknowledgments...... 5 List of Tables ...... 8 List of Figures ...... 9 Chapter 1: Introduction ...... 11 1.1: Overview on Monoamine Metabolism...... 11 1.2: Monoamines and Their Roles in Humans...... 14 1.2.1: Dopamine ...... 15 1.2.2: Serotonin ...... 17 1.2.3: Norepinephrine (Noradrenaline) and Epinephrine (Adrenaline) ...... 19 1.2.4: Tyramine and Octopamine ...... 20 1.3: Monoamine Oxidase in Humans...... 21 1.4: Monoamine Pathophysiology in Humans and Monoamine Oxidase Inhibitors. .... 25 1.5: Caenorhabditis elegans as a Model Organism...... 28 1.6: MA Trafficking in C. elegans...... 30 1.7: The Roles of MAs in C. elegans...... 32 1.8: Amine Oxidase (AO) Domain Containing Genes in C. elegans...... 34 1.9: Histone Demethylases in Vertebrates and C. elegans...... 37 Chapter 2: Background Experiments and Hypothesis ...... 42 2.1: Monoamine Dependent Behavior Assays...... 42 2.1.1: Sensitivity to Exogenous DA and 5HT...... 43 2.1.2: MA-dependent Movement ...... 46 2.1.3: Pharyngeal Pumping ...... 47 2.1.4: Egg-laying Behavior and Embryos in utero ...... 47 2.1.5: Effect of MAOIs ...... 48 2.2: amx Transgenic Studies ...... 48 2.3: In situ Monoamine Levels...... 51 7

2.4: Hypothesis...... 52 Chapter 3: Materials and Methods ...... 54 3.1: Bacterial Plasmid Construction ...... 54 3.1.1: Gateway-amx plasmids ...... 55 3.1.2: 2GFP-T-amx plasmids ...... 58 3.2: Yeast Plasmid Construction ...... 60 3.3: Protein Expression and Purification from Bacterial Constructs ...... 63 3.4: Protein Expression and Purification from Yeast Constructs ...... 65 3.5: Protein Expression Analysis by Western Blot ...... 70 3.6: Detection of FAD by Absorption Spectra Analysis...... 71 3.7: Preparation of Worm Protein Extracts ...... 71 3.8: Biochemical Assay for Monoamine Oxidase Activity ...... 72 3.9: Biochemical Assay for Histone Demethylase Activity ...... 73 Chapter 4: Results ...... 75 4.1: Cloning of amx cDNAs into Bacterial Expression Plasmids...... 75 4.2: Expression and Purification of AMX Proteins from Bacteria...... 78 4.3: Cloning of amx cDNAs into Yeast Expression Plasmids...... 81 4.4: Expression and Purification of AMX Proteins from Yeast...... 82 4.5: FAD Absorption Spectrum...... 85 4.6: Biochemical Activity of Wild-type and Mutant Worm Lysates...... 88 4.7: HDM Activity of AMX Proteins ...... 90 4.8: MAO Activity of AMX Proteins...... 94 Chapter 5: Discussion And Summary ...... 101 References ...... 108 Appendix A: Table 2 – Primer Information ...... 128 Appendix B: Table 3 – Expression Conditions Tested In E. Coli ...... 130 Appendix C: Nucleotide Sequencing Results ...... 131 Appendix D: Protein Domain Analyses Results ...... 135 Appendix E: Supplementary Figures ...... 137

LIST OF TABLES

Page

Table 1 - Monoamine Dependent Behavior Assays ...... 45

Table 2 - Primer Information ...... 128

Table 3 - Expression Conditions Tested in E. coli ...... 130

LIST OF FIGURES

Page

Figure 1: Simplified cartoon of a monoamine (MA) utilizing synapse ...... 13

Figure 2: Enzymatic degradation of MAs in vertebrates ...... 14

Figure 3: MA synthesis pathways in vertebrates ...... 15

Figure 4: Dopamine (DA) pathways in the human brain ...... 17

Figure 5: Serotonin (5HT) pathways in the human brain ...... 19

Figure 6: Structures of human MAO-A and MAO-B ...... 23

Figure 7: The basic mechanism of action of monoamine oxidases ...... 25

Figure 8: Life cycle of C. elegans at 22C ...... 29

Figure 9: Biosynthetic pathways for biogenic amines in C. elegans ...... 32

Figure 10: Amine oxidase (AO) domain containing genes in C. elegans ...... 36

Figure 11: Histone modifications...... 38

Figure 12: Mammalian flavin-dependent histone demethylases LSD1 and LSD2 ...... 41

Figure 13: Pamx-1::GFP (transcriptional reporter) expression in C. elegans...... 50

Figure 14: In situ MA levels ...... 52

Figure 15: amx-1 gene ...... 76

Figure 16: amx-2 gene ...... 76

Figure 17: Restriction enzyme analysis of the bacterial constructs ...... 78

Figure 18: PCR of amx constructs in E. coli expression strain ...... 80

Figure 19: AMX1 and AMX2 expression in bacteria ...... 81

Figure 20: Restriction enzyme analysis of the yeast constructs ...... 82 10

Figure 21: PCR of amx constructs in P. pastoris expression strain ...... 83

Figure 22: Purification of AMX1 and AMX2 from yeast ...... 85

Figure 23: Absorption spectra of AMX proteins ...... 87

Figure 24: HDM activity of worm lysates ...... 89

Figure 25: MAO activity of worm lysates ...... 89

Figure 26: HDM activity of purified AMX1 and partially purified AMX2 (L and S) ...91

Figure 27: HDM activity of worm lysates in presence of AMX1 ...... 92

Figure 28: HDM activity of worm lysates in presence of AMX proteins ...... 93

Figure 29: MAO activity of purified AMX proteins ...... 95

Figure 30: Substrate specificity of 1X purified AMX2L ...... 97

Figure 31: Substrate specificity of 1X purified AMX2S ...... 98

Figure 32: Substrate and inhibitor specificity of 2X purified AMX2L and AMX2S .....99

Figure 33: Effect of AMX2 on MAO activity of worm lysates ...... 100

CHAPTER 1: INTRODUCTION

In vertebrates and invertebrates, neurotransmission by biogenic amines or monoamines (MAs) controls and modulates a number of important physiological, behavioral and endocrine functions. MAs act on many, often overlapping targets in the central and peripheral nervous system. MAs include indoleamines [serotonin (5HT) and melatonin], catecholamines [dopamine (DA), epinephrine, norepinephrine], histamine, and other biogenic amines like tyramine (TYR), and octopamine (OCT). The regulation of MA levels in the body is achieved by regulation of their synthesis, packaging, release and degradation. Several proteins of the MA pathway are validated targets for the treatment of human neurological disorders like Parkinson’s disease, depression, etc. The

MA trafficking pathway has been studied in a number of model organisms, including the nematode Caenorhabditis elegans.

1.1: Overview on Monoamine Metabolism.

The monoamine neurotransmitters are produced in monoaminergic cells from amino acids like tryptophan and tyrosine in a multi-step enzymatic process. Following their synthesis, MAs are actively packaged into synaptic vesicles by vesicular monoamine transporters (VMAT) which use the proton concentration gradient set up by proton pumps in the vesicles to concentrate the MAs (Henry et al., 1994). Upon synaptic activation by membrane depolarization, a calcium mediated exocytosis process results in vesicular fusion with cell membrane and release of the MAs to the extracellular synaptic cleft (Stevens, 2003). At the synapse, the MA molecules may (1) bind to specific receptors (mostly G-protein coupled receptors) on adjacent post-synaptic cells and/or the 12 pre-synaptic cells, (2) travel as neurohormones to exert their action at a distal location,

(3) be removed by specific reuptake transporters back into the pre-synaptic cell, (4) be taken up and metabolized by distant cells, or (5) may also be metabolized in the extracellular space by non-specific oxidases. Following reuptake into the pre-synaptic cell, MAs may be repackaged into vesicles or may be metabolized. Within the cell, free

MAs are catabolized by enzymes like monoamine oxidase (MAO) or catechol-O- methyltransferase (COMT) ( Labrosse et al., 1958; Bach et al., 1988; Bortolato et al.,

2008; Eisenhofer et al., 2004) . Figure 1 illustrates a typical MA pathway.

Metabolism of MAs can occur by a number of different methods (Bortolato et al.,

2008). MAO may metabolize MAs to a cytotoxic deaminated aldehyde intermediate, releasing hydrogen peroxide and ammonia as byproducts. Subsequently, aldehyde dehydrogenase (ALDH) or aldehyde reductase (ALDR) or aldose reductase (ALR) acts on the deaminated aldehydes to form a more stable alcohol or acid. Alternatively, COMT can also act on MA substrates to form O-methylated products, which are thereafter acted upon by MAO and then by ALDH enzymes (reviewed in Eisenhofer et al., 2004). Figure

2A and 2B illustrates the MA degradation pathway in vertebrates, using dopamine (DA) and serotonin (5HT) as examples. In this project, I have focused on whether a similar

MAO is active in the MA degradation process in a model organism, the nematode C. elegans.

Figure 1: Simplified cartoon of a monoamine (MA) utilizing synapse. MA is synthesized from amino acid precursors, packaged into vesicles by vesicular monoamine transporters (VMAT), released into the synaptic cleft, taken up via specific reuptake transporters, and degraded by monoamine oxidase (MAO; mitochondrial outer membrane bound) and /or catechol-O-methyltransferase (COMT; cytoplasmic or membrane bound) or repackaged into vesicles.

Figure 2: Enzymatic degradation of MAs in vertebrates. (a) Serotonin degradation in vertebrates by the action of monoamine oxidase (MAO) and aldehyde dehydrogenase (ALDH) [Image courtesy: http://openi.nlm.nih.gov/]. (b) Dopamine degradation in vertebrates by the action of monoamine oxidase (MAO), catechol-O-methyltransferase (COMT) and aldehyde dehydrogenase (ALDH) [Image courtesy: Wikipedia].

1.2: Monoamines and Their Roles in Humans.

The proteins controlling synthesis, packaging and release of the monoamines are conserved among primates and rodents. Behaviors that are modulated by monoamine neurotransmitters in humans may be similarly modified across a variety of vertebrate species, as seen from a large number of rodent and non-human primate studies. Given the wide distribution and spectrum of effects of each of the MAs in the human brain, and their relevance in our subsequent discussion, I will briefly discuss some of the important 15 human MAs. Two of the trace MAs in the human brain – tyramine and octopamine – will also be discussed, keeping in view their relevance in my invertebrate model C. elegans.

Figure 3: MA synthesis pathways in vertebrates. (a) Synthesis of catecholamines [Image courtesy: pharmacology-online.blogspot.com]. (b) Synthesis of serotonin in vertebrates [Image courtesy: homepage.psy.utexus.edu].

1.2.1: Dopamine

Dopamine (3, 4-dihydroxyphenethylamine; DA), is a small molecule catecholamine neurotransmitter synthesized from the amino acid tyrosine [shown in

Figure 3(a) above]. DA is synthesized in several areas of the central nervous system

(CNS). Some of the dopaminergic neurons in the ventral tegmental area (VTA) and 16 substantia nigra pars compacta (SN) of the midbrain project to the caudate putamen in the striatum (nigrostriatal pathway) and regulate motor control of the body (reviewed by

Knab and Lightfoot, 2010). DA pathways from the VTA to the nucleus accumbens in the ventral striatum (mesolimbic pathway) and to the frontal cortex (mesocortical pathway) are involved in controlling cognitive alertness, reward, motivation, mood, food choice and feeding, and some forms of impulsive / risky decision making, like gambling

(reviewed by Hunt, 2006; Ikemoto, 2007; Knab and Lightfoot; 2010, Clark et al., 2012).

The activity of the DA neurons in the VTA region can in turn be regulated by several other neurotransmitters (e.g., glutamate, GABA) and neurohormones (e.g., leptin and ghrelin) to modulate motivational and cue aspects of food acquisition (de Araujo et al.,

2012; Narayanan et al., 2013). DA released from regions in the hypothalamus (arcuate nucleus, periventricular nucleus, zona incerta) travels to the pituitary gland via the tuberoinfundibular pathway and is involved in regulating secretion of several hormones like prolactin and gonadotrophin releasing hormone (Sinclair et al., 2014). DA release from amacrine cells in the human retina regulates a number of rhythmic processes induced by light and circadian rhythms (Doyle et al., 2002). The sympathetic nervous system also releases DA, leading to paracrine effects. Non-neuronal release of DA controls the activation states of T-lymphocytes in many immune organs (reviewed by

Sarkar et al., 2010), regulates salt-fluid reabsorption, renin-angiotensin levels and oxidative stress in kidney cells (Zhang et al., 2012), and have protective functions in the gastrointestinal mucosa (Mezey et al., 1996). Figure 4 below depicts some of the DA pathways in the human brain. 17

Figure 4: DA pathways in the human brain. [Image courtesy: Ben Best; http://www.benbest.com/science/anatmind/anatmd10.html]

1.2.2: Serotonin

Serotonin (5-hydroxytryptamine; 5HT) is an indoleamine neurotransmitter synthesized from the amino acid tryptophan [shown in Figure 3(b) above]. 5HT in the

CNS constitutes less than 10% of the total 5HT in the body and is produced in the raphe nuclei (shown in Figure 5 below), the reticular formation, and retinal amacrine cells and in trace amounts in the pineal gland. 5HT neurons in the CNS are broadly divided into two groups – rostral (85% of total 5HT neurons) and caudal (15% of total 5HT neurons) - based on the location of their cell bodies and regions innervated by their axons (reviewed by Frazer and Hensler, 1999). The rostral group has cell bodies in the mesencephalic and rostral pons (dorsal and medial raphe nuclei and the reticular formation) and their axons 18 innervate the cingulum, hippocampus, amygdala, hypothalamus, lateral and medial cortex, basal ganglia, basal forebrain and the septum. The caudal group has cell bodies in the rostral pons and the medulla oblongata (raphe nuclei and ventral reticular formation), and their axons innervate laterally into the cranial motor nuclei and caudally along the spinal cord. Based on the presence or absence of specialized synaptic contacts, the role of

5HT in the brain can be as a neurotransmitter or a neuromodulator of an ongoing synaptic activity initiated by another neurotransmitter. 5HT release is regulated by other neurotransmitters like acetylcholine (ACh), -amino butyric acid (GABA) and catecholamines (reviewed by Frazer and Hensler, 1999, Charnay and Leger, 2010). In the

CNS, serotonin is reported to affect wake-sleep cycles, nociceptive (pain) pathways, memory, cognition, learning, aggression, emotion and mood (particularly anxiety and depression) (reviewed by Charnay and Leger, 2010; Nordquist and Oreland, 2010).

Neuronal and humoral 5HT regulate both innate and adaptive immune responses by regulating pro-inflammatory responses, cytokine production/release, T-cell proliferation, etc. (reviewed by Baganz and Blakely, 2013). Non-neuronal 5HT is secreted mostly by the enterochromaffin cells in the digestive tract and by bone cells. Brain-derived 5HT stimulates, while peripherally produced 5HT inhibits, bone formation (reviewed by Ducy and Karsenty, 2010). Free and platelet bound 5HT can modulate blood pressure in humans through the regulation of contractions of smooth muscle lining the blood vessels

(reviewed by Watts et al., 2012). High plasma 5HT levels are known to inhibit insulin secretion in vertebrates (Zhang et al., 2013). Finally, humoral 5HT is reported to alter 19 carbohydrate metabolism in the liver, fetal growth, bowel movements, etc. (Sirek and

Sirek, 1970).

Figure 5: Serotonin (5HT) pathways in the human brain. [Image courtesy: Ben Best; http://www.benbest.com/science/anatmind/anatmd10.html]

1.2.3: Norepinephrine (Noradrenaline) and Epinephrine (Adrenaline)

Norepinephrine (4,5-β- trihydroxy phenethylamine; NE) is a neurohormone and is mostly produced by the action of dopamine-beta-hydroxylase on DA, derived in turn from the amino acid precursor tyrosine (see Figure 3a) and present both in central and 20 peripheral nervous system. In the CNS, NE is synthesized in the pons (locus coeruleus) and ventral tegmental area (VTA) and in the adrenal medulla. Efferent noradrenergic pathways from the locus coeruleus innervate the cerebral cortex, cerebellum, thalamus, hypothalamus, olfactory bulb, hippocampus and the spinal cord. Epinephrine (4,5-β- trihydroxy-N-methylphenethylamine; E) is a neurohormone produced primarily from NE by the action of phenylethanolamine-N-methyltransferase. E is synthesized mostly in the adrenal medulla and in low levels in the CNS. The epinephrine pathway originates in the

VTA and the dorsal medulla and extends to the hypothalamus, locus coeruleus and other parts of the brain. Release of NE and E from adrenergic nerves is regulated by several other classes of neurotransmitters such as DA, Acetylcholine (ACh), etc.

Epinephrine has major effects on metabolism and bronchodilation (Warren,

1986). NE and E, along with DA, modulate synaptic plasticity, fear conditioning, stress response (‘fight-or-flight’ response – increased heart rate, increased oxygen supply to brain, increased blood-flow to skeletal muscles, increased glucose release from cellular storage, etc.), and memory (reviewed by Tully and Bolshakov, 2010). NE can also act as a neuromodulator to modify behaviors like concentration, decision-making, working

(short-term memory) and processing of sensory information (Eckhoff et al., 2009).

1.2.4: Tyramine and Octopamine

Tyramine (4-hydroxyphenethylamine; TA) neurotransmitter is found in trace amounts in the human brain at the mediobasal hypothalamus, nigrostriatal DA neurons and in the NE neurons at the locus coeruleus. TA is synthesized by the decarboxylation of its amino acid precursor tyrosine. The trace amines including TA have poorly 21 characterized vesicular storage mechanisms in mammalian brain and a high turn-over rate

(reviewed by Burchett and Hicks, 2006). In humans, non-human primates and rodents, highly conserved TA-receptors (like TAAR1) have been identified with nanomolar specificities for TA in the cerebral cortex, olfactory bulb, spinal cord, thalamus, hypothalamus, hippocampus and amygdala. TA is reported to have tonic and phasic effects modulating the signaling efficacy of DA, 5HT and NE (reviewed by Burchett and

Hicks, 2006).

Octopamine (β,4-dihydroxyphenethylamine; OA) neurotransmitter is found in trace amounts in several areas of the human brain including the thalamus, amygdala, hippocampus, raphe nucleus, VTA, locus coeruleus, etc. Like TA, the presence of OA at the synapse may sensitize and enhance the effects of the major MAs (reviewed by

Burchett and Hicks, 2006). It is synthesized from TA. While OA has well defined functions in invertebrates [fruit-fly (Drosophila melanogaster), nematode

(Caenorhabditis elegans), octopus, honeybee, etc. (Roeder et al., 2003; Roeder, 2005)], its physiological role in vertebrates is not clearly understood. Unlike TA, no OA-specific receptors have been identified in the mammalian brain.

1.3: Monoamine Oxidase in Humans.

Humans have two monoamine oxidase (MAO) genes – MAO-A and MAO-B – with 70% sequence similarity in the coding region. Both MAOs have fifteen exons with identical exon-intron composition and both genes are located on the X-chromosome

(Xp11.23) (reviewed by Shih et al., 1999). MAO-A and MAO-B proteins belong to the family of flavin containing oxidoreductases (PFAM: PF01593) with a characteristic 22 amine oxidase (AO) domain. MAO-A and MAO-B vary in their substrate and inhibitor specificities. While DA and TA are substrates for both enzymes, MAO-A has higher affinity for 5HT, NE and E, while MAO-B has a higher affinity for benzylamine (BA) and phenylethylamine (PEA). MAO-A is specifically inhibited by clorgyline, while

MAO-B is specifically inhibited by deprenyl (Selegiline) (reviewed by Remick and

Froese, 1990).

Histochemical, immunocytochemical, and more recently positron emission tomography (PET) imaging of human brains have revealed detailed information about the distribution of these two MAOs in human brain. In primates, MAO-A is more abundant than MAO-B in infants (in neurons and astrocytes), whereas the trend was largely reversed after one year of age (Willoughby et al., 1988). Although MAO-A is more selective for 5HT as a substrate, it is present predominantly in the DA and NE neurons of the brain, whereas the serotonergic neurons in the raphe nuclei contain MAO-B (which has lower selectivity for 5HT). This could be due to a lack of substrate specificity of the

MAOs at high substrate concentrations or the MAOs may eliminate the incorrect MA in the corresponding cells (Levitt et al., 1982). In the periphery, human placenta contains predominantly MAO-A while human platelets, liver, and kidney contain MAO-B

(reviewed by Shih et al., 1999).

The crystal structures of MAO-B (Binda et al., 2002) and MAO-A (De Colibus et al., 2005) have helped to elucidate their reaction mechanisms. As shown in Figure 6, both

MAO-A and MAO-B monomers have three distinct domains – a C-terminal membrane binding domain (by which the enzymes associate with the mitochondrial outer 23 membrane), a flavin binding domain (for covalent attachment of the cofactor FAD) and a hydrophobic substrate binding amine oxidase (AO) domain. MAO-A and MAO-B exhibit slight differences in all three of these domains (Edmondson et al., 2009). Although human MAO-A crystallizes as a monomer (De Colibus et al., 2005), both human MAO-

A and MAO-B form a homodimer when active (Binda et al., 2002, Edmondson et al.,

2004, Edmondson et al., 2009).

Figure 6: Structures of human MAO-A and MAO-B. Three dimensional ribbon diagrams of human MAO-A (a) and MAO-B (b). MAO-A crystallized as a monomer with three distinct domains – a flavin binding domain (blue with the yellow covalently bound FAD), a substrate binding amine oxidase (AO) domain (red) and a C-terminal membrane binding domain (green) [Image courtesy: De Colibus et al., 2005, PNAS]. MAO-B crystallized as a dimer with each monomer having the same three distinct domains – a C- terminal membrane binding domain (green), flavin binding domain (covalently bound FAD shown in yellow), and a substrate binding amine oxidase (AO) domain [Image courtesy: Binda et al., 2002, Nature Structural Biology]

Results of in vitro studies show that MAOs catalyze the oxidative deamination of some primary, secondary and a few tertiary amines, converting them to the corresponding aldehydes. The proposed mechanism is shown in Figure 7 below. FAD acts as an essential cofactor by transiently accepting electrons, which are then taken up by oxygen to form hydrogen peroxide as a byproduct. An imine intermediate is formed, which undergoes spontaneous hydrolysis to form a toxic aldehyde and an ammonium ion

(Edmondson et al., 2009). The complete catabolism of MAs is a multistep process, usually involving MAO, COMT and ALDH. The end product is a less toxic acid or alcohol. Figure 2 depicts the catabolic processes of DA and 5HT in vertebrates. Briefly,

MAO first acts on DA to form 3,4-dihydroxyphenylaldehyde (DOPAD) which is then converted to 3,4-dihydroxyphenylaceticacid (DOPAC) by ALDH and thereafter to homovanillic acid (HVA) by COMT. HVA can also be formed from the sequential actions of MAO and ALDH on 3-methoxytyramine (3MT), formed by the action of

COMT on DA (reviewed by Gesi et al., 2001; Bortolato et al., 2008). Similarly, epinephrine and norepinephrine may be oxidized to DOMA or DOPEG by the sequential action of MAO and ALDH (or ALDR). COMT then converts DOMA to VMA and

DOPEG to MHPEG. When 5HT is metabolized, MAO first converts it to 5- hydroxyindoleacetaldehyde, which is thereafter converted to 5HIAA by ALDH. The

5HIAA is rapidly eliminated from the body by diffusion and excretion through the kidneys (reviewed by Bortolato and Shih, 2011).

Figure 7: The basic mechanism of action of monoamine oxidases, using FAD as the cofactor. When the monoamine substrate is oxidized to an unstable aldehyde, ammonia and hydrogen peroxide are formed as byproducts. [Image courtesy: Dorset et al., 1994, Journal of Neural Transmission]

1.4: Monoamine Pathophysiology in Humans and Monoamine Oxidase Inhibitors.

Monoamines have a widespread presence in the body; therefore it is not surprising that their dysregulation is implicated in a large number of pathological conditions.

Reduced activities of both MAOs have been reported to highly elevate the levels of 5HT,

DA and NE in the human brain, with associated severe developmental delays, episodic hypotonia, stereotypical hand movements and autistic features in male human subjects

(Whibley et al., 2010; Saito et al., 2014). A selective loss of function mutation in MAO-

A was reported in a Dutch kindred; affected male members were socially withdrawn and sporadically exhibited extremely aggressive and violent behavior (Brunner et al., 1993).

Human subjects with only MAO-B deficiency exhibited no apparent developmental or 26 behavioral abnormalities (Lenders et al., 1996). In cigarette smokers and type-II alcoholics, reduced MAO-B activity has been reported, although it is considered an effect rather than a cause (Fowler et al., 1996; Shih et al., 1999; Ou et al., 2009; Rendu et al.,

2011).

Studies in rodents have reveal ed a number of changes associated with reduced or loss of function mutations of MAO-A and/or MAO-B. Studies found that MAO A/B knock out (KO) mice had enhanced emotional and motor learning and fear conditioning compared to their wild-type littermates (Singh et al., 2013). Decreased function of MAO-

A in mice resulted in impaired impulse control, social deficits and increased perseverative behaviors while MAO-A KO mice showed had markedly higher aggressive behavior (Bortolato et al., 2011). MAO-B deficient mice had increased stress-induced hyperactivity and were also resistant to the neurodegenerative effects of the neurotoxin

MPTP (Grimsby et al., 1997). The MAO-A/B KO mice also had altered expression of

NMDA-receptor subunits in their hippocampus and elevated adult hippocampal neurogenesis (Singh et al., 2013).

A number of human neurological disorders are associated with abnormal MA metabolism. Patients with Parkinson’s disease (PD) exhibit severe loss of the dopaminergic neurons in the nigrostriatum and drastically lower levels of DA in the brain. The causative role of DA cell loss in PD was highlighted in experiments using

MPTP, a drug of abuse, which is converted by MAO-B to the neurotoxin MPP+, which in turn causes Parkinson -like symptoms in normal human subjects (Robottom, 2011).

Schizophrenia patients also exhibit decreased brain DA levels (Sinclair et al., 2014). 27

Different types of depressive disorders are associated with significantly reduced levels of

5HT and NE in the brain (Bortolato and Shih, 2011). A decrease in MA levels in these cases suggests that an inhibition in the rate of degradation of MAs might ameliorate the pathological conditions.

Several classes of monoamine oxidase inhibitors (MAOIs) are prescribed as monotherapies or in conjunction with other drugs for a variety of human neurological disorders. Marketed MAOIs in United States include (1) non-specific irreversible MAOIs

(tranylcypromine, isocarboxazid, and phenelzine), (2) MAO-A specific reversible inhibitors (moclobemides), and (3) MAO-B specific irreversible inhibitors (selegiline, rasagiline). Chemotherapeutic intervention with selective MAO-B inhibitors has been shown to increase DA levels in the brains of PD patients. Further, pretreatment with

MAO-B inhibitors may delay the need for levodopa treatment for PD patients (Shih et al.,

1999; Riederer and Laux, 2011; Teo and Ho, 2013). MAOIs were the first class of antidepressant developed for treating depressive disorders (Bortolato et al., 2008).

Currently, selective serotonin reuptake inhibitors (SSRI) are the preferred initial treatment for depression; MAOIs are the second line of treatment, preferred over tricyclic antidepressants (TCA) and lithium, for depressive disorders (Fiedorowicz and Swartz,

2004; Bortolato et al., 2008; Bortolato and Shih, 2011). Newer reversible inhibitors of

MAOs (RIMA) like moclobemide have been identified as the most efficacious treatment of anxiety disorders like obsessive compulsive disorder (OCD) and post-traumatic stress disorder (PTSD) (Bortolato et al., 2008). Safety and tolerability of these MAOIs remains an issue, making them a second line of treatment in most cases. The adverse side effects 28 of MAOI treatments include weight gain, sexual dysfunction, orthostatic hypotension, daytime sedation, toxic drug interactions, and insomnia, arising mainly due to a dysregulation of brain neurotransmitter levels (Fiedorowicz and Swartz, 2004; Remick and Froese, 1990). However, increased research addressing the specificities of interactions of these molecules and the diverse role of their targets in the body may lead to improved therapeutics.

1.5: Caenorhabditis elegans as a Model Organism.

First introduced by Dr. Sydney Brenner in his seminal paper in 1974 (Brenner,

1974), the free-living soil nematode Caenorhabditis elegans has emerged as an exceptional model organism in research on genetics, developmental biology, neuroscience, ageing, metabolism, cell-cycle, cell polarity, toxicology, etc. Its virtues include a small size (~1.1 mm), three-day generation cycle with more than 200 potentially genetically identical offspring, average life-span of three weeks (at room- temperature), and growth on a diet of E. coli bacteria on agar plates. There are two sexes in C. elegans – the self-fertilizing hermaphrodite (XX) and the male (XO; ~0.1% natural frequency). Figure 8 below illustrates the life-cycle of a C. elegans hermaphrodite. The adult hermaphrodite has 959 somatic cells, including 302 neurons with well characterized and essentially invariant connectivity. The translucent body of the nematode allows use of non-invasive means of following biological processes in living cells across all developmental stages. Almost 80% of human genes have homologs in C. elegans and more than 70% of the known signal transduction pathways are conserved between humans and C. elegans (Leung et al., 2008, Shaye and Greenwald, 2011). The worm is 29 thus an established model for studying signaling pathways important for human neurological diseases, as well as for cancer, ageing, diabetes, obesity, etc. Research in C. elegans is supported by excellent resources including the C. elegans Gene Knockout

Consortium, Caenorhabditis Genetics Center, EST collections and shared information on the worm interactome, ORFeome, transcriptome, RNAi studies, genome-wide gene expression studies, etc. available in the centralized on-line resources WormBase and

WormAtlas (reviewed by Kaletta and Hengartner, 2006, Dimitriadi and Hart, 2011).

Figure 8: Life cycle of C. elegans at 22C. Fertilization is set as 0 minutes. Numbers in blue along the arrows indicate the length of time the animal spends at a certain stage. The length of the animal at each stage is marked next to the stage name in micrometers. [Image and legend courtesy: WormAtlas]

1.6: MA Trafficking in C. elegans.

Monoamine metabolism in many invertebrates, including in C. elegans, is similar to that in humans, particularly in the synthesis, packaging, release, receptor-binding and reuptake mechanisms. Many invertebrates use monoamines and their metabolites as neurotransmitters and neurohormones, as well as in processes such as pigmentation and exoskeleton formation (reviewed by Sloley, 2004). There are four major MA neurotransmitters found in C. elegans – dopamine (DA), serotonin (5HT), tyramine (TA) and octopamine (OA) (reviewed by Chase and Koelle, 2007). These MAs are formed in neuronal and non-neuronal cells from the amino acids tyrosine and tryptophan, as shown in Figure 9 below. Hermaphrodites have about 25 neurons containing MAs, including eight dopaminergic (Sulston et al., 1975), eight serotonergic (Horvitz et al., 1982; Desai et al., 1988; Duerr et al., 1999; Anderson et al., 2013), two tyraminergic and two octopaminergic (Alkema et al., 2005) neurons. Some neurons contain MAs as well as other neurotransmitters like acetylcholine and neuropeptides (Duerr et al., 2001; Taghert and Nitabach, 2012; Komuniecki et al., 2012; Hapiak et al., 2013; Holden-Dye and

Walker, 2013). The MAs are packaged into vesicles by the vesicular monoamine transporter (VMAT), encoded by the cat-1 gene (a homolog of mammalian VMAT1)

(Duerr et al, 1999). Upon depolarization of the neuron, vesicular fusion releases MAs and the released MAs bind to specific pre-synaptic or post-synaptic receptors. The MA receptors in C. elegans are mostly G-protein coupled receptors but a number of MA- gated chloride-ion channels are also present (Suo et al., 2004; Wragg et al., 2007; Chase and Koelle, 2007; Ringstad et al., 2009). The mechanisms of removal of the MAs from 31 the extracellular space in invertebrates vary amongst different invertebrate species as well as with different MAs. In C. elegans, the major method of terminating 5HT transmission is reuptake by a serotonin reuptake transporter (SERT), encoded by the mod-5 gene (a homolog of human serotonin transporter SLC6A4) (Ranganathan et al., 2001; Jafari et al., 2011). Extracellular DA is largely removed by a cocaine-sensitive dopamine reuptake transporter (DAT), encoded by the dat-1 gene [a homolog of the human catecholamine transporter SLC6A2 (a NE transporter) and SLC6A3 (human dat-1 gene)] (Jayanthi et al., 1998; Nass et al., 2002; McDonald et al., 2007). In some invertebrates including C. elegans, MAs may be degraded by oxidative deamination as well as by N-acetylation, indicating the presence of MAO and arylalkylamine-N-acetyltransferase enzyme activities (reviewed by Isaac et al., 1990; Isaac et al., 1996, Sloley, 2004). Recently, succinylation has been identified as an important pathway of degradation for MAs like

OA, 5HT, DA, and TA; the activity is highest in the L1 larval stage of C. elegans. The succinylated MAs are used as building blocks for small signaling molecules called ascarosides (Artyukhin et al., 2013). The enzymes responsible for MA degradation by oxidative deamination have not yet been identified and characterized in C. elegans.

Figure 9: Biosynthetic pathways for biogenic amines in C. elegans. [Image courtesy: Chase and Koelle, 2007]

1.7: The Roles of MAs in C. elegans.

The MAs affect a number of behaviors in C. elegans. These behaviors are often regulated by multiple types of MAs acting antagonistically to each other and can be also modulated by other neurotransmitters and specific neuropeptides (Sawin et al., 2000,

Hills et al., 2004, Wragg et al., 2007, Mills et al., 2012, Hapiak et al., 2013, Flavell et al.,

2013).

In C. elegans hermaphrodites, DA is secreted by eight mechanosensory neurons and is involved in modulating the locomotory behavior of the animal in response to environmental cues. C. elegans shows distinct locomotory behavior patterns including

‘area restricted searching’ (food search in immediate vicinity of recently exhausted food source by high angled turns), and a ‘basal slowing response’ (reduced locomotion on encountering bacterial lawn in well-fed worms) which are regulated by DA as well as by 33 glutamate (Hills et al., 2004, de Bono and Maricq, 2005, Chase and Koelle, 2007).

Swimming-induced paralysis (SWIP) is also regulated by DA (McDonald et al., 2007).

DA is implicated in the control of two different forms of learning – state-dependent olfactory habituation and mechanical tap habituation (Chase and Koelle, 2007). In C. elegans, exogenous DA causes rapid and reversible paralysis of movement, slowed defecation and a transient decrease in the egg-laying behavior (reviewed by McDonald et al., 2007).

5HT is secreted from eight neurons in C. elegans. 5HT from the NSM neurons regulates pharyngeal pumping (feeding), while 5HT from the HSN and VC neurons regulates egg laying behavior. 5HT-stimulated egg-laying and feeding behavior in C. elegans is suppressed by DA, OA and TA (Shyn et al., 2003; Chase and Koelle, 2007).

5HT regulates the ‘enhanced slowing response’ (starved worms slow down or stop on encountering bacterial lawn) (Sawin et al., 2000; Chase and Koelle, 2007). This 5HT induced slowing response, which ensures that a starved nematode stalls on encountering a food source, adds on to the DA-induced slowing response in a well-fed animal.

Exogenous 5HT suppresses locomotion and defecation in wild-type C. elegans but stimulates egg-laying and pharyngeal pumping.

Overall, OA and TA act antagonistically to 5HT in modulating behavior (Chase and Koelle, 2007). TA is synthesized from tyrosine in the RIC, RIM, UV1 neurons and in gonadal cells. The RIC interneurons and gonadal cells contain enzymes that can use TA to make OA, while the RIM and UV1 neurons only have the TA-synthesizing enzymes and use TA as a neurotransmitter. OA and TA inhibit egg-laying behavior, serotonin- 34 stimulated pharyngeal pumping (feeding) and food- and serotonin- dependent aversive responses by independent mechanisms (Wragg et al., 2007; Chase and Koelle, 2007).

Endogenous TA is involved in inhibiting head oscillations during retraction after mild head touch and in modulating spontaneous reversals. Exogenous OA causes uncoordinated movements and decreased defecation (Horvitz et al., 1982).

1.8: Amine Oxidase (AO) Domain Containing Genes in C. elegans.

In C. elegans and other nematodes, biochemical assays indicate the presence of oxidative deamination as well as acetylation and succinylation of MAs (Isaac et al.,

1996). However, these oxidative enzymes have not been identified. The vertebrate MAOs contain a characteristic amine oxidase (AO) domain using flavin as a cofactor (Interpro:

IPR002937). The AO domain containing genes belong to the oxidoreductase class of genes and include monoamine oxidases, polyamine oxidases, spermine oxidases, histone demethylases, etc. A homology search for AO domain containing genes in the nematode

C. elegans identifies seven different genes – amx-1, amx-2, amx-3, F55C5.6, hpo-15, lsd-

1 and spr-5 (illustrated in Figure 10 below). The spr-5, lsd-1 and amx-1 genes also possess a characteristic SWIRM domain (Interpro: IPR007526) – a specialized multifunctional protein module often found in chromatin remodeling complexes (Da et al., 2006). Two of the AO domain containing proteins in C. elegans, SPR5 and LSD1 have been studied biochemically and have been found to be lysine-specific histone demethylases (H3K4me2 demethylases), with high amino acid sequence identity to the human LSD1 (KDM1A) protein in the SWIRM and AO domains (Shi et al., 2004, Katz et al., 2009, Nottke et al., 2011, Alvares et al., 2013). The hpo-15 and amx-3 are putative 35 homologs of the human isoform-1 of peroxisomal N-(1)-acetyl-spermine/spermidine oxidase and isoform-4 of spermine oxidase proteins respectively (Wormbase 2014).

F55C5.6 has no predicted biochemical function. AMX1 has highest amino acid sequence identity to the human isoform-1 of lysine specific histone demethylase 1B (KDM1B or

AOF1 or LSD2), while the AMX2 has highest amino acid sequence identity to the human

MAO-A (Wormbase 2014) in all the functional domains (see Appendix). AMX1protein has a conserved AO domain between residues 331-804. For AMX2, the conserved AO domain is present between residues 55-472. In this project, I have investigated the biochemical roles of the proteins encoded by amx-1 and amx-2. 36

Figure 10: Amine oxidase (AO) domain containing genes in C. elegans. Top line indicate size (hash=100 bp), gene models (turquoise = putative exons), protein domains or signal sequence (orange). [Image and legend courtesy: Dr. Janet Duerr]

1.9: Histone Demethylases in Vertebrates and C. elegans.

AO domains, similar to those in human MAO-A and MAO-B, are also present in an important class of histone modifying enzymes in vertebrates and invertebrates – the lysine specific histone demethylases (HDMs). HDM mediated demethylation of specific tri-, di-, or mono-methylated lysine residues on core histones is an important mechanism of epigenetic modification and may have profound influence on the development and physiology of an organism.

Multiple post-translational changes like acetylation, deacetylation, methylation, demethylation, ubiquitinylation, phosphorylation, etc. can occur on the flexible N- or C- terminal tails of the highly conserved core histones (see Figure 11 below) (Martin and

Zhang, 2005). These modifications may lead to changes in the chromatin architecture leading to slight or dramatic modulation of gene expression. Specific DNA sequences

(e.g. Polycomb group response elements, Trithorax group response elements), numerous long non-coding RNAs, small non-coding RNAs, and DNA methylation have been identified as important for the recruitment of histone modifying enzymes to their target sites (reviewed by Greer and Shi, 2012). The recognition and recruitment of histone modifying enzymes to the target site is also regulated by the reader domains of the histone modifying complex or adaptors (e.g. bromodomain, chromodomain, Tudor domain, WD40-repeat domains) (reviewed by Martin and Zhang, 2005, Yun et al., 2011).

Histone methylation dynamics alone can influence biological processes like DNA damage repair, cell-cycle regulation, stress response, etc. (reviewed by Kouzarides, 2007, 38

Pederson and Helin, 2010, Eissenberg and Shilatifard, 2010, Greenberg, 2011, Greer and

Shi, 2012). We will briefly discuss only the histone demethylation process here.

Figure 11: Histone modifications. (a) Known post-translational modifications and the amino acid residues they modify. (b) Residues that can undergo different forms of post- translational modification. Specific modifications may inhibit subsequent modification. Histone amino acid sequence is from humans unless otherwise indicated; asterisk indicates that either the histone amino acid sequence or the modification is from S. cerevisiae. ac, acetylation; bio, biotinylation; cit, citrullination; me, methylation; su, SUMOylation. [Image and legend courtesy: Latham and Dent, 2007]

Histone methyl transferases (HMTs) and histone demethylases (HDMs) are responsible for adding and removing methyl groups on specific lysine (or arginine) residues on core histones. Regulation of gene expression by histone methylation depends on the location of the lysine residue on the histone tail and the degree of methylation

(mono, di or tri). Two classes of histone lysine demethylases have been identified to date

– the flavin dependent AO-domain containing HDMs and the Fe(II)-dependent jumonji

C-domain containing HDMs. Epigenetic regulation by histone modification has been extensively studied in vertebrate and invertebrate. In C. elegans, as in mammals, transcriptionally inactive heterochromatin is associated with H3K9me3, H3K9me2 and

H3K27me2, while transcriptionally active euchromatin is enriched in H3K4me2

(reviewed by Upadhyay and Cheng, 2011, Wenzel et al., 2011). There are 38 predicted

HMTs and 15 predicted HDMs in C. elegans. They are thought to regulate vulval cell- fate specification, life-span, embryonic and germline development and genome stability

(reviewed by Wenzel et al., 2011, Greer and Shi, 2012). Transgeneration inheritance of histone methylation marks in multicellular organisms was observed for the first time in C. elegans (Katz et al., 2009). The HDM proteins SPR-5 and LSD-1 are homologs of human

LSD1 and can demethylate mono- and di-methylated H3K4. SPR-5 has also been found to have a role in meiotic double-strand break repair (Nottke et al., 2011). No homologs of human LSD2 (KDM1B or AOF1), which can preferentially demethylate mono or di methylated H3K4, have yet been reported in C. elegans. Note however that the AMX1 amino acid sequence bears higher homology to the human-LSD2 (35-38% overall identity) than to human-LSD1 (32% overall identity). Both LSD1 and LSD2 in mammals 40 are known to demethylate the mono- and di-methylated lysine K4 in histone H3, by forming an imine intermediate using FAD as the transient electron acceptor. Neither human LSD1 nor human LSD2 can use tri-methylated lysine residues as substrates due to lack of a protonated amine (Shi et al., 2004, Upadhyay and Cheng, 2011). Figure 12 illustrates the model for demethylation by mammalian LSD1 and LSD2 and a comparative analysis of their structures.

Given that many of the enzymes, complexes, and epigenetic landscape are highly conserved between invertebrates and the vertebrates, detailed study of epigenetic process in C. elegans may help us understand the mechanisms in mammals.

Figure 12: Mammalian flavin-dependent histone demethylases LSD1 and LSD2. (a) Reaction catalyzed by LSD1 and LSD2. The sequence of the N-terminal 21-residue histone H3 peptide used as substrate is shown. R is a hydrogen atom and a methyl group for mono- and di-methylated Lysine-4, respectively. (b) Domain organization of LSD1 and LSD2. Both proteins contain a SWIRM domain and the catalytic amine oxidase domain. LSD2 contains an N-terminal zinc finger domain (Zn-CW) that is not present in LSD1. The amine oxidase like (AOL) domain of LSD2 does not include the insertion (tower) domain that in LSD1 provides the binding site for the corepressor protein CoREST. [Image and legend courtesy: Karytinos et al., 2009]

CHAPTER 2: BACKGROUND EXPERIMENTS AND HYPOTHESIS

This project is partly based on data obtained from experiments performed in the lab by former lab members (Nanda Filkin, Setu Kaushal, Jessica Rhominski, Nathan

Kuhn, Tami Coursey, and Melissa LaBonty). As discussed in chapter one, C. elegans have a number of MA-dependent behaviors and may also be affected by exogenous MAs.

Our lab investigates several aspects of the monoamine circuitry in the worm, including the genes for putative monoamine degradation enzymes – amx-1 and amx-2. I have summarized below some of the observations and inferences from behavior assays, experiments using transgenic lines and estimation of in situ MA levels, comparing differences between the wild-type (N2) and amx-1, amx-2 and amx-2;amx-1 mutants.

Most of the data have been presented in poster format or published in the Master’s thesis of Setu Kaushal (2008).

2.1: Monoamine Dependent Behavior Assays.

Two amine oxidase domain containing genes – amx-1 and amx-2 – are putative

MAO genes in C. elegans (Dr. Janet Duerr, personal communication). If they encode

MAOs, then the corresponding mutants are expected to have defects in MA-dependent behaviors. Also, these mutants are expected to show increased sensitivity to exogenous

MAs and resistance to the effects of MAOIs. The four MAs (DA, 5HT, OA and TA) found in C. elegans bind to specific receptors on neurons and muscles and affect physiological processes and behaviors like egg-laying, pharyngeal pumping (feeding), movement, etc. (reviewed in Chase and Koelle, 2007). Generally DA, OA and/or TA have been found to act in opposition to 5HT to regulate MA-dependent behaviors. 43

However, before predicting an expected behavior in the amx-1 or amx-2 mutants, the following points need to be considered. All of the behaviors discussed here depend upon multiple neurotransmitters besides MAs. Also, we do not know whether different MAs are metabolized differently in the worm (e.g. N-acetylation vs. succinylation vs. oxidative deamination). Hence, if there is decrease in a putative MAO protein in the worm, as is expected in the amx-1 or amx-2 mutants, it is not possible to predict a similar increase in the levels of all MAs. Therefore, the experiments with MA-dependent behaviors in wild- type and amx mutant worms are likely to provide a complex pattern of changes. The pattern may be better resolved by taking into account all other aspects of MA circuitry including synthesis, package, release and different types of homeostatic mechanisms involved in the maintenance of MA levels in the animal. A variety of behaviors were compared between N2 and putative nulls for amx-1(ok659) (which has a deletion in a coding exon), amx-2(ok1235) (which has a complex substitution in the coding exons), and amx-2 (ok1235);amx-1(ok659) double mutants. Table 1 below lists some of the important behaviors assayed, the known action of MA neurotransmitters on these behaviors and the experimental observations in wild-type versus the amx-1, amx-2, or the amx-2;amx-1 mutants (Kaushal, 2008).

2.1.1: Sensitivity to Exogenous DA and 5HT

DA and 5HT are important for switching between swimming and crawling behavior; exogenous DA and 5HT cause decrease in movement (Schafer and Kenyon,

1995; Schafer et al., 1996; Vidal-Gadea et al., 2011). If the amx genes encode MAOs, then the amx mutants are expected to have higher than normal levels of MAs and might 44 be hypersensitive to exogenous MAs. The short-term and long-term effects of a range of

DA and 5HT concentrations was measured by counting the number of body-bends per minute (‘thrashing’ or ‘swimming’) of well-fed young adult hermaphrodite worms in DA or 5HT solutions.

As observed by Kaushal in her thesis (Kaushal, 2008), wild-type, amx-1, and amx-2 worms moved at the same rate in absence of exogenous MAs and movement decreased with increasing concentrations of DA and 5HT. The amx-2 mutants were found to be slightly but significantly slower than wild-type in 60 mM and 90 mM exogenous

DA or 4 and 8.5 mM 5HT, compared to N2 and amx-1after 2 min acclimatization. amx-2 mutants immobilized significantly faster (15 min) than N2 (25 min) in 8.5 mm 5HT.

Compared to N2 and amx-2, the amx-1 mutants were slightly but significantly resistant to the long-term paralyzing effects of 15 mM DA (Kaushal, 2008).

Table 1: Monoamine Dependent Behavior Assays (done by Setu Kaushal; Kaushal, 2008)

BEHAVIOR MONOAMINE OBSERVED ACTION (done by Setu Kaushal; Kaushal, 2008) (known) 1. Movement DA decreases movement amx-2 worms moved significantly slower in DA than N2 at 60 and 90 mM DA solution 2. Movement 5HT decreases movement amx-2 worms moved significantly slower in 5HT than N2 at 4 and 8.5 mM 5HT solution 3. Immobiliza DA causes paralysis amx-1 worms significantly resistant to 15 tion in 15 mM DA mM DA solution 4. Immobiliza 5HT causes paralysis amx-2 worms immobilized significantly tion in 8.5 sooner than N2 and amx-1 mM 5HT solution 5. Movement DA decreases movement amx-2 and amx-2;amx-1 mutant worms in food (well-fed worms), 5HT moved significantly slower than N2 and decreases movement amx-1 worms (starved worms) 6. Movement DA decreases movement amx-2 worms moved significantly faster in sephadex than N2 and amx-1 bead suspension 7. Pharyngeal 5HT increases while DA, amx-2 worms had significantly lower pumping TA and OA decreases pharyngeal pumping rates than N2 and pharyngeal pumping rate amx-1 8. Number of 5HT increases while DA, amx-2 and amx-2;amx-1 mutant worms eggs laid TA and OA decreases egg laid significantly lesser number of eggs laying than N2 and amx-1 worms 9. Number of 5HT decreases while DA, amx-2 and amx-2;amx-1 mutant worms embryos in TA and OA increases had significantly lower number of eggs utero number of embryos in utero in utero than N2 and amx-1 worms 10. Effect of DA, 5HT decrease MAOIs decreased thrashing rate in N2; MAOIs on movement amx-2 and amx-2;amx-1 mutant worms thrashing were significantly resistant to selegiline; amx-1 and amx-2;amx-1 worms were significantly resistant to tranylcypromine. 46

2.1.2: MA-dependent Movement

Food provides mechanosensory and chemosensory stimuli while sephadex beads

(20-50 um) provide only a mechanosensory stimulus (Sawin et al., 2000). DA mediates a decrease in movement in food in well-fed worms (basal slowing response) and 5HT mediates a decrease in movement in food in starved worms (enhanced slowing response).

TA and OA can independently inhibit 5HT dependent aversive responses (Wragg et al.,

2007), while TA (and OA at higher concentrations) is known to affect head oscillations, reversal rates, and length of reversals during escape (Pirri et al., 2009). In the case of movement in a sephadex bead suspension, a basal slowing response is mediated by DA in worms (Sawin et al., 2000). 5HT appears to have no effect and the effect of TA or OA on movement under this type of stimulus is unknown. An increase in the concentration of all

MAs may be expected in amx mutants. Note that the cat-1 mutant, which has defective packaging of DA and 5HT and a consequent decrease in the release and levels of DA and

5HT, was found to move faster than the wild-type counterpart in food as well as in sephadex beads (Janet Duerr, personal communication).

The movement in food assay compared the number of body-bends per minute of well-fed young adult hermaphrodites (N2 or mutants) while moving in an E. coli lawn on an NGM (nematode growth media) plate. cat-1 mutants (with decreased release of DA and 5HT) have normal movement off food but have higher number of body-bends per minute than wild-type worms in food. If amx-2 mutant worms have higher level of DA and 5HT, they would be expected to exhibit slower movement in food. The amx-2 and 47 the double mutants moved normally off food, and showed slightly but significantly decreased movement in food compared to N2 (Kaushal, 2008). However the amx-2 mutants moved significantly faster than N2 in sephadex bead suspensions, contrary to expectation. Thus these observations were inconsistent with the simplest hypothesis of increase in MA levels in the amx-2 or amx-1 mutants.

2.1.3: Pharyngeal Pumping

The rate of pharyngeal pumping (feeding) is increased by endogenous and exogenous 5HT and decreased by OA and TA, while DA does not appear to directly regulate this behavior (reviewed in Chase and Koelle, 2007). Pharyngeal pumping per minute in well-fed young adult hermaphrodite worms was measured on a uniform E. coli lawn on an NGM plate. In this experiment, amx-2 mutants were observed to have a decreased pumping rate compared to N2 (Kaushal, 2008), consistent with higher levels of

OA and TA relative to 5HT.

2.1.4: Egg-laying Behavior and Embryos in utero

C. elegans lays eggs (fertilized embryos) in short 1-2 minute bursts separated by about 20 minute intervals (Waggoner et al., 1998). Exogenous 5HT stimulates while exogenous OA TA, and DA inhibit egg-laying (Schafer and Kenyon, 1995; Chase and

Koelle, 2007). The total number of eggs laid in 2.5 hours was counted, as well as the total number of embryos in utero in a fully mature adult (48 hr post L4 larval stage). The amx-

2 and amx-2;amx-1 mutants laid significantly fewer eggs and also had significantly fewer embryos in utero (Kaushal, 2008), which is consistent with an increase in OA, TA and

DA relative to 5HT in those mutants. 48

2.1.5: Effect of MAOIs

If amx-1 and amx-2 encode MAOs, then we predict the mutants will be at least partially resistant to the effects of MAOIs. However, note that MAOIs, which often are

MA analogues, can have targets besides MAOs, such as specific MA receptors in vertebrates. Thrashing of worms in liquid was assayed in the presence of the MAOIs tranylcypromine (non-specific irreversible MAO-A/B inhibitor), selegiline (MAO-B specific reversible inhibitor) or clorgyline (MAO-A specific reversible inhibitor).

The amx mutants showed altered sensitivity to some of the MAOIs at specific concentrations. amx-1 mutants were significantly but partially resistant to tranylcypromine but had normal responses to selegiline or clorgyline. amx-2 mutants were found to be very resistant to selegiline but not significantly resistant to tranylcypromine (Table 1) or clorgyline (data not shown). The amx-2;amx-1 mutant was partially resistant to tranylcypromine and very resistant to selegiline (Table 1) but not to clorgyline (data not shown) (Kaushal, 2008). Note that the sensitivity of the N2 and amx mutant worms to other concentrations of MAOIs was not examined. The variation in sensitivity to different classes of MAOIs suggests that there could be varied mechanisms of action of tranylcypromine vs. selegiline on AMX1 vs. AMX2.

2.2: amx Transgenic Studies

In our lab, the transcriptional reporter Pamx-1::GFP DNA construct was made by a PCR-fusion method (Hobert, 2002). It included 1.8 kb of nucleotide sequence upstream of the start of amx-1 coding region (the putative promoter) followed by the coding region of the green fluorescence protein (GFP) gene (Kaushal, 2008). GFP expression was 49 observed in almost all cells in the early embryo. In the adult, GFP expression was localized in ~30 cells in the head and tail regions. Using immunocytochemical methods and staining with the lipophilic dye DiI, some of these cells were identified. These include one pair of ASJ sensory neurons (part of the amphid; contain neuropeptide NLP-

3), three pairs of IL2 neurons (contain acetylcholine), and one pair of PHB sensory neurons (part of the phasmid; contain 5HT and neuropeptide NLP-1). Altogether almost twenty cholinergic neurons (including the six IL2 neurons) in the head region showed

GFP expression. Figure 19 below illustrates some of the previous results obtained from the Pamx-1::GFP transcriptional reporter expression in the transgenic worms.

Figure 13: Pamx-1::GFP (transcriptional reporter) expression in C. elegans. (a) Embryo - left - GFP; middle – DAPI staining of DNA in nuclei; right – merge of GFP and DAPI signal show that most embryonic cells have GFP expression. (b) Left – GFP expression in head region of an adult hermaphrodite; right – merge of DAPI and GFP showing that only a few cells in the head region express GFP. (c) and (d) are individual confocal sections of a nematode with DiI staining of cell membranes (red) and GFP expression. (c) Lateral view to show the overlap between GFP and DiI in a pair of IL2 neurons (arrow). GFP is present predominantly in the cell body (nucleus and cytosol) and DiI stains the plasma membrane of some head and tail sensory neurons. (d) A single section (ventral view) to show overlap between GFP and DiI in some amphid somas. A = anterior end and P = posterior end of the nematode. Images in (a) and (b) are 50 um wide while images in (c) and (d) are 120 um wide. [Image courtesy: Janet Duerr, personal communication; Kaushal, 2008]

The ASJ and IL2 neurons are sensory neurons and control entry to and exit from as well as characteristic behaviors in the dauer stage (an alternative larval stage that can withstand harsh environmental conditions) (reviewed in WormAtlas, 2014). The PHB neurons are chemosensory and important for regulating aversive behavior in the worm 51

(reviewed in WormAtlas, 2014). Thus the amx-1 gene seems to have a widespread role in the embryonic stage and a more specialized function in a subset of sensory neurons in the adult worm. Unfortunately, Setu Kaushal was unable to generate stable lines using the promoter for amx-2, so we are uncertain of its normal pattern of expression.

2.3: In situ Monoamine Levels.

If amx-1 and amx-2 encode MAOs, then the in situ levels of MAs are predicted to be higher in the amx mutants than in N2. Due to lack of available antibodies and appropriate techniques, in situ levels of OA and TA were not analyzed. For characterizing the DA and 5HT levels in specific cells, anti-serotonin antibody staining and glyoxylic acid induced fluorescence of DA and 5HT (Duerr et al., 1999) were used.

Using glyoxylic acid induced fluorescence (Figure 14), a higher but not statistically significant level of DA and 5HT were observed in amx-1 and amx-2 and amx-2;amx-1 mutants compared to N2. Similar results were obtained with antibody staining of 5HT.

As with the behavioral results, the staining results do not give unequivocal support for neuronal MAO activity of either AMX1 or AMX2 protein.

Figure 14: In situ MA levels. DA and 5HT visualized by glyoxylic acid induced fluorescence in N2 and amx mutant worms. The fluorescence signal indicates localization and approximate levels of DA (green) in the nerve ring and 5HT (red) in the NSM neurons in the head of the nematode. A normal nematode and three mutant strains are shown. Anterior is up, each image is 200 microns wide. [Image and legend courtesy: Dr. Janet Duerr]

2.4: Hypothesis.

Bioinformatics analyses of amx-1 and amx-2 genes predict amx-1 to encode a histone demethylase and amx-2 to encode a monoamine oxidase (Wormbase, 2014). Both are predicted to be flavin dependent oxidases. The behavior and staining results were consistent with varied effects on MA metabolism, but the results were not conclusive. To understand the functions of these proteins, direct biochemical analysis was required. That is the focus of this thesis. Based on the information discussed above, I hypothesized the following. 53

1. The amx-2 gene encodes a flavin dependent monoamine oxidase homolog in C.

elegans which may act to degrade some or all of the MAs found in the worm –

DA, 5HT, OA and TA.

2. The amx-1 gene encodes a homolog of the lysine specific histone demethylase

(reasoning discussed further below).

Accordingly, my project was designed for cloning, expression and purification of AMX1 and AMX2 in a heterologous expression system in adequate amounts and purity so that the proteins could be used for biochemical assays aimed at establishing their roles in C. elegans.

CHAPTER 3: MATERIALS AND METHODS

3.1: Bacterial Plasmid Construction

Two types of bacterial expression constructs were made – one using the two-step

Gateway cloning vectors pENTR/SD/D-TOPO and pET-DEST42 (Life Technologies,

Carlsbad, CA), and another using the pET GFP LIC cloning vector 2GFP-T (Addgene plasmid 29716; constructed by Dr. Scott Gradia).

The cDNAs for amx-1 and amx-2 genes were chosen based on the sequence information on WormBase (2011). WormBase includes several expressed sequence tags

(ESTs) of publically available cDNAs. The cDNAs for amx-1 (yk1390d04 and yk1627f09) and amx-2 (yk1054h04, yk1056g04, yk1326e11, yk285d9, and yk334g2), which were predicted to cover the full length of the genes, were chosen. They were a kind gift from Dr. Yuji Kohara, National Institute of Genetics, Japan. The cDNAs were obtained either as agar stabs of E. coli strain DH5, containing constructs in the pME18S-FL3 vector or as 10 uL of  ZAPII phage suspensions. The presence of amx-1 and amx-2 cDNAs in the constructs (yk plasmids) were confirmed first by PCR with outer (on vector backbone) and inner (on the inserts) primers (see Table 2 for sequences) and then by Sanger sequencing of the complete insert lengths (of yk1390d04, yk1627f09, yk1054h04, yk1056g04, and yk1326e11) at the Ohio University Genomics Facility

(OUGF; using an Agilent2100 Bioanalyzer). The sequences were analyzed by aligning against the respective gene sequences in Wormbase and against each other, using the nucleotide BLAST program (NCBI). For cloning, specific amx-1 and amx-2 inserts were amplified from the respective cDNAs, from the start to stop codons, using high fidelity 55 proof-reading Platinum Taq DNA Polymerase (Life Technologies, Carlsbad, CA).

Plasmid preparations were done with Promega Wizard Plus Miniprep kit (Promega,

Madison, WI) or MO-BIO Ultra Clean Miniprep Kit (MO-BIO, Carlsbad, CA), following manufacturers’ protocols.

3.1.1: Gateway-amx plasmids

The Gateway cloning technique uses the site specific recombination properties of

 bacteriophage to create plasmids (reviewed by Katzen, 2007). First, an Entry vector is prepared containing the gene-of-interest (amplified as a blunt end product) correctly positioned between the attL1 and attL2 sites on the vector backbone. From there it can be moved in vitro to the attR1 and attR2 sites of multiple Gateway expression (Destination) vectors, using the manufacturer supplied integrase, integration host factor and excisionase

(LR Clonase; Life Technologies, Carlsbad, CA). The ccdB (toxin) gene serves as the negative control, eliminating the propagation of the byproduct plasmid formed by the LR

Clonase reaction.

Gateway cloning of the confirmed cDNAs for amx-1 (yk1390d04) and amx-2

(yk1054h04 referred to as amx-2L and yk1326e11 referred to as amx-2S), was done following the manufacturer’s protocol, with minor modifications (Life Technologies,

Carlsbad, CA). The entry vector was pENTR/SD/D-TOPO (2601 bps; kanamycin resistant; phage T7 expression system, IPTG inducible). Briefly, the yk-plasmids

(ampicillin resistant) were used as templates and 2475 bps of amx-1, 2045 bps of amx-2S or 2145 bps of amx-2L were amplified by PCR, using primers 280/281 for amx-1;

282/286 for amx-2S; and 282/284 for amx-2L (see Table 2 for sequences). PCR was 56 performed using a Bio Rad DNA Engine thermal cycler, set at 95C for 5 minutes, followed by 35 cycles of 92C, 58C, 72C for 30 sec, 60 sec, 4 min respectively, and a final elongation step of 7 minutes at 72C. The PCR products were gel purified using the

Novagen Spin Prep Gel DNA kit (Novagen, Darmstdt, Germany), following manufacturer’s protocol and the concentration and quality were checked by running the

DNA on a 1% agarose gel. Purified PCR product was then mixed with the TOPO entry vector at a 1:1 molar ratio and incubated at room temperature (RT) for 30 min, followed by 4C for 30 min. Transformation (heat-shock at 42C, 30 sec) of 50 uL One Shot

Chemically Competent E. coli cells (Life Technologies, Carlsbad, CA) was done using 2 uL of the TOPO cloning reaction. Cells were then grown at 37C, 220 rpm for 60 min in

250 uL SOC media (Life Technologies, Carlsbad, CA), and then 50 or 100 uL was plated on LB (Luria Broth)-agar (1.5%) plates containing 50 ug/mL kanamycin, and incubated at 37C for 14-16 hr. The next day, 8-10 isolated colonies were chosen, streaked in duplicate on separate LB-agar plates containing either 50 ug/mL kanamycin or 50 ug/mL ampicillin, and incubated at 37C for 14-16 hr. This step was added in order to check for and eliminate any carryover of ampicillin resistant yk-plasmids. The ampicillin sensitive and kanamycin resistant colonies were chosen and grown in 3 mL LB-broth containing

50 ug/mL kanamycin at 37C, 220 rpm for 14-16 hr. A plasmid prep was done, followed by restriction enzyme analysis using NotI and Eag1 restriction enzymes (New England

Biolabs, Ipswich, MA) and PCR analysis using amx-1 (280/281) and amx-2 (282/284 and

282/286) specific primers. The positive entry clones were confirmed by Sanger sequencing at OUGF. 57

The destination (expression) vector was generated using the LR Clonase enzyme mix (Life Technologies, Carlsbad, CA), following the manufacturer’s instructions with minor modifications. The Gateway destination vector was pET-DEST42 Gateway vector

(for E. coli; 7440 bps; ampicillin resistant, chloramphenicol resistant without insert). The vector was designed to express a 6X-Histidine tag at the C-terminal of the insert. The entry clone and destination vector at a 1:1 molar ratio were mixed with 2 uL LR Clonase mix (total mixed volume 5-6 uL) and incubated at 25C for 60 min. The reaction was stopped by adding 1 uL Proteinase-K and incubating at 37C for 10 min. Transformation

(heat-shock at 42C, 30 sec) of 50 uL One Shot Chemically Competent E. coli cells (Life

Technologies), was done using 2 uL of the LR Clonase reaction. Cells were then grown at 37C, 220 rpm for 60 min with 250 uL SOC media; then 50 or 100 uL were plated on

LB-agar plates containing 50 ug/mL ampicillin and incubated at 37C for 14-16 hr. The next day, 8-10 isolated colonies were chosen, streaked in triplicate on LB-agar plates containing 50 ug/mL kanamycin or 35 ug/mL chloramphenicol or 50 ug/mL ampicillin, and incubated at 37C for 14-16 hr. This step was added in order to check for and eliminate any carryover of kanamycin resistant entry-plasmid and/or chloramphenicol resistant empty destination vector. The kanamycin and chloramphenicol sensitive and ampicillin resistant colonies were chosen and grown in 3 mL LB broth containing 50 ug/mL ampicillin at 37C / 220 rpm / 14-16 hr. Finally, plasmid preparations were done, followed by restriction enzyme (RE) analysis using BglII and EagI restriction enzymes

(New England Biolabs, Ipswich, MA) and PCR analysis using amx-1 (280/281) and amx- 58

2 (282/284 and 282/286) specific primers. The positive clones were confirmed by Sanger sequencing at OUGF.

3.1.2: 2GFP-T-amx plasmids

The ligase independent cloning (LIC) method was designed to eliminate the use of a ligase in the cloning process and instead exploits the 3’  5’ exonuclease activity of the T4 DNA polymerase to create complementary overhangs between the vector and the insert (Li and Elledge, 2007). Linearized vector is supplied with dGTP, while the amplified insert is supplied with dCTP as the only nucleotides. The exonuclease processing of the T4 DNA Pol is limited to the first complementary C residue for the vector and at the first complementary G residue for the insert, balancing the polymerization and exonuclease activities of the enzyme. Thus the sticky ends created for the vector and insert ligate spontaneously without the requirement of any DNA ligase.

Ligase independent cloning of the confirmed cDNAs for amx-1 (yk1390d04) and amx-2 (yk1054h04 referred to as amx-2L and yk1326e11 referred to as amx-2S), were done as described below. The expression vector was Addgene plasmid 29716 (2GFP-T;

5518 bp; ampicillin resistant; modified pET vector backbone; phage T7 expression system, IPTG inducible) from Dr. Scott Gradia (University of California, Berkeley). It was designed to have a TEV (Tobacco Etch Virus protease)-cleavable N-terminal

6xHistidine-GFP tag. The GFP can improve the solubility of expressed protein as well as serve as an easily observable reporter of expression. The Gateway entry-amx plasmids

(kanamycin resistant) were used as templates and the gene of interest was amplified by

PCR, using specially designed primers containing LIC V1 cleavable tags at their 5’-ends. 59

The primers 320/322 for amx-1; 321/323 for amx-2L; and 327/323 for amx-2S (see Table

2 for sequences) were used (same program as in previous section). The PCR products were purified using the Novagen Spin Prep PCR Cleanup kits (Novagen, Darmstadt,

Germany) following manufacturers’ protocol and concentration and quality was checked by running on an agarose gel. About 20-25 ug of p29761 vector was digested with SspI enzyme (New England Biolabs, Ipswich, MA) in NEB buffer 2 at 37C for 2 hr. SspI was inactivated by heating at 65C for 30 min and digestion was checked by running on a 1% agarose gel against the uncut vector. The insert (PCR product) and the digested vector were treated with T4 DNA Pol and dCTP and dGTP respectively (35 uL DNA + 5uL

10X T4DNA Pol reaction buffer + 2 uL T4DNA Pol + 1.5 uL 100 mM dGTP or dCTP +

0.5 uL 1 M DTT + 6 uL nuclease free water) at 25C for 45 min. Reactions were stopped by incubating at 75C for 20 min. Both reactions were cleaned by Novagen Spin Prep

PCR Cleanup kits (Novagen, Darmstadt, Germany) following manufacturers’ protocol, and the cleaned products were checked on a 1% agarose gel. Vector and insert were mixed in a 1:1 molar ratio in 1X TE buffer, and incubated at 25C for 30 min. About 4 uL of this mix was used to transform (heat-shock at 42C, 30 sec) 100 uL chemically competent DH5 cells, which were grown at 37C, 220 rpm for 60 min in 750 uL SOC media. Then 50 or 200 uL were plated on LB-agar plates containing 50 ug/mL ampicillin and incubated at 37C for 14-16 hr. The next day, 8 isolated colonies (for each construct) were chosen, streaked in duplicate onto LB-agar plates containing either 50 ug/mL kanamycin or 50 ug/mL ampicillin and incubated at 37C for 14-16 hr. This step was added in order to check for and eliminate any carryover of kanamycin resistant template 60 plasmid. Kanamycin sensitive and ampicillin resistant colonies were checked by colony

PCR (half colony boiled with 10 uL water at 100 C for 10 min, and 5 uL used as template) using 340/341 and 343/341 primer pairs (see Table 2 for primer sequences).

The PCR positive clones were grown in 3 mL LB-broth containing 50 ug/mL ampicillin at 37C, 220 rpm, 16 hr. Plasmid preps were done, followed by restriction enzyme (RE) analysis using EcoR1 restriction enzyme (New England Biolabs, Ipswich, MA) and PCR analysis using amx-1 (280/281) and amx-2 (282/284 and 282/286 ) specific primers. The positive clones were confirmed by Sanger sequencing at OUGF.

3.2: Yeast Plasmid Construction

The methylotropic yeast (Pichia pastoris) expression system has several advantages over bacterial expression systems for eukaryotic protein expression. This yeast system is superior in protein processing, folding and posttranslational modifications, and reportedly has higher eukaryotic protein expression levels than E. coli and S. cerevisiae (reviewed in Cereghino and Craig, 2000; Daly and Hearn, 2005). P. pastoris is a methylotropic yeast, capable of metabolizing methanol as the sole carbon source using the alcohol oxidase (AOX) enzyme. Heterologous protein expression in this system is driven by the promoter of the alcohol oxidase gene (Paox1). The pPIC3.5 vector (7751 bps; HIS4+; Ampicillin resistant; for intracellular expression) was chosen as the expression vector. In histidine-deficient growth media, it allows HIS4 (histidinol dehydrogenase) based selection of the transformants in his-4 mutant Pichia expression strains (like KM71). It also allows insertion of the gene-of-interest in either the AOX1 or

HIS4 loci of the Pichia genome. Insertion is by homologous recombination of the 61 linearized plasmid. The expression of heterologous proteins in this vector requires an initiating ATG codon in a Kozak consensus sequence for proper translation, included in the forward primers used for cloning. The genes of interest were inserted at the multiple cloning site between 5’-BamH1 (at pPIC3.5 bp 938) and 3’-AvrII (at pPIC3.5 bp 956) restriction sites, using the corresponding restriction sequences incorporated at the respective ends in the primers. The primers were designed to have an N-terminal 6X-

Histidine tag and a C-terminal Myc-tag for analysis and purification purposes. The cloning was done using the Pichia Expression Kit (Invitrogen, Carlsbad, CA), following manufacturers’ protocol.

The Gateway entry-amx plasmids (kanamycin resistant) were used as templates and the gene of interest was amplified by PCR, using primers pairs 315/317 for amx-1;

328/329 for amx-2L; and 316/326 for amx-2S (see Table 2 for primer sequences). PCR was performed using a Bio Rad DNA Engine thermal cycler, as follows: 95C for 5 min, followed by 35 cycles of 92C, 70C, 72C for 30 sec, 60 sec, 4 min respectively and a final

7 min incubation at 72C. The PCR products were purified using the Novagen Spin Prep

PCR Cleanup kit (Novagen, Darmstadt, Germany) following manufacturers’ protocol; concentration and quality was checked on a 1% agarose gel. 25 ug of pPIC3.5 vector was double digested with AvrII and BamHI enzymes (sequentially; with NEB buffer 4 and

1X BSA) and the PCR products were digested with BssHI, AvrII and BamHI (New

England Biolabs, Ipswich, MA) with NEB buffer 4 and 1X BSA, for a total of 4 hr at

37C. (BssHI was added to linearize the entry-amx plasmid template, since the size of its supercoiled band was same as the PCR products. Also, BssHI had no common site in the 62 amplicons.) The digests were gel purified using Novagen Spin Prep Gel DNA kit

(Novagen, Darmstadt, Germany) using manufacturers’ protocol, and concentration and quality were checked by running on a 1% agarose gel. The ligation reaction was set up using T4 DNA Ligase (New England Biolabs, Ipswich, MA) with 1:1 molar ratio of insert and vector, at 16C for 12-14 hours. About 3 uL of the ligation reaction was used to transform (heat-shock at 42C, 30 sec) 100 uL chemically competent DH5 cells, and grown at 37 C, 220 rpm for 60 min in 750 uL SOC media. Subsequently 50 or 200 uL were plated on LB-agar plates containing 50 ug/mL ampicillin and incubated at 37C for

14-16 hr. Isolated colonies were chosen and streaked in duplicate on LB-agar plates containing either 50 ug/mL kanamycin or 50 ug/mL ampicillin and incubated at 37C for

14-16 hr. This step was added in order to check and eliminate any carryover of kanamycin resistant template plasmid. Kanamycin sensitive and ampicillin resistant colonies were checked by colony PCR (half colony boiled with 10 uL water at 100C for

10 min, and 5 uL used as template) using 338/208 and 204/339 primer pairs (see Table 2 for primer sequences). All PCR positive colonies were grown in 3 mL LB-broth containing 50 ug/mL ampicillin at 37C, 220 rpm for 14-16 hr. Minipreps were done, followed by restriction enzyme analysis with BamHI, AvrII, NotI, StuI and SacI restriction enzymes (New England Biolabs, Ipswich, MA) and PCR analysis with amx-1

(280/105) and amx-2 (338/207 and 275/339; see Table 2 for primer sequences) specific primers. The positive clones were completely sequenced at OUGF for further confirmation. 63

3.3: Protein Expression and Purification from Bacterial Constructs

The induction system used for expression was the IPTG (Isopropyl--D-1- thiogalactopyranoside) inducible lac operon system in E. coli (Oehler et al., 1990). In the engineered expression strains, the -phage T7 Polymerase gene (expressed by the  DE3 lysogen) is placed under the lac promoter. IPTG, a lactose analog, drives expression of the T7 Polymerase, which in turn binds to the T7 promoter region in the expression plasmid (amx-pDEST42) and drives high level expression of the gene of interest.

Expression optimization was done using different E. coli expression strains

[BL21(DE3), BLR(DE3)pLysS, BLR(DE3)CodonPlus, and RosettaGami pLysS – all were kind gifts from Dr. Tomohiko Sugiyama, Ohio University, Athens, Ohio] and growth media [Luria Bertani (LB) vs. Terrific broth (TB)]. Induction conditions included varied inducer concentrations [0, 100, 200, 500, 1000, and 2000 mM IPTG], induction points [OD600 = 0.3, 0.6, and 1.2], post-induction temperatures [25C, 30C, 37C], and post-induction growth times [1, 2, and 6 hr at 30C and 37C; 6, 12, and 24 hr at 25C]. The effects of absence or presence of antibiotic (50 ug/mL ampicillin) during induction were also analyzed. Each expression condition is discussed in Table 3 in the Appendix.

For protein expression, both Gateway-amx and 2GFP-T-amx constructs were freshly transformed into competent E. coli cells using heat-shock (Froger and Hall,

2007). For transformation, 2-3 uL of plasmids were added to 100 uL of chemically competent expression strains and kept on ice for 30 min. Heat shock was given at 42C

(water bath) for 35-40 sec, followed by incubation for 2-3 min on ice. Cells were recovered by adding 700 uL sterile LB media and grown at 37C, 220 rpm for 60 min. 50- 64

200 uL cells were plated on LB-Amp (50 ug/mL) plates and incubated at 37C for 16 hr.

The next day, one isolated colony was picked and inoculated in 2 mL LB containing 50 ug/mL ampicillin and grown at 37C, 220 rpm for 12-14 hr to make a starter culture.

Starter culture was used as inoculum at a 1:500 – 1:1000 ratio (1 mL overnight starter culture for 500 mL or 1000 mL fresh Terrific broth). Cultures were grown at 37C until the induction point of OD600 = 0.6 was reached (growth was monitored every 30 min using a spectrophotometer). Induction was with 1 mM IPTG and growth at 25C, 220 rpm for 12 hr. Cells were harvested by centrifuging at 5000 x g , 4C, 10 min. Cell pellets (8 g/L) were stored at -80C if not used immediately. Cell pellets were resuspended in lysis buffer [50 mM sodium phosphate pH 7.4 + 5% glycerol + 1 mM PMSF + 1X HALT

Protease Inhibitor cocktail (Thermo Scientific, Rockford, IL)] by vortexing and 2 cycles of freeze-thaw [at -80C for 10 min and room temperature (25C) for 5 min] followed by 4 cycles of sonication (1 minute @ 30% duty cycle, output = 3, on ice) using a Branson

250 sonicator. Unlysed cells and cell debris were removed by centrifuging at 5000 x g,

4C, 10 min. Soluble and insoluble phases were separated by subsequent centrifugation at

25,000 x g, 4C, and 60 min. The supernatant contained the soluble proteins, while the insoluble pellet fraction contained hydrophobic proteins, membrane proteins and inclusion body proteins. The pellet fraction was resuspended in 10 mM CHAPS in 50 mM sodium phosphate pH 7.4 buffer. Both fractions were analyzed separately.

The AMX-1 and AMX-2 (L and S) proteins have either a C-terminal 6X-Histidine tag (in the Gateway constructs) or an N-terminal 6X-Histidine tag (in the 2GFP-T vector constructs), which could be used to bind positively charged metal ions (like Ni2+ or Co2+), 65 attached via a ligand to an immobilized matrix (like sepharose or agarose) in a column. A

Ni-NTA resin (Qiagen, Venlo, Limburg, Netherlands) was used in these experiments.

Following lysis of the cells, the soluble and insoluble phases were separately incubated with 0.5 mL Ni-NTA resin, washed sequentially with >10 column volumes (CV) of 10 mM imidazole then >10 CV of 30 mM imidazole (until no further protein eluted, as measured by the Bradford assay). Protein was eluted with 3-4 CV of 200 mM imidazole.

The eluateswere concentrated 10-fold with 30 kDa Centriprep concentrators following manufacturers’ protocols (EMD Millipore, Darmstadt, Germany) and dialyzed overnight against storage buffer (50 mM sodium phosphate pH 7.4 + 20% glycerol) using 10

MWCO 16 mm Snakeskin dialysis tubing (Thermo Scientific, Rockford, IL).

3.4: Protein Expression and Purification from Yeast Constructs

The strain chosen for heterologous expression in P. pastoris was KM71 (genotype: Muts,

Arg+, His-; Invitrogen, Carlsbad, CA), where the wild type ARG4 gene (2kb) replaces codons 16-227 of the AOX1 gene. This AOX1::ARG4 construct has a functional argininosuccinate lyase gene, allowing growth even in absence of arginine in the medium

(unlike the parent strain). Due to disruption of the AOX1 gene, the strain is Muts

(methanol utilization slow), meaning it can use methanol as the carbon source only via the AOX2 gene product. This lengthens the doubling time in methanol (from 2 hours to

18 hours), so no screening for Mut+ or Muts is necessary following transformation and growth in methanol. The KM71 strain is His- while the expression vectors have a functional HIS4 gene which allows successfully transformed KM71 to grow on minimal media without histidine supplementation. 66

Prior to insertion, the plasmids were linearized at specific sites (StuI for amx-1 and SacI for amx-2L and amx-2S) to allow integration into the Pichia genome by homologous recombination. Gene insertion events in KM71 (after Sac1 digestion of the construct) could arise from a single crossover event between the AOX1::ARG4 locus and any three of the AOX1 regions on the vector – the AOX1 promoter, the AOX1 transcription termination region or sequences further downstream of AOX1, leading to insertion of one or more copies of the vector upstream or downstream of the AOX1 locus.

Multiple gene insertion events occur spontaneously at a frequency of 1-10% of the single insertion events (Pichia Expression Kit manual; Invitrogen, Carlsbad, CA). The HIS4 locus on KM71 can also be used for gene insertion (by StuI digestion) in the HIS4 locus of the yeast chromosome.

The constructs and the parent vector (negative control for expression) were linearized with the respective enzymes and gel purified using Novagen Spin Prep Gel

DNA kit (Novagen, Darmstadt, Germany) and stored at -30C at a concentration of 50-80 ug/mL in 2 mg/mL denatured salmon sperm DNA (as carrier DNA). Transformation was done using the PEG-1000 method, as described in the manufacturers’ protocol

(Invitrogen, Life Technologies, Carlsbad, CA). First, KM71 (from agar stab) was streaked on a YPD-agar (1% yeast extract + 2% peptone + 2% dextrose + 2% agar) plate and incubated at 30C for two days. About 10 mL of YPD culture media was inoculated with a single colony from the plate and the culture was grown overnight at 30C in a 220 rpm shaker incubator. An aliquot of the overnight culture was used to inoculate a 100 mL

YPD culture which was grown to an OD600 of 0.5-0.8. The cells were harvested by 67 centrifugation at 3000 x g at RT and washed once with 50 mL buffer A (1M sorbitol + 10 mM bicine pH 8.35 + 3% ethylene glycol). The cells were resuspended in 4 mL of buffer

A and distributed in 200 uL aliquots in sterile 1.5 mL microcentrifuge tubes. Next 11 uL

DMSO was added, and tubes were vortexed and flash frozen in dry ice and used immediately or stored at -80C. About 20 uL of construct (~1 ug) plus carrier DNA (40 ug), was added to a frozen tube of competent cells and incubated at 37C (water bath) for

5 min. The samples were mixed once or twice (by tapping the tube) during this period.

The tubes were removed from the water bath and 1.5 mL of buffer B (40% PEG1000 +

0.2M bicine pH 8.35) was added and mixed thoroughly by pipetting twice. Tubes were incubated in a 30C water bath for 1 hour. Tubes were then centrifuged at 2000 x g for 10 minutes at room temperature (RT); supernatant was decanted and cells were resuspended in 1.5 mL buffer C (0.15 M NaCl + 10 mM bicine pH 8.35). The samples were centrifuged a second time and gently resuspended in 0.2 mL of buffer C. The contents of each tube were plated on RDB-agar (1 M sorbitol + 2% dextrose + 1.34% yeast nitrogen base + 4 × 10-5% biotin + 5 x 10-3% amino acids + 2% agar) plates and incubated at 30C for 4-5 days until colonies appeared. To confirm the presence of amx genes in the transformants, genomic DNA extraction from the colonies was done using zymolyase as described in the manufacturer’s protocol and/or by using the LiOAc-1% SDS (as described in Looke et al., 2011).

Protein expression and purification was performed as described in Wand and

Edmondson (2010), with some modifications. One isolated colony was picked and inoculated in 1L BMGY-media (buffered glycerol complex media - 1% yeast extract + 68

2% peptone + 100 mM potassium phosphate pH 6.0 + 1.34% yeast nitrogen base + 4 ×

10-5% biotin + 1% glycerol) and grown in a 4L flask at 30C, 220 rpm for 20-24 hr. The yeast culture was harvested by centrifuging at 1500 x g for 10 min at 4C. The pellets were resuspended in 100 mL of BMMY-media (buffered methanol-complex media - 1% yeast extract + 2% peptone + 100 mM potassium phosphate, pH 6.0 + 1.34% yeast nitrogen base + 4 × 10-5% biotin + 0.5% methanol) and grown in a 500 mL baffled flask at 25C, 220 rpm for 120 hr with supplementation with 1% methanol every 24 hr. [Note that before large scale expression, a brief expression optimization was done for each yeast construct using 0.5%, 0.75% or 1% methanol supplementation, growth at 30C or

25C, growth +/- 2.5% (v/v) DMSO in final growth media, and growth +/- 1 mM ligand

(benzylamine), as described in Andre et al., 2006]. At the end of the induction period, cells were harvested by centrifuging at 1500 x g for 10 min at 4C. Cell pellets were washed with lysis buffer [50 mM sodium phosphate buffer pH 8.0 (for AMX1) or pH 7.4

(for AMX2L or AMX2S) + 5% glycerol + 1 mM PMSF + 1X HALT protease inhibitor cocktail (Thermo Scientific, Rockford, IL)] and stored at -80C if not used immediately.

Cell pellets were resuspended in lysis buffer [50 mm Na-phosphate pH 8.0 or pH 7.4 +

5% glycerol + 1 mM PMSF + 1X HALT Protease Inhibitor cocktail (Thermo Scientific,

Rockford, IL)] and lysed by vortexing at high speed with an equal volume of 500 micron acid-washed glass beads (Sigma Aldrich, St. Louis, MO) in 8 cycles of 1 min vortexing then 4 min chilling on ice. Glass beads, unlysed cells and cell debris were removed by centrifuging at 1000 x g for 10 min at 4C. For AMX2 (L and S) purification, a 90 min incubation with 10 mM CHAPS (in lysis buffer) with gentle stirring at 4C was 69 performed, to extract membrane bound fractions into solution. Soluble and insoluble phases were separated by centrifugation at 15,000 x g for 20 min at 4C. The supernatant was incubated for 30 min with 0.5 mL pre-equilibrated Ni-NTA resin (Qiagen, Venlo,

Limburg), then washed sequentially with >10 column volumes (CV) of 10 mM imidazole followed by >10 CV of 30 mM imidazole until no more protein was eluted (as measured by Bradford assay). The 6-His tagged protein was eluted with 3-4 CV of 200 mM imidazole. The eluateswere concentrated 10-fold with 30 kDa Centriprep concentrators following manufacturers’ protocols (EMD Millipore, Darmstadt, Germany) and dialyzed overnight at 4C against dialysis buffer 1 (50 mM Tris-Cl pH 7.5 + 10% glycerol + 1 mM

EDTA + 5 mM beta-mercaptoethanol + 100 mM NaCl) using 16 mm Snake Skin 10

MWCO dialysis tubing (Thermo Scientific, Rockford, IL).

One mL anion exchange Q-Sepharose columns (HiTrap Q-FF column, GE

Healthcare BioSciences, Pittsburgh, PA) were equilibrated by sequentially washing with

10 mL distilled water, then 10 mL dialysis buffer 2 (50 mM Tris-Cl pH 7.5 + 10% glycerol + 1 mM EDTA + 5 mM beta-mercaptoethanol + 1M NaCl), then 10 mL distilled water, and then 10 mL dialysis buffer 1. Following dialysis, the Ni-NTA eluateswere loaded onto the equilibrated anion exchange columns using a 5.5 mL loading loop. A fraction collector was used to collect 0.5 mL fractions while changes in salt concentration and protein concentration were recorded using a conductance detector and a UV detector respectively (Single Path Monitor UV-1; Pharmacia Biotech, Stockholm, Sweden).

AMX-1 (predicted pI = 5.34; Wormbase 2014) eluted at around 600 mM NaCl, while

AMX-2L and AMX2S (predicted pI = 5.97; Wormbase 2014) eluted at around 450 mM 70

NaCl. The eluates were checked by SDS-PAGE; fractions containing purified protein were pooled and concentrated 10-fold with 30 kDa Centriprep concentrators following manufacturers’ protocols (EMD Millipore, Darmstadt, Germany) and dialyzed overnight at 4C against storage buffer [50 mM Na-phosphate, pH 8.0 (for AMX!) or 7.4 (for

AMX2) + 20% glycerol] using 16 mm Snake Skin 10 MWCO dialysis tubing (Thermo

Scientific, Rockford, IL). Protein was stored in 100 uL aliquots at -80C.

3.5: Protein Expression Analysis by Western Blot

Either the whole cell lysate (WCL) or the Ni-NTA eluatesof the soluble and insoluble fractions of all conditions were checked by SDS-PAGE (8% gel, acrylamide:bis

= 29:1) analysis and Coomassie blue staining using Precision Plus (250-10 kDa) standard dual color protein marker (BioRad, Hercules, CA). Proteins were transferred onto a nitrocellulose membrane (Pall Corporation, Port Washington, NY) using Western blot transfer (12 hr wet transfer at 70 mA; BioRad western transfer apparatus model # 179-

3930). Blocking of the membrane was done using 1% non-fat dry milk (NFDM) in phosphate buffered saline (PBS - 137 mM NaCl + 2.7 mM KCl + 10 mM Na2HPO4 + 1.8 mM KH2PO4, pH 7.4) for 4 hr at RT. Staining was performed by incubating overnight

(12-14 hr) in 1:2000 (final concentration = 0.5 ug/mL in 0.1% NFDM in PBS) anti-

6XHis primary antibody (mouse polyclonal, Aviva Systems Biology, San Diego, CA) or

1:5000 (final concentration = 0.5 ug/mL in 0.1% NFDM in PBS) anti-Myc primary antibody (mouse polyclonal, kind gift of Dr. Mark Berryman, Ohio University, Athens,

Ohio) at 4C. This was followed by three washes in PBS followed by incubation with

1:2000 (final concentration = 0.4 ug/mL in 0.1% NFDM in PBS) HRP-tagged donkey 71 anti-mouse IgG (H+L) as the secondary antibody (Jackson Immunoresearch, West Grove,

PA). The bands were detected using the TMB Membrane Peroxidase system (KPL

Biosciences, Gaithersburg, MD) using manufacturers’ protocol and photographed using

Quantity One software in a Gel Doc analyzer (BioRad, Hercules, CA).

3.6: Detection of FAD by Absorption Spectra Analysis

The presence of FAD in purified proteins was analyzed using 150 uM tranylcypromine (in 50 mM Na-phosphate, pH 7.4), as previously described (Schmidt and

McCafferty, 2007; Karytinos et. al., 2009). Briefly, flavoenzymes give a characteristic dual absorbance maxima at 375-382 nm and 456-458 nm. To check for the presence of

FAD in the enzyme, 10 uM protein was incubated with excess tranylcypromine in a 0.5 mL quartz cuvette and absorbance was analyzed from 300 – 600 nm over 60-90 min at

RT. The MAOI tranylcypromine forms a covalent adduct with the FAD cofactor, thereby bleaching the protein, resulting in a flattening of the FAD-specific peaks until they form a single shallow maximum at ~415 nm. Absorbance was measured in a Cary 50 Bio

(Varian, Palo Alto, CA) UV-visible spectrophotometer using Cary WinUV software.

Two different preps were checked for each protein.

3.7: Preparation of Worm Protein Extracts

For each genotype of nematodes, two 60 mm growth plates were completely washed with 10 mL of M9 (0.3% KH2PO4 + 0.6% Na2HPO4 + 0.05% NaCl + 0.1%

NH4Cl), collected in a tube, and centrifuged at 1000 x g for 2 min. The supernatant was discarded and the worms were washed twice more with fresh M9. After the third wash, supernatant was discarded and the tube was incubated on ice for 3 min and 5 mL of 35% 72 sucrose was added and the tube was centrifuged at 1500 x g for 5 min at 4C. The worms from the top of the fluid were rapidly aspirated to a fresh tube and 10 mL of double distilled water was added to it to dilute the residual sucrose. After centrifuging at 1000 x g, 2 min, 4C, the supernatant was removed to leave the pellets in ~100 uL of fluid, which was transferred to a 1.5 mL microcentrifuge tube and washed 3 times using M9 and centrifugation at 1000 x g for 2 min. Finally the pellets were retained in 120-150 uL of

M9 and an equal volume of 2X worm lysis buffer [100 mM sodium phosphate buffer pH

7.4 + 40% glycerol + 2 mM PMSF + 0.5 mM EDTA + 0.5 mM EGTA + 2 mM benzamidine + 2X HALT protease inhibitor cocktail (Thermo Scientific, Rockford, IL)] was added and incubated at -80C for 30 min and then thawed rapidly. Next the worms in the lysis buffer were subjected to six 30 sec cycles of sonication with 3 min incubation on ice between each cycle. Centrifugation at 1500 x g for 2 min was done and the supernatant was collected in a fresh tube and stored at -80C. At least four different sets of preps were made from N2, amx-1, spr-5, spr-5;amx-1 and amx-2 mutants.

3.8: Biochemical Assay for Monoamine Oxidase Activity

The monoamine oxidase (MAO) activity of worm lysates as well as of heterologously expressed proteins was done using an HRP-coupled fluorometric assay

(Zhou et. al., 1997). For each protein, a minimum three different worm lysate preps were made and each was tested more than three times in separate experiments. Oxidation of monoamine substrates by enzymes that use FAD as the cofactor creates stoichiometrically equivalent amounts of hydrogen peroxide (H2O2) as a by-product. This can react with equivalent amounts of 10-acetyl-3,7-dihydroxyphenoxazine or Ampliflu 73

Red (Sigma Aldrich, St. Louis, MO) to form a fluorescent product, resorufin (excitation

= 560 nm; emission = 589 nm). The assay format followed the manufacturers’ protocol described for the Amplex Red Monoamine Oxidase Assay Kit (Molecular Probes,

Eugene, Oregon). Briefly, 200 uL reaction was done in a 96 well clear bottom flat plate

(Corning, Tewsbury, MA) by adding a 100 uL mix of 200 uM Ampliflu Red (in DMSO)

+ 1 U/mL horseradish peroxidase (Sigma Aldrich, St. Louis, MO) + 1 mM substrate

[benzylamine (BA), dopamine (DA), octopamine (OA), tyramine (TA), serotonin (5HT)

– all purchased as hydrochloride salts from Sigma Aldrich, St. Louis, MO] to a 100 uL solution containing purified protein in 1X assay buffer (50 mM sodium phosphate buffer, pH 7.4) and incubated at RT for 6 hr. To detect H2O2 production, an Yvon-Horiba fluorescent detection system with a MicroMax 96/384 microwell plate reader (Horiba

Scientific, Edison, NJ) was used (MicroMax software). A human MAO-A (Promega,

Madison, WI) was used as the positive control. When an MAOI (tranylcypromine or selegiline or clorgyline; purchased as hydrochloride salts from Sigma, St. Louis, Mo) was used, it was preincubated for 30 min at RT with the enzyme.

3.9: Biochemical Assay for Histone Demethylase Activity

The histone-3 lysine-4 (H3K4me2) specific demethylase (HDM) activity of worm lysates or purified proteins were checked using an LSD1 Demethylase Activity/Inhibition

Colorimetric Assay kit (Epigentek, Farmingdale, NY) following manufacturers’ protocol.

For each protein, a minimum of three different preps were made and each was tested more than three times in separate experiments. Briefly, worm lysates or purified enzymes were incubated with di-methylated histone (H3K4me2) substrates (in a 50 uL reaction 74 volume) for 180 min at 25C. Mono-methylated histone (H3K4me) products were detected after washing by incubation for 60 min (at RT) with a capture antibody against

H3K4me, followed by washing and 30 min (at RT) incubation with a secondary detection antibody. After the final wash, 100 uL developer solution was added for 5-10 min at RT.

Then 100 uL stop solution was added and absorbance at 450 nm (with reference wavelength of 655 nm) was measured using a Quant 96-well plate reader (KC-Junior software). Human LSD1 (Epigentek, Farmingdale, NY) was used as the positive control.

CHAPTER 4: RESULTS

4.1: Cloning of amx cDNAs into Bacterial Expression Plasmids.

Following the preliminary analysis of amx-1 and amx-2 cDNAs using primers from the outer sequences (on the vector plasmid) and inner sequences (on the gene of interest), complete sequencing and analysis of the cDNA sequences was done. One confirmed cDNA for amx-1 (yk1390d04; Wormbase, 2012) and two for amx-2

(yk1054h04 referred to as amx-2L and yk1326e11 referred to as amx-2S) were successfully cloned first into the Gateway entry vector and then into the Gateway destination (expression) vector (Life Technologies, Carlsbad, CA). The complete sequencing of the requested amx-1 cDNAs, including yk1390d04, showed that the corresponding coding transcript for amx-1 was 24 bp (8 amino acids) shorter in the seventh exon (in the AO domain) than predicted in WormBase (highlighted in orange in

Figure 15 below; the complete sequences are in the Appendix). The complete sequencing of five different amx-2 cDNAs confirmed that there were two types of cDNAs. yk1326e11 differed in length and sequence from the other cDNAs (including yk1054h04) in the last (15th) exon (highlighted in red in Figure 16 below). The yk1054h04 sequence was identical to the amx-2 predicted coding sequence in Wormbase, with 123 bp in the last exon (41 amino acids) whereas the yk1326e11 sequence had only 27 bp (9 different amino acids) in the last exon (complete sequences are in the Appendix). This appeared to reflect alternate splicing that might lead to variation in function or location of the proteins. Hence both cDNAs were cloned separately into Gateway bacterial constructs.

Chromosome III

Figure 15: amx-1 gene. Top line indicate size (hash=100 bp), top red box indicates gene model (modified from Wormbase, 2011), bottom red box indicates protein domains or signal sequence, green boxes indicate partially sequenced ESTs (modified from Wormbase, 2011), while the yellow boxes indicate my completely sequenced cDNAs. The transparent vertical purple bar highlights the differences (described in the text) between the gene model and the cDNAs.

Figure 16: amx-2 gene. Top line indicates size (hash=100 bp), top red box indicates gene model (modified from Wormbase, 2011), bottom blue box indicates protein domains or signal sequence, green boxes indicate partially sequenced ESTs (modified from Wormbase, 2011), while the yellow boxes indicate my completely sequenced cDNAs. The transparent vertical purple bar highlights the differences (described in the text) between the gene model and the cDNAs.

In addition to the cloning into the Gateway vector, amx-1 and amx-2L cDNAs were also cloned into the 2GFP-T expression vector (Scott Gradia; Addgene plasmid

29716, Addgene, Cambridge, MA). Cloning of the amx-2S cDNA into the 2GFP-T vector was attempted once and was not successful. All the constructs were checked by PCR and restriction enzyme digestion. Figure 17 below shows the results of the restriction digestion analysis. The constructs were then completely sequenced and the presence of the intact genes and tags [C-terminal 6-Histidine (6-His) tag for Gateway constructs and

N-terminal dual 6-His and GFP tags for 2GFP-T construct] were confirmed. The 6-His tag was added for purification by affinity chromatography and the GFP tag was added to increase the solubility of the proteins. Supplementary Figures S1- S8 (in Appendix) illustrates the plasmid maps of all the bacterial constructs of amx-1, amx-2L and amx-2S.

Figure 17: Restriction enzyme analysis of the bacterial constructs. (a) In the Gateway entry constructs, NotI cuts on the vector backbone and EagI on the insert once. The double digestion gave the expected bands - 3535 and 1514 bp for amx-1(lane 3), 3905 and 871 bp for amx-2L (lane 5) and 3905 and 781 bp for amx-2S (lane 7). The supercoiled uncut constructs for amx-1 (5049 bp), amx-2L (4776 bp) and amx-2S (4686 bp) are seen in lanes 2, 4, and 6 respectively. [NB: the top bands in lanes 2, 4 and 6 are the relaxed conformations of the respective plasmids.] (b) BglII-EagI double digestion of the amx-1 plasmid construct and BglII digest of the amx-2L and amx-2S destination constructs. In the Gateway destination constructs, BglII cuts the vector backbone once; while EagI cuts both the vector backbone and the amx-1 insert once. This digestion yields three bands - 4908, 1750 and 1575 bp (lane 3). For amx-2, BglII cuts once in the vector backbone and once in the insert, to give 7347 and 685 bp bands for amx-2L (lane 5) and 7347 and 595 bp bands for amx-2S (lane 7). The uncut destination constructs for amx-1 (8233 bp), amx-2L (8032 bp) and amx-2S (7942 bp) are seen in lanes 2, 4, and 6 respectively. [NB: the top band in lanes 2 – 7 is the relaxed conformation of the respective plasmids.] (c) EcoRI digestion of 2GFP-T-amx-1 constructs. EcoRI cuts once in the vector backbone and once in the insert to give 7000 and 969 bp (lane 3). The supercoiled uncut 2GFP-T-amx-1 plasmid (7969 bp) is seen in lane 2.

4.2: Expression and Purification of AMX Proteins from Bacteria.

Transformation of the expression plasmids into bacterial expression strain

BL21(DE3) CodonPlus was verified by PCR analysis (Figure 18). The predicted molecular weight of AMX1 is 93.4 kDa, AMX2L is 79.4 kDa and AMX2S is 77 kDa [6-

His tag adds ~0.8 kDa and GFP tag adds ~26 kDa to yield these predicted molecular 79 weights]. The optimum conditions for protein expression were found to be using

BL21(DE3) CodonPlus bacteria, induced at OD600 = 0.6 with 1 mM IPTG and grown at

25C for 12 hr. The growth medium used was Luria-Bertani (LB) broth with 50 ug/mL ampicillin. Protein expressions were determined by western blot with anti-6-His antibodies. Using the Gateway plasmids, AMX1 expression was relatively low and the protein was in the insoluble fraction (possibly in inclusion bodies). I was unable to purify

AMX1 using a Ni-NTA column, which binds soluble 6-His tags. Attempts to solubilize the protein using 10 mM CHAPS as well as by denaturation using 6M urea and renaturation by dialysis, followed by binding to the Ni-NTA column, were unsuccessful.

AMX1 expression from the 2GFP-T construct was higher but the protein was again insoluble (Figure 19).

The AMX2L Gateway plasmid had little and only insoluble protein expression and the protein appeared to be significantly degraded (Figure 24). No detectable protein was expressed from the AMX2L-2GFP-T plasmid. AMX2S expression was not detectable in either constructs.

Figure 18: PCR of amx constructs in bacterial expression strain BL21(DE3)CodonPlus. The primer pairs are given at the top of each lane. Primer sequences are described in the Appendix. The expected 2475 and 3000 bp bands for amx-1 (lanes 2 and 5 respectively), the 2100 and 2700 bp bands for amx2L (lanes 3 and 6 respectively), 1900 bp band for amx2S (lane 4), and for the empty 2GFP-T vector (lane 8) are shown.

Figure 19: AMX1 and AMX2 expression in bacteria. (a) Total protein (lanes 1 and 2; stained with Coomassie blue) and western blot with anti-6-His antibodies (lanes 4 and 5) show low levels of insoluble AMX1 and AMX2 using the Gateway vector. (b) and (c) AMX1 and AMX2L expressions in 2GFP-T constructs. (b) Top to bottom: AMX1-GFP and AMX2-GFP expression show fluorescence of GFP in culture under blue light. (c) Total protein (lanes 1 and 2) and western blot with anti-6-His antibodies (lanes 4 and 5) show insoluble expression of AMX1 using the 2GFP-T construct. (Red arrows indicate the expected protein bands)

4.3: Cloning of amx cDNAs into Yeast Expression Plasmids.

The amx-1, amx-2L, and amx-2S cDNAs were cloned into the pPIC3.5 vector for expression in the yeast species Pichia pastoris (Invitrogen, Carlsbad, CA). All the constructs were checked by PCR and by restriction enzyme digestion (Figure 20). The constructs were then completely sequenced and the presence of the tags (N-terminal 6-

His tag and C-terminal Myc tag) and full length genes were confirmed. Supplementary

Figure S9- S11(in Appendix) illustrates the plasmid maps of all the yeast constructs. 82

Figure 20: Restriction enzyme analysis of the yeast constructs. (a) Restriction enzyme digestion of pPIC3.5-amx-1 constructs. AvrII, BamHI and StuI linearize the plasmid to expected 10253 bp band (in lanes 3, 4 and 6 respectively) while the AvrII-BamHI double digestion releases a 2.5 kbp insert from the 7.8 kbp vector (lane 5) backbone. The uncut supercoiled pPIC3.5-amx-1 plasmid is seen in lane 1 (top band is the relaxed conformation of the plasmid). (b) Restriction enzyme digestion of pPIC3.5-amx-2L and pPIC3.5-amx-2S constructs. AvrII-BamHI double digestion releases the expected 2 kbp and 1.9 kbp inserts from the 7.8 kbp vector backbone for pPIC3.5-amx-2L (lane 3) and pPIC3.5-amx-2S (lane 5) respectively. SacI linearizes the plasmids to expected 9974 bp for pPIC3.5-amx-2L (lane 8) and 9881 bp for pPIC3.5-amx-2S (lane 10). The uncut pPIC3.5-amx-2L can be seen in lanes 2 and 7; while the uncut pPIC3.5-amx-2S can be seen in the lanes 4 and 9. [Top bands in lanes 4, 7 and 9 represent the relaxed conformation of the respective plasmids]

4.4: Expression and Purification of AMX Proteins from Yeast.

Transformation of the expression plasmids into the KM71 Pichia expression strain

(Invitrogen, Carlsbad, CA) was verified by PCR analysis (Figure 21). The optimum conditions for maximum expression were determined to be growth at 25C with 1% methanol induction every 24 hr for 120 hr. Soluble AMX1 was partially purified using

Ni-NTA column chromatography (“1X purification”; one major contaminating band in the gel) and to >95% by subsequent anion exchange column chromatography (“2X 83 purification”; Figure 22b). AMX1 was observed near its expected size of 95.4 kDa region

(AMX1 + 6-His tag + Myc tag = 93.4 kDa + 0.8 kDa + 1.2 kDa = 95.4 kDa) and was confirmed by western blot using antibodies to the N-terminal 6-His tag or the C-terminal

Myc tag. The final yield of pure AMX1 protein was ~1.0 mg per liter.

Figure 21: PCR of amx constructs in Pichia pastoris expression strain (KM71). The primer pairs are given at the top of each lane (see Appendix). The expected 550 and 2800 bp bands for amx-1 (lanes 4 and 6 respectively), the 550 and 2400 bp bands for amx2L (lanes 1 and 7 respectively), and 200 and 2300 bp bands for amx2S (lanes 3 and 8 respectively) are shown.

AMX2L and AMX2S had very low (not detectable by Coomassie protein staining) and insoluble expression under the same conditions. AMX2L and AMX2S were extracted from the insoluble phase using 10 mM CHAPS. AMX2L and AMX2S were partially purified (<10% purity) first by Ni-NTA chromatography and then further purified with anion exchange chromatography. The final yield of AMX2 (L and S) was

~200 – 400 ug per liter. When analyzed by western-blot using an anti-6-His antibody, bands for both AMX2L and AMX2S were detected near the 100kDa protein marker 84 band, which was higher than the expected molecular weight of 81.4 kDa for AMX2L or

79 kDa for AMX2S [AMX2L (or AMX2S) + 6-His tag + Myc tag = 79.4 (or 77) kDa +

0.8 kDa + 1.2 kDa = 81.4 (or 79.4) kDa]. Note that many proteins migrate in SDS-PAGE at higher or lower speeds than predicted from their molecular weights, for reasons such as associated membrane fractions, presence of bound detergent, presence of covalently attached cofactors, post-translational modifications, amino acid composition, etc.

(Klenova et al., 1997, Iakoucheva et al., 2001, Rath et al., 2009). The identity of AMX2L and AMX2S fusion proteins were confirmed by western blot using an anti-Myc antibody.

Figure 22 below illustrates the results of the Coomassie staining and Western blot analysis of the soluble proteins after Ni-NTA (1st round of purification) and Q-sepharose

(2nd round of purification) elution.

Figure 22: Purification of AMX1 and AMX2 from yeast. (a) AMX1 (95.4 kDa) purified using Ni-NTA (lane 2) followed by Q-sepharose columns (lane 3) respectively, showing protein (Coomassie staining) and Western blot using either anti-6-His (lane 5) or anti- Myc (lane 6) antibodies. Green arrows indicate the predicted protein band for AMX1. (b) AMX2L and AMX2S (predicted size 81.4 and 79.4 kDa; band at 100kDa) purified using Ni-NTA (lanes 1 and 4 respectively) followed by Q-Sepharose columns (lanes 5 and 6) respectively, showing protein (Coomassie staining) and western blot using either anti-6- His (lanes 9 and 8) or anti-Myc (lanes 11 and 10) antibodies. Yellow arrows indicate the observed bands for AMX2L and AMX2S. An additional unknown band close to ~55 kDa is also picked up by anti-6-His antibody (lanes 8 and 9), but not by the anti-Myc antibody (lanes 10 and 11).

4.5: FAD Absorption Spectrum.

The AO domain is predicted to bind the cofactor FAD. Tranylcypromine is known to form a covalent adduct with the FAD and displace it from the active site of the protein (Schmidt and McCafferty, 2007; Karytinos et al., 2009). On incubating the purified AMX proteins with tranylcypromine for 60 to 90 minutes, changes in the spectrum consistent with binding of an FAD cofactor were observed. AMX1 showed a significant peak characteristic of FAD at ~456 nm (and 375 nm) that decreased after incubation with tranylcypromine (see Figure 28). AMX2L and AMX2S proteins were 86 less pure and showed only a shallow peak at ~415 nm; this peak decreased slightly after incubation with tranylcypromine (see Figure 23).

Figure 23: Absorption spectra of AMX proteins. (a) AMX1, (b) AMX2L and (c) AMX2S after 0 or 60 minutes in 150 uM tranylcypromine. [ Inset: The absorption spectra at 0, 5, 10, 20, 40, 60 and 90 min after adding Tranylcypromine.] Tranylcypromine forms a covalent adduct with FAD cofactor resulting in a flattening of the FAD-specific peaks. [The experiments were done with two different preps for every protein.]

4.6: Biochemical Activity of Wild-type and Mutant Worm Lysates.

Sequence homology data in Wormbase suggested that AMX1 was a histone demethylase (HDM) homolog and AMX2 (L and S) was a monoamine oxidase (MAO) homolog. Accordingly, mutant worm lysates were assayed for differences in HDM and

MAO activities. The HDM assay used di-methylated H3K4 as the substrate, followed by antibody based detection of mono-methylated products (Epigentek, Farmingdale, NY).

This substrate could also be affected by other histone demethylases like SPR5 and/or

LSD1. MAO activity was assayed by incubating lysates with MAs and detecting the formation of hydrogen peroxide by a peroxidase coupled system using a fluorogenic substrate.

Wild-type (N2), amx-1(ok659) and amx-2 (ok1235) mutant worm lysates were tested for HDM (Figure 24) and MAO (Figure 25) activity. For each type of worms, four different worm lysate preps were made and tested with a single batch of purified AMX1

(or AMX2) protein. amx-1(ok659) and amx-2(ok1235) mutants had significantly lower

HDM activity than N2 worms. amx-2(ok1235) had significantly lower MAO activity than N2 and amx-1 worms. Tranylcypromine is a known inhibitor of vertebrate HDM and

MAO activities (Schmidt and McCafferty, 2007; Karytinos et. al., 2009) and had also shown to significantly affect some MA-dependent behaviors in wild-type worms, as discussed in Chapter 2. Accordingly, amx mutant worms are expected to be at least partially tranylcypromine resistant. However, note that in the biochemical assay using crude worm lysates, several other proteins with overlapping functions (like SPR5 and 89

LSD1) might be present, partially masking the HDM and MAO activity differences in the mutants vs. N2 as well as the inhibitory effects of Tranylcypromine.

Figure 24: HDM activity of worm lysates. Activity of amx-1(ok659) and amx-2(ok1235) lysates was significantly lower than N2. [n = 4; * indicates p<0.05, ** indicates p<0.01, Wilcoxon signed rank test. Error bars indicate standard deviation from the mean.]

Figure 25: MAO activity of worm lysates. Activity of amx-2 (ok1235) mutants was significantly lower than N2. [n = 4; * indicates p<0.05, ** indicates p<0.01, Wilcoxon signed rank test. Error bars indicate standard deviation from the mean.] 90

4.7: HDM Activity of AMX Proteins

Partially purified AMX1, AMX2L and AMX2S (Ni-NTA eluates) were examined for HDM activity using H3K4me2 as a substrate (the expected substrate for LSD1 type demethylases) (see Figure 26). Unfortunately, all showed very little activity in vitro

(measured as an increase in absorbance at 450 nm). It is possible that the purified proteins needed a cofactor or other cellular constituent for their activity. Therefore, we next tested whether the partially purified proteins could increase the HDM activity of worm lysates

To analyze the effects of adding AMX1 to worm lysates, several different lysates were used. SPR5 is a known HDM for H3K4me and H3K4me2 in C. elegans (Katz et al.,

2009) thus the effects of AMX1 on HDM activities of spr-5 and spr-5; amx-1 mutant lysates were analyzed in addition to the amx-1 and amx-2 mutants. Pure AMX1 significantly increased the HDM activity in all of the worm lysates (except in amx-2 where the increase was non-significant) (Figure 27b). The relative increase in HDM activity after addition of AMX1 was significantly higher in the amx-1 and spr-5;amx-1 mutants in comparison to the other genotypes tested (Figure 27). The experiments were performed again with more highly purified AMX1 (Q-sepharose eluate) and even more (3 fold) stimulation of HDM activity in amx-1 lysate was seen (Figure 28a).

The effects of partially purified AMX2L and AMX2S (from Ni-NTA and Q- sepharose eluates) on HDM activity of worm lysates were also analyzed. Surprisingly, the AMX2 proteins caused a significant increase in the HDM activity of the amx-1 worm lysates (there was non-significant increase in the N2 and amx-2 lysates; Figure 28). This was true even though AMX2 lacks the SWIRM domain that is characteristic of LSD-like 91

HDMs. However, the results are consistent with the fact that the amx-2 worm lysates had lower than normal HDM activity (Figure 25) and that AMX2 might have some HDM activity of its own. Three types of MAOIs – tranylcypromine (non-specific MAO inhibitor), clorgyline (MAO-A specific inhibitor) and selegiline (MAO-B specific inhibitor) - were tested in the HDM assay. Only tranylcypromine significantly inhibited

HDM activity (150 uM tranylcypromine caused ~65% inhibition of 20 uM AMX1)

(Figure 28a).

Figure 26: HDM activity of purified AMX1 and partially purified AMX2L and AMX2S (two column purifications). H3K4me2 was used as the substrate. Absorbance values at 450 nm (minus the blank) are shown. 20 ug of purified AMX1, AMX2L and AMX2S were used.

Figure 27: HDM activity of worm lysates in presence of AMX1. H3K4me2 was used as the substrate. (a) Partially purified AMX1 (1st column purification) significantly increased the HDM activity in N2, amx-1, and spr-5 mutants. (b) The percentage increase in HDM activity of worm lysates after addition of AMX1. The increase in amx-1, spr-5 and spr-5;amx-1 lysates was significantly more than in N2. [n = 4; * indicates p<0.05, ** indicates p<0.01, Wilcoxon signed rank test]

Figure 28: HDM activity of worm lysates in presence of AMX proteins. H3K4me2 was used as the substrate. 1X pure indicates purification by only Ni-NTA column and 2X pure indicates purification using Ni-NTA followed by Q-sepharose column. (a) More highly purified AMX1 significantly increased the HDM activity in amx-1 mutant lysates (~3 times more than 1X pure AMX1). Of the MAOIs tested, only tranylcypromine inhibits the increase in HDM activity by >50% (Tran = tranylcypromine, Sele = Selegiline, Clor = Clorgyline). 2X pure AMX2L or AMX2S also caused a significant increase in HDM activity in the amx-1 lysates. (b) In N2 lysates there was lower but still significant increase in HDM activity due to addition of AMX1. (c) HDM activity was not significantly affected by AMX1 addition to amx2 mutant lysates. 2X pure AMX2L or AMX2S caused no significant increase in HDM activities in N2 or amx-2 worm lysates. [n = 4; * indicates p<0.05, ** indicates p<0.01; Wilcoxon signed rank test]

4.8: MAO Activity of AMX Proteins.

Initial assays of MAO activity of AMX2L and AMX2S proteins were done with

1X purified proteins (only Ni-NTA column). The results were confirmed with 2X purified AMX2 proteins (Ni-NTA followed by Q-sepharose column) (see Figure 29).

Benzylamine, a naturally occurring monoamine substrate of human MAO-A and –B, that is used in standard MAO assays (Molecular Probes, Eugene, OR) was used in preliminary MAO assays of the AMX proteins. AMX1 (1X or 2X purified) had very low

MAO activity; while AMX2L and AMX2S showed strong MAO activity (significantly higher in more purified samples). This activity was strongly inhibited by the MAOIs tranylcypromine (non-specific MAO inhibitor) and selegiline (MAO-B specific inhibitor) but not by clorgyline (MAO-A specific inhibitor).

Figure 29: MAO activity of purified AMX proteins. 1 mM benzylamine (BA) was used as substrate and 40 ug 1X or 2X purified AMX1 (a), AMX2L (b) or AMX2S (c) proteins were used. For MAOIs, 100 uM tranylcypromine (nonspecific MAOI), selegiline (MAO- B inhibitor) and clorgyline (MAO-A inhibitor) were checked. AMX1 has negligible MAO activity which is not significantly inhibited by tranylcypromine. AMX2L and AMX2S exhibit significant MAO activity which increased with increased purification. Tranylcypromine and selegiline significantly inhibit the MAO activity of AMX2L and AMX2S. (Tran = tranylcypromine, Sele = Selegiline, Clor = Clorgyline). [For each protein, three different preps, each prep tested at least in duplicate; * indicates p<0.05; Wilcoxon signed rank test]

AMX2L and AMX2S were tested with other MA substrates – dopamine (DA), serotonin (5HT), octopamine (OA), and tyramine (TA) – in the absence and presence of

MAOIs. AMX2L and AMX2S had similar orders of substrate specificities - DA > BA =

OA = TA > 5HT. AMX2S had a much higher specific activity than AMX2L with DA, but a lower specific activity with TA. The added round of purification (Q sepharose column) increased the activity (by three to four times) of both AMX2L and AMX2S (See

Figures 30a, 31a and 32a).

Of the three MAOIs tested for inhibition of activity of AMX2 proteins, only tranylcypromine and selegiline showed significant effects. For the 2X purified AMX2 96 proteins, tranylcypromine caused maximum inhibition, followed by selegiline (Figures

30b, 31b, 32b). Clorgyline did not significantly inhibit either of the proteins.

Finally the AMX2 proteins were checked for their ability to rescue MAO activity in amx-2 (ok1235) lysates (Figure 25). amx-2 lysates had significantly lower MAO activity compared to N2 lysates. Addition of AMX2L or AMX2S significantly increased the MAO activity in both N2 and amx-2 mutant lysates in vitro (Figure 33) making them more similar.

Figure 30: Substrate specificity of 1X purified AMX2L. (a) MAO activity on different MA substrates with and without MAOIs (100 uM tranylcypromine, selegiline and clorgyline). The MA specificity of AMX2L was in the order of DA>BA=OA=TA>5HT. (b) Percentage inhibition of MAO activity of by 100 uM selegiline, clorgyline or tranylcypromine. Tranylcypromine and selegiline significantly inhibited AMX2L. [Two different protein preps, assay performed in triplicate; * indicates p<0.05; Wilcoxon signed rank test]

Figure 31: Substrate specificity of 1X purified AMX2S. (a) MAO activity on different MA substrates with and without MAOIs (100 uM tranylcypromine, selegiline and clorgyline). The MA specificity of AMX2S was in the order of DA>>BA=OA=TA>5HT. (b) Percentage inhibition of MAO activity of by 100 uM selegiline, clorgyline and tranylcypromine. Tranylcypromine and selegiline significantly inhibited AMX2S. [Two different protein preps, assay performed in triplicate; * indicates p<0.05; Wilcoxon signed rank test]

Figure 32: Substrate and inhibitor specificity of 2X purified AMX2L and AMX2S. (a) Substrate specificity of the Q-sepharose eluatesof AMX2L and AMX2S. (b) Percentage inhibition of the three different MAOIs tested, with different substrates. [One protein prep, assay performed in duplicate; * indicates p<0.05; Wilcoxon signed rank test]

100

Figure 33: Effect of AMX2 on MAO activity of worm lysates. 2X purified AMX2L and AMX2S significantly increased the MAO activity when added to worm lysates [N2 and amx-2 (ok1235)]. [One protein prep, assay performed in duplicate; * indicates p<0.05; Wilcoxon signed rank test]

101

CHAPTER 5: DISCUSSION AND SUMMARY

This project provides the first expression, purification and biochemical characterization of two AO domain containing genes – amx-1 and amx-2 – from C. elegans. The amx-1and amx-2 (both the ‘L’ and ‘S’ variants as named in this project) genes were successfully expressed in the P. pastoris system and were purified using Ni-

NTA and Q-sepharose columns. AMX1 was purified to >95% and AMX2 (L and S) were partially purified. The proteins showed enzymatic activity in vitro.

My first experiments used bacteria to express all of the proteins. Unfortunately

AMX1was insoluble and purification of sufficient active protein from the inclusion bodies was not possible. AMX2S protein was not significantly expressed, while AMX2L expression was low and insoluble and highly denatured (Figure 18) despite adjustments of numerous aspects of inducing expression (Table 3 in Appendix). This poor expression may be due to the numerous differences between eukaryotes and prokaryotes, including absence of post-translational modifications and sub-cellular membranes in prokaryotes, and different codon usages in prokaryotes vs. eukaryotes. Thus eukaryotic proteins, especially membrane proteins, are often improperly folded or poorly expressed in bacteria (Terpe, 2006, Bernaudat et al., 2011). AMX2L and AMX2S are homologs of human MAOs, which are outer mitochondrial membrane associated proteins. The expression and purification of human MAO from E. coli was attempted by others in the past, but purification was only partial and yield was low (Lu et al., 1996). The lack of expression of soluble AMX2L and AMX2S led to the decision to change to the system 102 that has been successfully used for the expression of human MAO – the yeast Pichia pastoris.

AMX protein expression was done in the yeast Pichia pastoris, using a methanol inducible expression vector (Newton-Vinson et al., 2000, Li et al., 2002; Hubalek et al.,

2003, Upadhyay and Edmondson, 2008, Arslan and Edmondson, 2010). AMX1 showed reasonable levels of expression of soluble protein and >2 mg/L of pure (>95%) and active protein was purified. AMX2L and AMX2S protein expression in P. pastoris was low (<

1 mg/L) and the proteins could only be partially purified. Future research may lead to an increase in the expression and yield of C. elegans AMX2L and AMX2S in P. pastoris.

Purified AMX1 protein has negligible histone demethylase (HDM) activity with

H3K4me2 as a substrate. But purified AMX1 significantly increased the HDM activity of worm lysates using the same substrate. Lysates from amx-1 mutants had significantly lower HDM activity than N2 lysates. Purified AMX1 increased HDM activity of amx-1 lysates significantly more than of wild-type lysates. This suggests that AMX1 may be part of a HDM process (H3K4me2 substrate specific). Purified AMX1 also has very little monoamine oxidase (MAO) activity with either of benzylamine, dopamine, octopamine, serotonin or tyramine as substrates. The lack of MAO activity was consistent with the lack of consistent changes in MA-dependent behaviors in the amx-1 mutants and no significant difference in MAO activity between N2 and amx-1 lysates.

The fact that heterologously expressed AMX1 had no HDM activity of its own but increased the activity of worm lysates, is consistent with observations that many histone modifiers act in conjunction with different types of proteins (Bernstein et al., 103

2005; Upadhyay and Cheng, 2011; Greer and Shi, 2012; Fang et al., 2013). Varied proteins may assist in genomic targeting, chromatin-binding, docking, recruitment and activation of demethylase proteins. Recent examples include (1) the several MBT

(Malignant Brain Tumor) domain containing proteins (like SFMBT1) which recognize mono-, di-, and tri-methylated lysine and assist in Snai1 mediated recruitment of LSD1 demethylase (Tang et al, 2013), (2) the HP1a assisted demethylation of H3K36me3 by

KDM4a demethylase in Drosophila (Lin et al, 2012), (3) human LSD2 requires the protein NPAC/GLYR1 for demethylation process (Fang et al., 2013). Previous data indicates that amx-1activity is redundant with spr-5 as the spr-5;amx-1 mutants had a more severe phenotype than spr-5 or amx-1 alone and also the amx-1 mRNA levels go up in the spr-5 mutants (Katz et al., 2009). The absence of AMX1 in the amx-1 mutant might cause increased expressions of SPR5, LSD1 or other proteins involved in HDM

(H3K4me2 specific). This would explain the higher than wild-type (N2) HDM activity in equivalent amount of amx-1 mutant lysates when exogenous AMX1 is added. Another possible explanation for the lack of HDM activity in vitro is that the HDM activity of

AMX1 requires the presence of cofactors besides FAD that are present in the worm lysate. Since HDMs often act as complexes, I favor the hypothesis that AMX1 might be a part of a HDM complex in C. elegans. Other techniques like immunoprecipitation can be useful to understand the role of AMX1 in C. elegans better.

In general, histone demethylases can act on mono-, di- or tri-methylated lysine residues on a particular histone residue (Greer and Shi, 2012). For example, both SPR5 and LSD1 act on both mono- and di-methylated H3K4 residue (Upadhyay and Cheng, 104

2011). Therefore future research should examine if AMX1 can demethylate other substrates such as mono-methylated H3K4, H3K9, H3K27, etc. in vitro and in vivo.

The HDM activity of AMX1 was significantly inhibited by the MAOI tranylcypromine, but not by selegiline and clorgyline. This was consistent with the results of behavior assays, where the amx-1 mutant was partially resistant to tranylcypromine but not to selegiline or clorgyline. This is unsurprising since tranylcypromine removed the

FAD cofactor from the active site of homologous mammalian LSD1 and LSD2 proteins

(Edmondson et al., 2004, Schmidt and McCafferty, 2007, Karytinos et al., 2009).

AMX1 shares sequence identities with its paralogs – CeSPR5 and CeLSD1- which in turn are homologous to the huLSD1 (KDM1A) (see sequence identity analyses in Appendix). However AMX1 is more similar to huLSD2 than to CeSPR5 or CeLSD1.

AMX1 possesses three conserved domains – the SWIRM domain (residues 251-337; found mostly in histone binding proteins), an NAD(P) binding Rossman-like domain

(residues 356-422), and the Amine Oxidase domain (residues 361-804). AMX1 is most similar to huLSD2 in each of these domains (see Appendix). LSD2 expression was found in oocytes in adult mice, with a possible role in establishing maternal DNA methylation imprints during oogenesis (Ciccone et al., 2009). Previous work in the lab found that the expression of the amx-1::GFP transcriptional fusion in all the cells in the embryo and a few neurons in the adult (Kaushal, 2008). This suggests that AMX1 may have a more general role in the embryo and more specific roles in the adult.

Despite the similarities between huLSD2 and AMX1, huLSD2 has an additional domain not present in AMX1 - an N-terminal CW type zinc finger domain. AMX1is even 105 less similar to huLSD1, which has a 100 amino acid ‘tower’ domain inserted in the AO domain (see Figure 12 for structures of human LSD1 and LSD2). Therefore AMX1 may have a different role than either human homologs.

C. elegans AMX2L and AMX2S proteins were also expressed and partially purified for the first time. They show MAO oxidase activity in vitro with different MA substrates. While both proteins had similar specific activity and relative substrate specificity (DA > BA = OA = TA > 5HT), AMX2S showed significantly higher activity on DA and significantly lower on TA compared to AMX2L. However both AMX2L and

AMX2S had the least activity with 5HT as the substrate. Note that in C. elegans, succinylation was recently found to be a significant pathway for 5HT and OA metabolism (Artyukhin et al., 2013). Thus 5HT degradation may not generally use a

MAO dependent pathway. It is not known where and when AMX2L and AMX2S are expressed. Future investigation using transgenes and AMX2L and AMX2S-specific antibodies should be helpful in that regard.

AMX2L and AMX2S were significantly inhibited by tranylcypromine, followed by selegiline, but not by clorgyline. This was consistent with the observation that amx-

2(ok1235) mutants were significantly resistant to selegiline and slightly resistant to tranylcypromine but not resistant to clorgyline. In behavior assays in this lab, amx-2 mutants were found to show abnormalities in some MA dependent behaviors like movement in food, movement in a sephadex bead solution, pharyngeal pumping, etc. consistent with amx-2 being an MAO. In this project, the in vitro biochemical assays provide direct support for a role of AMX2 in oxidative deamination of MAs. The result 106 should be further validated under in vivo conditions. My results here indicate that

AMX2L and AMX2S are similar types of MAOs in terms of substrate and MAOI specificity. Note that AMX2 (L and S) have high sequence identities in both FAD- binding (50% to MAO-A and 47% to MAO-B) and AO domains (19% to both MAO-A and –B) to the human MAO proteins (see Appendix for sequence identity analyses).

Surprisingly I found that AMX2L and AMX2S, like AMX1, could significantly increase the HDM activity in amx-1 worm lysates, but had no in vitro HDM activity.

Also amx-2 lysates showed a significant decrease in HDM activity compared to wild-type

(although not as low as in amx-1 mutants). This suggests that AMX2L and AMX2S have

HDM activity in vivo. It is consistent with the fact that although AMX2 does not have a

SWIRM domain, it shares comparable identity in FAD-binding and AO domains with huLSD1 as with huMAO (see Appendix).

Together, my results establish the first in vitro biochemical activities for C. elegans AMX1, AMX2L and AMX2S proteins. AMX1 is inhibited by tranylcypromine and may be an important part of a histone demethylase complex, since the protein significantly increases H3K4me2 specific demethylase activity in worm lysates. The partially purified AMX2L and AMX2S also appeared to have significant HDM activity in the assay. AMX2L and AMX2S (and AMX1 to a much lower extent) were able to oxidatively deaminate BA, DA, 5HT, OA and TA, with highest effect for DA. AMX2L and AMX2S were selectively inhibited by tranylcypromine and selegiline. These newly purified proteins can be used for future biochemical investigations, generation of specific 107 antibodies which can significantly broaden our understanding of the roles that these AO domain containing proteins play in C. elegans.

108

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APPENDIX A: TABLE 2 – PRIMER INFORMATION

Primer Name Position Purpose Sequence 105(rev) 179 bp upstream of end of Sequencing TACGTAGCATCACCATCCG amx1 cDNA; RC 108(fwd) 1484 bp downstream of start Sequencing GACTTGGACTCTCTCTACAC (ATG) of amx1 cDNA 174(fwd) 323 bps downstream of start Sequencing TCCAACGGACACCCAATTAT (ATG) of amx2 cDNA 176(fwd) 534 bp downstream of start Sequencing TCTCAGCAAATGGACACTGC (ATG) of amx2 cDNA 204(fwd) 858 bp downstream of start Sequencing GCTGTGCCAATTCCGACAC (ATG) of amx2 cDNA 207(rev) 787 bps upstream of end of Sequencing CCCACCCAAATTGTGCGAG amx2 cDNA 208(rev) 915 bps upstream of end of Sequencing CAAGTCGTCCGTGGCAAAG amx2 cDNA 272(fwd) flanks on yk-plasmid Sequencing of yk-clones CTTCTGCTCTAAAAGCTGCG (ME774FW) 273(fwd) flanks on yk-plasmid Sequencing of yk-clones GGATGTTGCCTTTACTTCTA (ME735FW) 274(rev) flanks on yk-plasmid Sequencing of yk-clones TGTGGGAGGTTTTTTCTCTA (ME1250RV) 275 760 bp downstream of start Sequencing GAATATAGTCAACGTGTCGT (fwd) (ATG) of amx2 cDNA 276(fwd) 202 bps downstream of start Sequencing AATTGTAAGAAAACTGCAAG (ATG) of amx1 cDNA 278(fwd) 1025 bps downstream of start Sequencing TCAG AAT TGA TCC ATT GAA (ATG) of amx1 cDNA 279(fwd) 1658 bp downstream of start Sequencing CTAGTTCACAGTACCCGACT (ATG) of amx2 cDNA 280(fwd) CACC-ATG-….start of amx-1 GATEWAY Entry CACCATGACAACAGAACTTGAAATA cDNA cloning 281(rev) from last aa (except STOP GATEWAY Entry TTCGAAAGTGGCCGAATCCCGCTT codon) into amx1 cDNA cloning 282(fwd) CACC-ATG-….start of amx2 GATEWAY Entry CACCATGTTGCGTGGGTGGTGGATA cDNA cloning 283(rev) from last aa (except STOP GATEWAY Entry TGCTCGTGAGTCGATTCTATTTC codon) into 1326 cDNA cloning 284(rev) from last aa (except STOP GATEWAY Entry CTGTAGACGCAAAGTCTGTAAGAG codon) into amx2 cDNA cloning 286 (rev) from last aa (except STOP GATEWAY Entry AAAATTCAGGAAAAAAGTTAGATGTGCAGGTG codon) into amx2 cDNA cloning TTGC 315(fwd) BamHI. Kozak seq, 8XHis tag, Pichia pastoris cloning GAAGGATCCAAAAAAATGCACCACCACCACC first 7 aa of amx1 cDNA seq ACCACCACCACATGACAACAGAACTTGAAATA

316(fwd) BamHI. Kozak seq, 8XHis tag, Pichia pastoris cloning GAAGGATCCAAAAAAATGCACCACCACCACC first 7 aa of amx2 cDNA seq ACCACCACCACATGTTGCGTGGGTGGTGGATA

317(rev) AvrII, Myc tag, last 8 aa (no Pichia pastoris cloning AAGCCTAGGAAGATCTTCTTCAGAAATAAGTT STOP codon) of amx1 cDNA TCTGTTCTTCGAAAGTGGCCGAATCCCGCTT seq 318(rev) AvrII, Myc tag, last 8 aa (no Pichia pastoris cloning AAGCCTAGGAAGATCTTCTTCAGAAATAAGTT STOP codon) of amx2 cDNA TCTGTTCCTGTAGACGCAAAGTCTGTAAGAG seq 319(rev) AvrII, Myc tag, last 8 aa (no Pichia pastoris cloning STOP codon) of 1326 cDNA AAGCCTAGGAAGATCTTCTTCAGAAATAAGTT seq TCTGTTCGTTGCTCGTGAGTCGATTCTATTT 129

Primer Name Position Purpose Sequence 320(fwd) LIC cloning seq, first 7 aa of p29716 LI cloning TACTTCCAATCCAATGCAATGACAACAGAACT amx1 cDNA TGAAATA 321(fwd) LIC cloning seq, first 7 aa of p29716 LI cloning TACTTCCAATCCAATGCAATGTTGCGTGGGTG amx2 cDNA GTGGATA 322(rev) LIC cloning seq, last 8 aa (no p29716 LI cloning TTATCCACTTCCAATGTTATTATTCGAAAGTGG STOP codon) of amx1 cDNA CCGAATCCCGCTT 323(rev)

324(rev) LIC cloning seq, last 8 aa (no p29716 LI cloning TTATCCACTTCCAATGTTATTACTGTAGACGCA STOP codon) of amx2 cDNA AAGTCTGTAAGAG 325(rev) LIC cloning seq, last 8 aa (no p29716 LI cloning TTATCCACTTCCAATGTTATTAGTTGCTCGTGA STOP codon) of 1326 cDNA GTCGATTCTATTT 326(rev) second reverse primer with a Pichia pastoris cloning AAGCCTAGGAAGATCTTCTTCAGAAATAAGTT STOP codon for AMX2long TCTGTTCTTACTGTAGACGCAAAGTCTGTAAG AG 327(rev) second reverse primer with a Pichia pastoris cloning AAGCCTAGGAAGATCTTCTTCAGAAATAAGTT STOP codon for AMX2short TCTGTTCTTAAAAATTCAGGAAAAAAGTTAGA TGTGCAGGTGTTGC 330(fwd) Second primer with STOP p29716 LI cloning TTATCCACTTCCAATGTTATTATTAAAAATTCA codon for AMX2short GGAAAAAAGTTAGATGTGCAGGTGTTGC

331(rev) 5' AOX1 sequencing primer in Sequencing GACTGGTTCCAATTGACAAGC P. pastoris 332(fwd) 3' AOX1 sequencing primer in Sequencing GCAAATGGCATTCTGACATCC P. pastoris 333(rev) fwd primer for RNA Pol Sequencing (cell lysis TTTGGAAATTAAAGTTGGCTACAGAA subunit in P. pastoris control) 334(fwd) reverse primer for RNA Pol Sequencing (cell lysis TTAATTACCGGTAACAGTCTGTAACT subunit in P. pastoris control) 335(rev) 477th nt in pENTR/SD/D- Sequencing CCTGGCAGTTCCCTACTCTC TOPO backbone 336(fwd) 883rd nt in pENTR/SD/D- Sequencing CCAGGAAACAGCTATGACCA TOPO backbone 337(rev) 216th nt in pET-DEST42 Sequencing GTGATGTCGGCGATATAGG backbone 338(fwd) 2547th nt in pET-DEST42 Sequencing GCCCTTTCGTCTTCAAGAAT backbone 339(rev) 761th nt in pPIC3.5 backbone Sequencing AACCCCTACTTGACAGCAAT

340(fwd) 1237th nt in pPIC3.5 backbone Sequencing CTCGAATGATTTTCCCAAAC

341(rev) 4564th nt in p29716 backbone Sequencing GGCATCGACTTCAAGGAG

343(fwd) 5194th nt in p29716 backbone Sequencing ACTTGGAGCCACTATCGACT

GFP region in p29716 Sequencing CAGCTCGCCGACCACTA

130

APPENDIX B: TABLE 3 – EXPRESSION CONDITIONS TESTED IN E. COLI

Expression Discussion Condition (1) Expression Strain (1a) BL21(DE3) Derived from wild type E. coli B cells with the T7 Polymerase from the lambda-DE3 lysogen under the lacUV5 promoter, lon and OmpT deficiency. May have leaky expression of T7 RNA Polymerase.

(1b) Derived from recA (recombination deficient) E. coli B cells with the T7 Polymerase from BLR(DE3)pLysS the lambda-DE3 lysogen under the lacUV5 promoter, lon and OmpT deficiency. Expresses T7 lysozymeto decrease leaky expression of T7 RNA Polymerase. Plasmids are more stable in BLR than BL21. 34 ug/mL Chloramphenicol used for selection (but not during induction).

(1c) Derived from recA (recombination deficient) E. coli B cells with the T7 Polymerase from BLR(DE3)CodonPlus the lambda-DE3 lysogen under the lacUV5 promoter, lon and OmpT deficiency. Encodes tRNAs that recognize codons AGA and AGG (arginine), AUA (isoleucine), and CUA (leucine) for enhanced eukaryotic expression. 12 ug/mL Tetracycline used for selection (but not during induction).

(1d) RosettaGami Derived from BLR, Rosetta and Origami strains. Has T7 RNA Pol expression under pLysS lacUV5 promoter, lon and OmpT deficiency, six to seven rare codon tRNAs for enhanced eukaryotic expression, and trxB/gor mutations to allow di-sulfide bond formation in the cytoplasm. 12 ug/mL Tetracycline and 34 ug/mL Chloramphenicol used for selection (but not during induction). (2) Inducer (IPTG) IPTG concentrations of 100 uM, 200 uM, 500 uM, 1000 uM and 2000 uM, were checked. Concentration (3) Induction Points Log phase induction, at OD600 (optical density at 600 nm wavelength) = 0.6, as well as higher (OD600 = 1.0-1.2) and lower (OD600 = 0.2-0.3) induction points were tested. (4) Post-induction Prior to induction, the E. coli cells are grown at 37C, with ~20-30 minutes/duplication Temperature cycle. Usual post-induction temperature is 37C. Since nematodes grow at 15-25C, the optimum temperature for expressing nematode proteins may be lower than 37C. Therefore, post induction temperatures of 30C and 25C were also checked.

(5) Post-induction Post-induction times of 1, 2, 4, or 6 hours at 37 C and 30 C; or 6, 12 and 24 hours at 25 C Time were checked. Longer time may increase the expression at 25C; higher temperatures may cause lysis of the cells.

(6) Growth Media Luria-Bertani (LB) broth (yeast extract 5 g + tryptone 10 g + NaCl 10 g / liter of media) versus the Terrific broth (12 g tryptone + 24 g yeast extract + 0.4% glycerol + 9.4 g K2HPO4 + 2.2 g KH2PO4/ liter of media) were checked. Terrific broth gave higher cell mass, but no change in the level of protein expression.

(7) Antibiotic during Ampicillin (@ 50 ug/mL) was the selection antibiotic for Gateway expression plasmids. Induction Since removing the antibiotic load during induction could favor expression of some proteins. Expression levels +/- ampicillin were checked. No difference in either amx-1 or amx-2 (L and S) expression levels were observed.

131

APPENDIX C: NUCLEOTIDE SEQUENCING RESULTS

Nucleotide sequence of amx-1 as obtained by sequencing of yk1390d04 cDNA: (Start codon is highlighted in green and stop codon is highlighted in red) Length = 2451 bp

ATGACAACAGAACTTGAAATAGACGATAGAAAAGAAGAAGAAGCTCAAATTCCTGGAGAAACGAGTGA AAGTGAAGAGGGAGATGAACCTGTAAAAATGAATCGCGTAAAACGCGCCTGTGCAATGGCAGCCGAATC TTTAAAAGAGGCAAAAAGAATAAAGAACATGTTGATGGAAGAAGAATGTGTTTTACAATTGGGTAATTG TAAGAAAACTGCAAGTTATGGGCATAGATTGAGTAAAACTGAAGCATGTTGTCCaTCGTGCTTTAGTGTT GCATTTCGTGGAAAGGATTATGAGAATGAGTTTACAATTTGGAAGCAGAAGGCAATGGATGGACAGACA CATGTTCGAAGTGAACAATTTGTTAAACGATATGTTAATAGCTGCTTCCTACCATACTACGCAAAATGCC ACCAATGCTCAAAATACTCCAAATTGATAACCTCCGACTCTCTCTCTGCTCAACAATTATCCGACTTCAAG TGCGACTGTGCTTCTACAATTGAATCCCCCAAGATTGAACGAGTTCGTGAAGACTCAGAATGGTGTTTCA ATGAATTTGGACATCCTCCTTTACTTCAAAATAATATTTCCTATGATCTACTAGTTGATCACTACGTTACA CGGACCACTGGAATGGATGCCACGTGTCAGGAGAAGGCGGCGTTGATTGACAATGGTGGCATCGAATTT CGAGATACAAGAAGAATTATGAACATGTTTTATGTTCCATTTACGGATGTTATTGCGAATATTGTACATCC CGAGTTTATGGAAACCGATGAGAAATTTGCATTCCCTAAATTTGCTGATGATCCCATTTCAATCTACTACC TCCAAGTCCGAAATACAATTATCGCAATGTGGCTGAAGCATCCATTCGTCGAGTTAACCGTGAAAATGAT TGAACCCCAGATTATTGTCAGAGGTCATGCGAGGATATTCTTTATTGAACACCTCATTCATCCAATTTTGG AATTTTTGACAATTAAAGGAGTTGTCAATTATGGAGCATTTGATTTCAGAATTGATCCATTGAATGGAAT GAGACCGAAAATTGCAATAATTGGAGCAGGaAtaTCTGGAATCTCAACAGCCCGCCACCTAAAACATTTGG GAATTGATGCTGTTCTCTTCGAAGCAAAGGATCGCTTCGGTGGACGTATGATGGATGATCAATCACTTGG AGTATCAGTTGGAAAGGGAGCTCAGATTATTGTTGGAAATATCAATAATCCAATTACTCTTTTGTGTGAA CAAATTGGTATTAAATACCGAAATTCGAACTTTTTCTGTCCACTTATTGATGAGAATGGACGGTGCTTTAC ATTGGAACGAAAGGAGTTGGATGATCAAGTTGATTTGCATTATAATAATGTTCTTGATGCAATTCGGAAT AAGTATCAGAGCGATCGGAATTTTCCGGATGTTCCTCTAGAAGAAATGTTCTCGAAAATGAGTTCCGGAT TGCTCTCGGCCGCCGACTTGGACTCTCTCTACACTCCTGAATTTGAAAAACTTTTGGATTTTCATCTAGGA AATCTGGAATTCTCTTGTGGAACCCATGTTTCAAATCTTTCGGCAAAAGACTATGATCATAATGAAAAGT TTGGAAACTTTGCCGGAGAGCATGCAGTTATCACTGATGGAGCACAGAGAATTATTGATTTTTTGGCAAC TGGATTGGATATTCGACTGAATTGCCCTGTTAAGTGCATTGATTGGGGCAGAGATGATCGAAAAGTCAAA ATATTCTTTGAAAATGCTGAACAAGCAGCGGAAGAGTTTGATAAAGTAGTTATCACAACTTCCCTATCAG TACTCAAATCAAATCATTCAAAAATGTTCGTTCCTCCACTTCCAATTGAAAAACAGAAGGCTATTGATGA TCTGGGCGCTGGATTAATTGAGAAGATCGCTGTGAAGTTTGACAGAAGATTTTGGGATACTGTAGATGCT GATGGATTGCGAACGGAATATTTCGGAAAGGTTTCCGATTGCAAGACTGATCGAAGTCTCTTCAATATAT TCTACGACTTTTCTGGAAAGGACCCCAATGGCGAGGACACCTTCGTCCTAATGTCCTATGTCACCGCTGA ACACGTGAATCTTGTCAATGTACTAACGGAATCGGAAGTCGCCGACAAATTCTGTGCAACCCTTCGGAAG ATGTTCCCATCAGCTGTTATTAACCCTCTAGGACATATGATGTCCCATTGGGGTGCTGATCGATTCGTCGG TATGTCGTACACTTTTGTCCCATTTGGATCGGATGGTGATGCTACGTATAATCAACTGAAAAAGTCAATCG ACGAGAAGCTCTACTTTGCGGGAGAACATACAATTGCAGCGGAACCGCAGACTATGGCAGGAGCCTATA TTTCAGGATTACGAGAAGCCGGGCAGATTGTGATGAGTTTGAAGCGGGATTCGGCCACTTTCGAATAA

Corresponding predicted amino-acid Sequence for amx-1: (Obtained using http://www.ebi.ac.uk/Tools/st/emboss_transeq/) Length = 816 amino acids

MTTELEIDDRKEEEAQIPGETSESEEGDEPVKMNRVKRACAMAAESLKEAKRIKNMLMEE ECVLQLGNCKKTASYGHRLSKTEACCPSCFSVAFRGKDYENEFTIWKQKAMDGQTHVRSE QFVKRYVNSCFLPYYAKCHQCSKYSKLITSDSLSAQQLSDFKCDCASTIESPKIERVRED SEWCFNEFGHPPLLQNNISYDLLVDHYVTRTTGMDATCQEKAALIDNGGIEFRDTRRIMN MFYVPFTDVIANIVHPEFMETDEKFAFPKFADDPISIYYLQVRNTIIAMWLKHPFVELTV KMIEPQIIVRGHARIFFIEHLIHPILEFLTIKGVVNYGAFDFRIDPLNGMRPKIAIIGAG ISGISTARHLKHLGIDAVLFEAKDRFGGRMMDDQSLGVSVGKGAQIIVGNINNPITLLCE QIGIKYRNSNFFCPLIDENGRCFTLERKELDDQVDLHYNNVLDAIRNKYQSDRNFPDVPL 132

EEMFSKMSSGLLSAADLDSLYTPEFEKLLDFHLGNLEFSCGTHVSNLSAKDYDHNEKFGN FAGEHAVITDGAQRIIDFLATGLDIRLNCPVKCIDWGRDDRKVKIFFENAEQAAEEFDKV VITTSLSVLKSNHSKMFVPPLPIEKQKAIDDLGAGLIEKIAVKFDRRFWDTVDADGLRTE YFGKVSDCKTDRSLFNIFYDFSGKDPNGEDTFVLMSYVTAEHVNLVNVLTESEVADKFCA TLRKMFPSAVINPLGHMMSHWGADRFVGMSYTFVPFGSDGDATYNQLKKSIDEKLYFAGE HTIAAEPQTMAGAYISGLREAGQIVMSLKRDSATFE*

Nucleotide sequence of amx-2L as obtained by sequencing of yk1054h04 cDNA: (Start codon is highlighted in green and stop codon is highlighted in red) Length = 2175 bp

ATGTTGCGTGGGTGGTGGATATTCATTTGGGCGGCTGTCTGCGTCTATGCACAGAATACACTCTCACAAC AGTCACAGCAACAACAGCAACCAACGTCTGCAAATATTAGCAGTTCTAATATTCAGCAAACAATATATGA TGTCATCGTTGTCGGAGCAGGACTTACCGGGCTCACAGCAGCCCGGAACATCCAGCAAAATCGACCAGG ACTATCCGTATTAGTACTGGAAGCACGTGGTCAAGTTGGTGGCAGAATTCGATATGCTACAATGCAGACA AGAAATGGTGTTGAATTCGTAGATACAGGATCTCAGTTCATTTCTCCAACGGACACCCAATTATTAAGTC TAATTCAAGAATTGAATGTTCGTACAACACAACAGTTGACATGTGGTAATAATACAGTATTCCAGCAAAC AAGGAAGAAGAGACAACTATCCCTCCAACAGCAATGGAGTACAACCCTCTTCACAGATCTTATCAATTCG CCAGAAACTCTCGGAAATCTTACAAATACTTCAGTTTCAGCAATGTCTCAGCAAATGGACACTGCTGACG CAGATTCGGTGAACCGAATGATGCAGACATTTTTCGATGCTCCCGGAGAACAAGTTCCAGAAATTCAGCT GGCCCTGACGTGTTCCAGTCAAAATGCGACAGCCGTTGAAATTCTCCGGAGATTTGGTCATGGGCAGAGT CTTTTGGCACAAGGTGGAATGAATGAAGTCGTGAGAAGGCTTGCTGATGGATTATTGATTGAATATAGTC AACGTGTCGTTTCGGTTAATGATGCCGCCTTCCCAGCTGTGGTCCAAACATCTGCCGGCCGAAGGTTCAG TGGCCGTCAAGTGATTGTAGCTGTGCCAATTCCGACACTGGAAAATATTGAGCTGGTACCAGCACCAGAA GCTCCCTTTCAGCAGCTTATTCAAAACTACGGTCCAACAGGCCACGCCTACTACTTCACAATGTCATTCCA ACGTGCCACGTGGCGGCTAAATGGGCGGAGCGGAAAAGTGATCTACACGTCGGCGACTGGTCCATTGGT ATGGCTGACCACATTTGATACCACTTTTGCGGCGAGTTGTGACAACTCCACATCAGCGTCATCAACACTTT GGGGTATCGCCCATTTCTCCTACGATGTTCCGTTCGAGACCCGCCGCAAATTGTACACTCAGGCGATTATG TACTCGCTGAGATTTGCTGATTTTTCGCCATTGGATGTTAGTGACGTCAACTTTGCCACGGACGACTTGGC GAAAGGAACAATTCCAACATTGAAGCTTAATATTCCATTAGAATCCCTGAAATATCTCAACGATTTCCAT ACTTTGTACCAAAATGTACATATTGCTACTGCGGATATTGCCTCGCACAATTTGGGTACCATGAACGGAG CCGTACACGCAGCTGGCTCTGTGAGCACATATGTGCTCCAAATGCTCTCAGCAGCAGATGCTCAAAGTAA CGGAGTCCTTAGGGATACACCAGTCGAGTCGACAACTCCATACGTCTACAGTACCTCCTCACACTACCCA CCATCGCTTTTCGCGGCTCGAAATGTGCAAAATGACTCTGGAAATAGAAATACCACTGGAAACAATTCTG TGAACTCTGGAAATACTGGAAATCAAACAAATTTCCAATATGAAACTAGTTCACAGTACCCGACTACTGT AGAAACACCAACAACTACTACTATGAAACATTTCAGTTTTGGAAATTTGGACAACTCCACAAATGAAGAA CCGGCTGCCGTGGCCTACGACAGCCCACAACAGGATGCCCCACCAACATTGAACTATTCAACGACATTTG CGTATTCCACTTCAAGTGTAATGCCACTAGTCGAGACGATCGAAACGACAACTTTTAAACATTTTAGTAA CGGAAATTTAGATCAAAATGCCACGTCAGCTGTTCAACGAGATGTGCCAGCAGTTCTCGTAAATCAACAG CAGAATGGTTACGCCTACACGACATCAACTCATTATCCAGTACAATTATCTTCAAATCGAAATAGAATCG ACTCACGAGCAACCCCATCTCCACAAGTTGTGCAGGAGTTGCAACAAGTTTCAGCAAATGCTTCCAACTC GACGGCTCTCCAATTGGCGTCAAGTCTGACGCAATTGGTGCAAACACTCTTACAGACTTTGCGTCTACAG TAA

Corresponding predicted amino-acid Sequence for amx-2L: (Obtained using http://www.ebi.ac.uk/Tools/st/emboss_transeq/) Length = 724 amino acids

MLRGWWIFIWAAVCVYAQNTLSQQSQQQQQPTSANISSSNIQQTIYDVIVVGAGLTGLTA ARNIQQNRPGLSVLVLEARGQVGGRIRYATMQTRNGVEFVDTGSQFISPTDTQLLSLIQE LNVRTTQQLTCGNNTVFQQTRKKRQLSLQQQWSTTLFTDLINSPETLGNLTNTSVSAMSQ QMDTADADSVNRMMQTFFDAPGEQVPEIQLALTCSSQNATAVEILRRFGHGQSLLAQGGM NEVVRRLADGLLIEYSQRVVSVNDAAFPAVVQTSAGRRFSGRQVIVAVPIPTLENIELVP APEAPFQQLIQNYGPTGHAYYFTMSFQRATWRLNGRSGKVIYTSATGPLVWLTTFDTTFA ASCDNSTSASSTLWGIAHFSYDVPFETRRKLYTQAIMYSLRFADFSPLDVSDVNFATDDL AKGTIPTLKLNIPLESLKYLNDFHTLYQNVHIATADIASHNLGTMNGAVHAAGSVSTYVL 133

QMLSAADAQSNGVLRDTPVESTTPYVYSTSSHYPPSLFAARNVQNDSGNRNTTGNNSVNS GNTGNQTNFQYETSSQYPTTVETPTTTTMKHFSFGNLDNSTNEEPAAVAYDSPQQDAPPT LNYSTTFAYSTSSVMPLVETIETTTFKHFSNGNLDQNATSAVQRDVPAVLVNQQQNGYAY TTSTHYPVQLSSNRNRIDSRATPSPQVVQELQQVSANASNSTALQLASSLTQLVQTLLQT LRLQ*

Nucleotide sequence of amx-2S as obtained by sequencing of yk1326e11 cDNA: (Start codon is highlighted in green and stop codon is highlighted in red) Length = 2079 bp

ATGTTGCGTGGGTGGTGGATATTCATTTGGGCGGCTGTCTGCGTCTATGCACAGAATACACTCTCACAAC AGTCACAGCAACAACAGCAACCAACGTCTGCAAATATTAGCAGTTCTAATATTCAGCAAACAATATATGA TGTCATCGTTGTCGGAGCAGGACTTACCGGGCTCACAGCAGCCCGGAACATCCAGCAAAATCGACCAGG ACTATCCGTATTAGTACTGGAAGCACGTGGTCAAGTTGGTGGCAGAATTCGATATGCTACAATGCAGACA AGAAATGGTGTTGAATTCGTAGATACAGGATCTCAGTTCATTTCTCCAACGGACACCCAATTATTAAGTC TAATTCAAGAATTGAATGTTCGTACAACACAACAGTTGACATGTGGTAATAATACAGTATTCCAGCAAAC AAGGAAGAAGAGACAACTATCCCTCCAACAGCAATGGAGTACAACCCTCTTCACAGATCTTATCAATTCG CCAGAAACTCTCGGAAATCTTACAAATACTTCAGTTTCAGCAATGTCTCAGCAAATGGACACTGCTGACG CAGATTCGGTGAACCGAATGATGCAGACATTTTTCGATGCTCCCGGAGAACAAGTTCCAGAAATTCAGCT GGCCCTGACGTGTTCCAGTCAAAATGCGACAGCCGTTGAAATTCTCCGGAGATTTGGTCATGGGCAGAGT CTTTTGGCACAAGGTGGAATGAATGAAGTCGTGAGAAGGCTTGCTGATGGATTATTGATTGAATATAGTC AACGTGTCGTTTCGGTTAATGATGCCGCCTTCCCAGCTGTGGTCCAAACATCTGCCGGCCGAAGGTTCAG TGGCCGTCAAGTGATTGTAGCTGTGCCAATTCCGACACTGGAAAATATTGAGCTGGTACCAGCACCAGAA GCTCCCTTTCAGCAGCTTATTCAAAACTACGGTCCAACAGGCCACGCCTACTACTTCACAATGTCATTCCA ACGTGCCACGTGGCGGCTAAATGGGCGGAGCGGAAAAGTGATCTACACGTCGGCGACTGGTCCATTGGT ATGGCTGACCACATTTGATACCACTTTTGCGGCGAGTTGTGACAACTCCACATCAGCGTCATCAACACTTT GGGGTATCGCCCATTTCTCCTACGATGTTCCGTTCGAGACCCGCCGCAGATTGTACACTCAGGCGATTATG TACTCGCTGAGATTTGCTGATTTTTCGCCATTGGATGTTAGTGACGTCAACTTTGCCACGGACGACTTGGC GAAAGGAACAATTCCAACATTGAAGCTTAATATTCCATTAGAATCCCTGAAATATCTCAACGATTTCCAT ACTTTGTACCAAAATGTACATATTGCTACTGCGGATATTGCCTCGCACAATTTGGGTACCATGAACGGAG CCGTACACGCAGCTGGCTCTGTGAGCACATATGTGCTCCAAATGCTCTCAGCAGCAGATGCTCAAAGTAA CGGAGTCCTTAGGGATACACCAGTCGAGTCGACAACTCCATACGTCTACAGTACCTCCTCACACTACCCA CCATCGCTTTTCGCGGCTCGAAATGTGCAAAATGACTCTGGAAATAGAAATACCACTGGAAACAATTCTG TGAACTCTGGAAATACTGGAAATCAAACAAATTTCCAATATGAAACTAGTTCACAGTACCCGACTACTGT AGAAACACCAACAACTACTACTATGAAACATTTCAGTTTTGGAAATTTGGACAACTCCACAAATGAAGAA CCGGCTGCCGTGGCCTACGACAGCCCACAACAGGATGCCCCACCAACATTGAACTATTCAACGACATTTG CGTATTCCACTTCAAGTGTAATGCCACTAGTCGAGACGATCGAAACGACAACTTTTAAACATTTTAGTAA CGGAAATTTAGATCAAAATGCCACGTCAGCTGTTCAACGAGATGTGCCAGCAGTTCTCGTAAATCAACAG CAGAATGGTTACGCCTACACGACATCAACTCATTATCCAGTACAATTATCTTCAAATCGAAATAGAATCG ACTCACGAGCAACACCTGCACATCTAACTTTTTTCCTGAATTTTTAA

Corresponding predicted amino-acid Sequence for amx-2S: (Obtained using http://www.ebi.ac.uk/Tools/st/emboss_transeq/) Length = 692 amino acids

MLRGWWIFIWAAVCVYAQNTLSQQSQQQQQPTSANISSSNIQQTIYDVIVVGAGLTGLTA ARNIQQNRPGLSVLVLEARGQVGGRIRYATMQTRNGVEFVDTGSQFISPTDTQLLSLIQE LNVRTTQQLTCGNNTVFQQTRKKRQLSLQQQWSTTLFTDLINSPETLGNLTNTSVSAMSQ QMDTADADSVNRMMQTFFDAPGEQVPEIQLALTCSSQNATAVEILRRFGHGQSLLAQGGM NEVVRRLADGLLIEYSQRVVSVNDAAFPAVVQTSAGRRFSGRQVIVAVPIPTLENIELVP APEAPFQQLIQNYGPTGHAYYFTMSFQRATWRLNGRSGKVIYTSATGPLVWLTTFDTTFA ASCDNSTSASSTLWGIAHFSYDVPFETRRRLYTQAIMYSLRFADFSPLDVSDVNFATDDL AKGTIPTLKLNIPLESLKYLNDFHTLYQNVHIATADIASHNLGTMNGAVHAAGSVSTYVL QMLSAADAQSNGVLRDTPVESTTPYVYSTSSHYPPSLFAARNVQNDSGNRNTTGNNSVNS GNTGNQTNFQYETSSQYPTTVETPTTTTMKHFSFGNLDNSTNEEPAAVAYDSPQQDAPPT LNYSTTFAYSTSSVMPLVETIETTTFKHFSNGNLDQNATSAVQRDVPAVLVNQQQNGYAY 134

TTSTHYPVQLSSNRNRIDSRATPAHLTFFLNF*

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APPENDIX D: PROTEIN DOMAIN ANALYSES RESULTS

The amino acid sequences obtained above were analyzed by PROSITE (ExPASy) freeware (http://prosite.expasy.org/) to identify conserved protein domains. The protein domains were then analyzed against C. elegans and H. sapiens protein databases using the protein-BLAST (NCBI) freeware (http://blast.ncbi.nlm.nih.gov/Blast.cgi). AMX1

C. elegans AMX1 possesses the following putative domains

- the SWIRM domain (residues 251-337):

- an NAD(P) binding Rossman-like domain (residues 356-422):

- an FAD binding domain (residues 354-382): Almost completely within the NAD(P) domain.

- the AO domain (residues 361-804): Overlaps with the NAD(P) and the FAD domains [FAD

Comparison of domain identity with AMX2, SPR5, LSD1 in C. elegans; and LSD1 (KDM1A / AOF2), LSD2 (KDM1B / AOF1), MAO(A,B) in humans.

AMX1 Worm Worm Worm Human Human Human domains AMX2 SPR5 LSD1 LSD1 LSD2 MAOs SWIRM Not found 23% 28% 23% 44% Not found domain FAD 30% Not found Not found 45% 41% 41% binding Domain NAD(P) 32% Not found <10% 37% 57% 45% binding domain AO 26% 28% 26% 25% 38% 25% domain

The nearest human homologs for each domain are highlighted.

AMX2 C. elegans AMX2 possesses the following putative domains

- an NAD(P) binding Rossman-like domain (residues 38-78):

136

- an FAD binding domain (residues 47-84): Almost completely overlaps with the NAD(P) domain.

- and the AO domain (residues 55-480): Overlaps partially with the FAD and NAD(P) domains.

Comparison of domain identity with AMX1, SPR5, LSD1, in C. elegans; and LSD1, LSD2, MAO-A, MAO-B, in humans.

AMX2 Worm Worm Worm Human Human Human Human domains AMX1 SPR5 LSD1 LSD1 LSD2 MAO-A MAO-B NAD(P)- 31% Not Not 51% Not 44% 44% bind found found found domain FAD- 30% Not Not 50% Not 50% 47% bind found found found domain AO 26% 29% 28% 19% 19% 19% 19% domain

The nearest human homologs for each domain are highlighted.

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APPENDIX E: SUPPLEMENTARY FIGURES

Figure S1: Plasmid map for Gateway entry vector clone containing amx-1 (yk1390d04) cDNA. The amx-1 sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto-annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S2: Plasmid map for Gateway entry vector clone containing amx-2L (yk1054h04) cDNA. The amx-2L sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto-annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S3: Plasmid map for Gateway entry vector clone containing amx-2S (yk1326e11) cDNA. The amx-2S sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto-annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S4: Plasmid map for Gateway destination (expression) vector clone containing amx-1 (yk1390d04) cDNA. The amx-1 sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto- annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S5: Plasmid map for Gateway destination (expression) vector clone containing amx-2L (yk1054h04) cDNA. The amx-2L sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto- annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S6: Plasmid map for Gateway destination (expression) vector clone containing amx-2S (yk1326e11) cDNA. The amx-2S sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto- annotating plasmid maps" Nucleic Acids Res. 2004).

143

Figure S7: Plasmid map for 2GFP-T (p29716) expression vector clone containing amx-1 (yk1390d04) cDNA. The amx-1 sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto- annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S8: Plasmid map for 2GFP-T (p29716) expression vector clone containing amx-2L (yk1054h04) cDNA. The amx-2L sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto- annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S9: Plasmid map for yeast expression vector (pPIC3.5) clone containing amx-1 (yk1390d04) cDNA. The amx-1 sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto- annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S10: Plasmid map for yeast expression vector (pPIC3.5) clone containing amx-2L (yk1054h04) cDNA. The amx-2L sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto- annotating plasmid maps" Nucleic Acids Res. 2004).

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Figure S11: Plasmid map for yeast expression vector (pPIC3.5) clone containing amx-2S (yk1326e11) cDNA. The amx-2S sequence is shown in magenta. Plasmid maps were prepared using the PlasMapper software (Version 2.0; Xiaoli Dong, Paul Stothard, Ian J. Forsythe, and David S. Wishart; "PlasMapper: a web server for drawing and auto- annotating plasmid maps" Nucleic Acids Res. 2004).

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