Regulation of NAD Kinase in Neutrophils

Regulation of NAD kinase in neutrophils

Razieh Rabani

Department of Medicine, Division of Experimental Medicine McGill University Montreal, Quebec, Canada

August 2015

A thesis submitted to the McGill University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Dedication

This work is dedicated to my wonderful parents.

Acknowledgements

First and foremost, I would like to thank my supervisor, Dr. William Powell for his inspiring knowledge and attitude concerning everything scientific and for his continual support and encouragement.

I am very grateful to my thesis committee advisors for their helpful advice on my project; Dr. Simon Rousseau, Dr. Elizabeth Fixman, Dr. Arnold Kristof and Dr. Martin Olivier. I would like to thank Dr. Denis Faubert for performing LC-MS/MS analysis and helping to interpret data.

I appreciate the time and effort of the members of the Meakins-Christie Laboratories in particular Dr. James Martin and Dr. Qutayba Hamid for providing a collaborative research environment with workshops and seminars.

I am truly thankful to our research assistant, Chantal Cossette for teaching me many techniques and taking care of orders. I am very grateful to our lab technician, Sylvie Gravel who makes everything work, and always with a smile!

I thank Julie Berube and Guy Martel for their advice on protein expression. I would like to thanks Di Xue for her moral support and being a great friend. I would like to give my thanks to Dr. Oleg Matusovsky for unwavering support and valuable advice on many aspects of my project.

I am very grateful to Leo and Kathy for taking time of their busy schedules to draw blood from patients for my project and I would like to give special thanks to all of the blood donors at the Meakins-Christie and the Montreal Chest Institute.

My deepest appreciation goes to my parents for their continual support over years and finally, I would like to give many special thanks to my love, Mehdi who is my greatest inspiration.

Table of Contents

Table of Contents ...... iii

List of Abbreviations ...... ix

List of Tables ...... xv

List of Figures ...... xvi Abstract ...... xix Résumé ...... xxi Contribution of Authors ...... xxiii

Chapter1. Introduction and Literature Review ...... 1

1.1 Biology of neutrophils ...... 1

1.1.1 Introduction ...... 1

1.1.2 Neutrophil activation and migration ...... 1

1.1.3 Phagocytosis ...... 2

1.1.4 Neutrophil granules ...... 2

1.1.5 Neutrophil extracellular traps (NETs) ...... 3

1.1.6 NOX family ...... 4

1.1.7 Neutrophil apoptosis ...... 6

1.1.8 Neutrophils and adaptive immunity ...... 6

1.1.9 Neutrophils and diseases ...... 7

1.2 Pyridine nucleotides ...... 8

1.2.1 Introduction ...... 8

1.2.2 Biosynthesis of NAD+ ...... 8

1.2.3 Production of NADP+ and NADPH ...... 10

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1.2.4 Pyridine nucleotides in metabolism and energy production ...... 12

1.2.5 Pyridine nucleotides in protein modification ...... 12

1.2.6 Pyridine nucleotides in calcium signaling ...... 13

1.2.7 Pyridine nucleotides in regulation of redox state ...... 13

1.3 Eicosanoid Biosynthesis ...... 13

1.3.1 Introduction ...... 13

1.3.2 5-Lipoxygenase pathway ...... 15

1.3.3 5-oxo ETE formation and regulation ...... 15

1.3.4 Biological functions of 5-oxo ETE in PMNs ...... 16

1.4 NAD kinase, a crucial enzyme for biosynthesis of NADP+ ...... 18

1.4.1 Introduction ...... 18

1.4.2 Structural properties of NADK ...... 18

1.4.3 Subcellular location of NADK...... 19

1.4.3.1 Yeasts ...... 19

1.4.3.1 Plants ...... 21

1.4.3.1 Human ...... 21

1.5 Enzymatic properties of NADK...... 22

1.5.1 Substrate specificity ...... 22

1.5.2 Modulation of NADK activity ...... 22

1.6 Human cytosolic NADK ...... 24

1.6.1 Properties of human NADK...... 24

1.6.2 Regulation of human NADK by divalent cations ...... 25

1.6.3 Role of human NADK in regulating intracellular NADPH ...... 25

1.6.4 Regulation of human NADK by Ca2+/ calmodulin ...... 26

1.6.5 Activation of NADK by PMA ...... 27

1.6.6 Potential binding partner of NADK ...... 28

1.6.7 Alternatively spliced isoforms of NADK ...... 28

1.7 Human mitochondrial NAD kinase ...... 30

1.8 Protein kinase C ...... 32

1.8.1 Introduction ...... 32

1.8.2 Classification of PKC enzymes ...... 33

1.8.3 Regulation of PKCs ...... 33

1.8.4 ATP-competitive Inhibitors of PKCs...... 35

1.9 14-3-3 proteins ...... 35

1.9.1 General properties ...... 35

1.9.2 Interaction of 14-3-3 proteins with other proteins ...... 36

1.9.2.1 Phosphorylation-dependent interactions ...... 36

1.9.2.2 Interaction with non-phosphorylated proteins ...... 37

Chapter 2. Aim of Study ...... 38

2.1 Activation of NADK by PKC in neutrophils ...... 38

Chapter 3. Material and Methods ...... 39

3.1 Materials ...... 39

3.2 Isolation of neutrophils ...... 39

3.3 Quantitation of NAD+ and NADP+ ...... 40

3.4 Measurement of NADPH ...... 40

3.5 Measurement of cytosolic calcium levels in PMA stimulated neutrophils...... 41

3.6 Differentiation of PLB-985 cells into neutrophil like cells ...... 41

3.7 Preparation of cell lysates ...... 41

3.8 Protein quantitation ...... 41

3.9 Measurement of NAD kinase activity...... 42

3.10 Western blotting ...... 42

3.11 Immunoprecipitation ...... 42

3.12 Production of recombinant NADK ...... 43

3.12.1 Cloning of cDNA of NADK ...... 43

3.12.2 Expression of NADK in E.Coli ...... 43

3.12.3 Purification of GST-NADK from bacterial extracts ...... 43

3.12.4 Cleavage of GST using PreScission protease ...... 44

3.13 In vitro phosphorylation of GST-NADK by PKC-δ ...... 44

3.14 Partial purification of NADK ...... 44

3.14.1 Size exclusion chromatography ...... 44

3.14.2 Hydroxyapatite chromatography ...... 45

3.14.3 Hydrophobic interaction chromatography ...... 45

3.14.4 Ion exchange chromatography ...... 45

3.15 LC-MS-MS Analysis ...... 46

3.15.1 Preparation of samples ...... 46

3.15.2 In-gel digestion of proteins ...... 46

3.15.3 LC-MS/MS ...... 46

3.15.4 Protein identification ...... 47

3.16 Data analysis ...... 47

Chapter 4: Results...... 47

4.1 PMA activates NADK in intact neutrophils ...... 47

4.1.1 PMA has a dramatic effect on pyridine nucleotide levels in intact neutrophils ...... 47

4.1.2 PMA-induced activation of NADK is independent of NADPH oxidase and calcium mobilization ...... 49

4.1.3 PKC is required for the activation of NADK by PMA ...... 51

4.2 NADK activity in cell lines ...... 54

4.3 NADK activity is elevated in lysates from PMA-stimulated neutrophils ...... 56

4.4 Detection and immunoprecipitation of NADK using various antibodies ...... 60

4.4.1 Monoclonal anti-NADK antibody ...... 60

4.4.2 Polyclonal (H300) anti-NADK antibody ...... 64

4.4.3 Polyclonal (A) anti-NADK antibody ...... 65 4.4.4 Polyclonal (B) anti-NADK antibody ...... 66

4.4.5 Polyclonal (C) anti-NADK antibody ...... 66

4.5 Post-translational modification of NADK ...... 67

4.5.1 Detection of NADK phosphorylation in neutrophils with a phospho-(Ser) PKC substrate antibody...... 68 4.5.2 PKC-induced modification of recombinant NADK ...... 69

4.5.2.1 Production of recombinant NADK in E.coli ...... 69

4.5.2.2 Characterization of recombinant NADK ...... 71

4.5.2.3 In vitro phosphorylation of recombinant NADK ...... 74

4.6 Partial purification of NADK from human neutrophils ...... 74

4.6.1 Size exclusion chromatography ...... 76

4.6.2 Hydroxyapatite chromatography ...... 78

4.6.3 Hydrophobic interaction chromatography ...... 78 4.6.4 Ion exchange chromatography ...... 81

4.7 Analysis of post-translational modification of NADK by LC-MS/MS ...... 85

4.7.1 Analysis of NADK purified from PMA- treated neutrophils by IP (MC-aNADK Ab) ...... 85

4.7.2 Analysis of NADK purified from vehicle- treated neutrophils by conventional methods ..87

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4.7.3 Analysis of NADK purified from vehicle treated neutrophils by a combination of chromatography and IP (Poly (B) aNADK) ...... 89 4.7.4 Analysis of NADK purified by IP (poly (B) aNADK Ab) ...... 92

4.7.5 Analysis of GST-NADK before and after treatment with PKC-δ ...... 96

4.8 Interaction of NADK with other proteins ...... 99

4.8.1 14-3-3 protein, a candidate for regulation of NADK ...... 99

4.8.2 PMA changes the interaction of NADK with 14-3-3 zeta protein ...... 99

Chapter 5. Discussion ...... 102

5.1 PMA activates NADK in intact neutrophils ...... 102

5.2 NADK activity is elevated in lysates from PMA-stimulated neutrophils ...... 104

5.3 Post translational modification of NADK ...... 106

5.4 Partial purification of human NADK from neutrophils ...... 108

5.5 LC-MS/MS analysis of NADK ...... 111

5.6 PMA changes the interaction of NADK with 14-3-3 zeta protein...... 117

5.7 Conclusion and implications in diseases...... 119

Chapter 6. Prospects for future studies ...... 120

Claims to original research ...... 122

References ...... 123

Appendix ...... 142

Figure S.1. MS/MS spectra of 43k NADK form vehicle-treated neutrophil ...... 142

Figure S.2. MS/MS spectra of 49k NADK form vehicle-treated neutrophils ...... 143

Figure S.3. MS/MS spectra of 43k NADK form PMA-treated neutrophils ...... 144

Figure S.4. MS/MS spectra of 49k NADK form PMA-treated neutrophils ...... 145

Figure S.5. MS/MS spectra of recombinant GST-NADK ...... 146

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List of Abbreviations

5-HEDH 5-hydroxyeicosanoid dehydrogenase

5-HETE 5S-hydroxy-6, 8, 11, 14-eicosatetraenoic acid

5-HpETE 5S-hydroperoxy-6, 8, 11, 14-eicosatetraenoic acid

5-LO 5-lypoxygenase

5-oxo-ETE 5-oxo-6,8,11,14-eicosatetraenoic acid

6PG 6-phosphogluconate

AA Arachidonic acid

AIR Autoinhibitory region

ANOVA Analysis of variance

AP-1 Activator protein 1

APAF-1 Apoptotic protease-activating factor 1

APS Acrylamide/bisacrylamide solution, ammonium persulfate

ARTs ADP - ribosyl transferases

AtNADK Arabidopsis thaliana NADK

BAD BCL-2-associated death promoter

BAX BCL2-associated X protein

BPI Bactericidal/permeability-increasing protein

BSA Bovine serum albumin cADPR Cyclic ADP-ribose

CaM Calmodulin

CAM Carbamidomethyl

CF Cystic fibrosis

CGD Chronic granulomatous disease

CHAPS 3-[(3-cholamidopropyl)-dimethylammonio]1-propanesulfonate

CkI Cyclin-dependent kinase inhibitor

COPD Chronic obstructive pulmonary disease cPLA2 Calcium-dependent phospholipase A2

DAG Diacyl-glycerol

DCs Dendritic cells

DEAE Diethylaminoethyl

DISC Death-inducing signaling complex

DMSO Dimethyl sulfoxide

DPI Diphenylene iodonium

DSS Disuccinmidyl suberate

DTT Dithiothreitol

DUOXA Dual oxidase enzyme

EcNADK Escherichia coli NADK

EDTA Ethylenediaminetetraacetic acid

FLAP 5-LO activating protein

G6P Glucose 6-phosphate

G6PDH Glucose 6-phosphate dehydrogenase

GCF Granulocyte-colony stimulating factor

GM-CSF Granulocyte macrophage colony-stimulating factor

GSH Glutathione reduced state

HA CHT hydroxyapatite hCAP-18 The human cationic antimicrobial protein

HsNADK Homo sapiens NADK

IDHc Isocitrate dehydrogenase, cytosolic

IDHm Isocitrate dehydrogenase, mitochondrial

IL-3 Interleukin 3

IFN-γ Interferon gamma

IP Immunoprecipitation

IPTG Isopropyl β-D-1-thiogalactopyranoside

IS Nicotinamide hypoxanthine dinucleotide

JNK c-Jun N-terminal protein kinase

LPS Lipopolysaccharide

LT(A-D) Leukotriene (A-D)

LTA4H Leukotriene A4 hydrolase

MEc Malic enzyme, cytosolic

Mem Malic enzyme, mitochondrial

MHC Major histocompatibility complex

MPO Myeloperoxidase

MtNADK Mycobacterium tuberculosis NADK

MTT Thiazolyl blue tetrazolium blue (MTT

NaAD Nicotinic acid adenine dinucleotide

NAADP Nicotinic acid adenine dinucleotide phosphate

NAD Nicotinamide adenine dinucleotide

NADPH Nicotinamide adenine dinucleotide phosphate

NADS NAD+ synthase

NaMN Nicotinic acid mononucleotide

NaMNAT Nicotinic acid mononucleotide adenylyl transferase

NET Neutrophil extracellular trap

NFAT Nuclear factor of activated T-cells

NGAL Neutrophil gelatinase-associated lipocalin

NMN Nicotinamide mononucleotide

NMNAT Nicotinamide mononucleotide adenylyl transferase

NOX NADPH oxidase

PAMPs Pathogen-associated molecular patterns

PARPs Poly (ADP) – ribosyl transferases

PB-1 Phox and Bem1 domain

PBS Phosphate buffered saline

PGP Proline-glycine-proline tripeptide

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PGs Prostaglandins

PIP3 Phosphatidylinositol 3,4,5-trisphosphate

PKC Protein kinase C

PLC Phospholipase C

PMA Phorbol myristate acetate

PMS Phenazine meta sulphate

PMSF Phenylmethylsulfonyl flouride

PPP Pentose phosphate pathway

PRR Proline rich region

PS Phosphatidylserine

PSGL-1 P-selectin glycoprotein ligand-1

PX N-terminal phox homology

QPRT Phosphoribosyltransferase

RA Rheumatoid arthritis

RACK Receptor for activated C-kinase

ROS Reactive oxygen species

RP-HPLC Reversed phase high-performance liquid chromatography

ScNADK Saccharomyces cerevisiae NADK

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SLE Systemic lupus erythematosus

SOD Superoxide dismutase

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TBAH Tetrabutylammonium hydroxide

TBST Tris-Buffered Saline and Tween 20

TCA Tricarboxylic acid cycle

TEMED N, N,N”,N’-tetramethylethylenediamine

TH Transhydrogenase

TLR Toll-like receptor

TMB-8 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester

TNF-α Tumor necrosis factor alpha

TRAIL TNF-related apoptosis-inducing ligand

Trx Thioredoxin

TX Thromboxanes

XIAP X-linked inhibitor of apoptosis

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List of tables

Table 1.1. Potential binding partner of human NADK based on BioGRID (database of molecular interactions)...... 29

Table 1.2. Alternatively spliced transcripts of NADK ...... 30

Table 4.1. The intensity of ion signal for the peptide carrying P-S46 in NADK purified from vehicle and PMA treated neutrophils by IP using poly (B) aNADK Ab. Phosphorylated residues are shown in red text ...... 95

Table 4.2. The intensity of ion signal for the peptide carrying P-S48 in NADK purified from vehicle and PMA treated neutrophils by IP using poly (B) aNADK Ab. Phosphorylated residues are shown in red text ...... 95

Table 4.3. The intensity of ion signal for the peptide containing P-S64 in NADK purified from vehicle and PMA treated neutrophils by IP using poly (B) aNADK Ab. Phosphorylated residues are shown in red text ...... 95

Table 4.4. Reference peptides and their ion signal intensity in NADK purified from vehicle and PMA treated neutrophils by IP using poly(B) aNADK Ab ...... 95

Table 4.5. Comparison of ion signal intensity of the phospho-peptides in NADK purified from vehicle and PMA-treated neutrophils by IP using poly(B) aNADK Ab ...... 96

Table 4.6. Summary of LC-MS/MS analysis ...... 98

List of Figures

Figure 1.1. Activation of NADPH oxidase ...... 5

Figure 1.2. Biosynthesis of pyridine nucleotides ...... 9

Figure 1.3. Biological functions of pyridine nucleotides ...... 10

Figure 1.4. Scheme of pentose phosphate pathway (PPP) ...... 11

Figure 1.5. Pyridine nucleotides in regulation of redox state ...... 14

Figure 1.6. 5-Lypoxygenase (5-LO) pathway...... 17

Figure 1.7.Biosynthesis of NADP+ by NADK ...... 19

Figure 1.8. Partial multiple alignment of amino acid sequences of various NADKs using Clustal W...... 20

Figure 1. 9. Multiple alignment of alternatively spliced transcripts of human NADK ...... 31

Figure 1.10. Regulation of BAD with 14-3-3 ...... 37

Figure 4.1. Effect of PMA on pyridine nucleotide levels ...... 48

Figure 4.2. Effects of the NADPH oxidase inhibitor DPI on PMA-induced changes in pyridine nucleotide levels in neutrophils ...... 50

Figure 4.3. Lack of effect of PMA on intracellular calcium levels in neutrophils ...... 51 Figure 4.4. Effects of PKC inhibitors on PMA-induced changes in pyridine nucleotide levels in neutrophils ...... 52 Figure 4.5. Concentration-response curves for PKC inhibitors ...... 53 Figure 4.6. Effects of PMA on NAD(P)+ levels in PLB-985 cell ...... 54 Figure 4.7. Effects of PMA on NAD(P)+ levels in monocytes and monocytic cell lines ...... 55 Figure 4.8. Effects of PMA on NAD(P)+ levels in lymphocytic cell lines ...... 55 Figure 4.9. NADK activity in neutrophil lysates ...... 57

Figure 4.10. Effect of endogenous NADP and NADPH on NADK activity assay ...... 58

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Figure 4.11. Effect of NADH on NADK activity ...... 59 Figure 4.12. Effect of cationic ions on NADK activity ...... 59

Figure 4.13. Time course for the generation of immunoreactivity to MC-aNADK in neutrophils treated with PMA ...... 60

Figure 4.14. Immunoprecipitation of NADK using MC-aNADK ...... 62

Figure 4.15. Effects of PKC inhibitors on PMA-induced immunoreactivity of NADK ...... 63

Figure 4.16. Western blotting of NADK using polyclonal H300 anti NADK antibody ...... 64

Figure 4.17. Immunoprecipitation of NADK using polyclonal antibody (poly (A) aNADK Ab) 65

Figure 4.18. Western blotting of NADK using polyclonal antibobdy (poly (B) aNADK Ab)...... 66

Figure 4.19. Western blotting of of NADK using polyclonal antibody (poly (C) aNADK Ab) ...67

Figure 4.20. Detection of phosphorylated NADK with anti-phospho (Ser) PKC substrates antibody...... 68

Figure 4.21. Production of recombinant NADK. A ...... 70

Figure 4.22. Characterization of recombinant NADK ...... 72

Figure 4.23. Size exclusion chromatography of recombinant NADK ...... 73

Figure 4.24. In vitro phosphorylation of recombinant GST-NADK ...... 75

Figure 4.25. Purification of NADK performing size exclusion chromatography (SEC) ...... 77

Figure 4.26 Purification of NADK by hydroxyapatite chromatography (HA) ...... 79

Figure 4.27 Purification of NADK doing hydrophobic interaction chromatography (HIC) ...... 80

Figure 4.28. Purification of NADK by anion exchange chromatography (SAX) ...... 82

Figure 4.29. Effect of staurosporine on activity of NADK partially purified by SAX...... 83

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Figure 4.30 Purification of NADK doing anion exchange chromatography using Q-STAT column...... 84

Figure 4.31 LC-MS/MS analysis of NADK from PMA-treated neutrophils purified by SEC and IP (MC-aNADK) ...... 86

Figure 4.32 LC-MS/MS analysis of NADK from vehicle-treated neutrophils purified by conventional methods ...... 88

Figure 4.33 Analysis of NADK from vehicle-treated neutrophils purified by HA-IP (poly(B) aNADK) ...... 90

Figure 4.34 LC-MS/MS analysis of NADK from vehicle-treated neutrophils purified by SAX (Rainin)-IP (poly(B) aNADK) ...... 91

Figure 4.35. Analysis of NADK from vehicle and PMA-treated neutrophils purified by IP (poly(B) aNADK) ...... 93

Figure 4.36. LC-MS/MS analysis of GST-NADK before and after PKC-δ...... 97

Figure 4.37. 14-3-3 proteins as interacting partners of NADK ...... 100

Figure 4.38. PMA-induced change in interaction of 14-3-3zeta with NADK PMA ...... 101

Figure 5.1. Regulation of pyridine nucleotides by NADK ...... 103

Figure 5.2. Alignment of 44k Da and 49kDa alternatively spliced transcripts of human NADK using Clustal W program ...... 114

Figure 5.3. Schematics mechanism of NADK activation by PKC ...... 118

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Abstract

NADP+ and NADPH are involved in many biosynthetic pathways and signal transduction mechanisms. NAD kinase (NADK) is the only known enzyme that catalyzes the de novo synthesis of NADP+ from NAD+. In neutrophils, NADK plays an essential role to provide sufficient levels of NADPH to support a robust respiratory burst. Additionally, it would provide the NADP+ that is required for the synthesis of the potent granulocyte chemoattractant, 5-oxo-ETE. Despite the critical role of NADK, very little is known about its regulatory mechanisms in mammals. Activation of NADPH oxidase-2 (NOX-2) in neutrophils by stimulators of PKC, such as PMA, results in a robust respiratory burst, in which superoxide is generated from oxygen at the expense of oxidation of NADPH to NADP+. In this study, we measured the levels of pyridine nucleotides following the addition of PMA to neutrophils. The expected rapid rise in NADP+ levels was observed, but, contrary to expectations, this was not accompanied by the loss of NADPH, which initially also increased, but rather by the depletion of NAD+. The time course for NADP+ formation was precisely mirrored by a comparable decline in NAD+ levels, suggesting that NADK had been activated. We confirmed that the PMA-induced depletion of NAD+ in intact neutrophils is not dependent on NOX-2, since it is not blocked by the NOX inhibitor, DPI. Western blotting of neutrophil lysates with a monoclonal anti-NADK antibody revealed an intense band in PMA- treated neutrophils but only a very weak band in vehicle-treated neutrophils, suggesting that PKC induces a post-translation modification on NADK leading to an increase in its immunoreactivity. We also showed that the PMA-induced increases in NADK enzymatic activity and immunoreactivity are mediated by PKC, as they were blocked by the PKC inhibitors staurosporine and GO6983. The immunoreactivity of recombinant GST-NADK was also shown to dramatically increase following treatment with PKC-delta. Exposure to PMA or PKC appears to induce the phosphorylation of neutrophil NADK and recombinant NADK, respectively. LC-MS/MS analysis of NADK revealed that it is partially phosphorylated on S46, S48, S64 and T62 in resting neutrophils and that phosphorylation of S46 and S64 is markedly increased following exposure of neutrophils to PMA. Similarly, PKC-delta induced phosphorylation of recombinant GST-NADK on S46, S55 and S64 and dramatically increased its immunoreactivity with the monoclonal antibody. These results suggest that the increased immunoreactivity of NADK following treatment with PMA is due to phosphorylation of S46 and/or S64. As in vitro phosphorylation of recombinant GST-NADK did not affect its enzymatic activity, we hypothesize that PKC regulates

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activation of NADK through phosphorylation-induced changes in its interaction with other proteins. We obtained preliminary evidence that in resting neutrophils NADK interacts with 14-3- 3 zeta protein, and that this interaction is reduced following treatment with PMA. This suggests that phosphorylation of NADK induces a conformational change leading to dissociation of 14-3-3 or a related inhibitory adapter protein, and activation of NADK. Phosphorylation of NADK by PKC could therefore be an important regulatory mechanism for providing sufficient levels of NADPH to support a robust respiratory burst in phagocytic cells.

Résumé

Le NADP+ et le NADPH sont impliqués dans plusieurs voies de biosynthèse ainsi que dans les mécanismes de signaux de transduction. La NAD Kinase (NADK) est la seule enzyme connue qui catalyse la synthèse de novo du NAD+ en NADP+. Dans les neutrophiles, la NADK joue un rôle essentiel à la formation de niveaux suffisants de NADPH nécessaires à une explosion respiratoire puissante. De plus, il procurerait le NADP+ nécessaire à la synthèse du chimioattractant de granulocytes, le 5oxo-ETE. Malgré son rôle majeur, très peu est encore connu sur son rôle de régulateur chez les mammifères. L’activation de la NADPH oxidase-2 (NOX-2) dans les neutrophiles par des activateurs de la PKC, comme le PMA, provoque une explosion respiratoire puissante, dans lequel un superoxide est formé à partir de l’oxygène provenant de l’oxydation du NADPH en NADP+. Dans cette étude, nous avons mesuré les niveaux de nucléotides pyridines par HPLC, après traitement de neutrophiles avec du PMA. Une augmentation rapide des niveaux de NADP+ fut observée, mais contrairement à ce qui était attendue, celle-ci n’était pas accompagnée d’une diminution de NADPH, qui initialement à aussi augmenté, mais plutôt par une diminution de NAD+. L’augmentation de la formation de NADP+ était exactement comparable à la diminution des niveaux de NAD+, suggérant que la NADK a été activée. Nous avons confirmé que la diminution de NAD+ induite par le PMA dans des neutrophiles intacts, n’est pas dépendante de NOX-2, puisqu’ il n’est pas bloqué par l’inhibiteur de NOX-2, le DPI. Des hybridations Western d’un lysat de neutrophiles avec un anticorps monoclonal anti-NADK a révélé une bande intense avec les neutrophiles traités au PMA mais seulement une bande très faible avec des neutrophiles traités avec le véhicule, suggérant que la PKC induit une modification post-traductionnelle sur la NADK menant à une augmentation de sa réactivité immunologique. Nous avons aussi démontré que la PKC influence l’augmentation de l’activité enzymatique et immunologique de la NADK induite par le PMA, puisqu’elle est bloquée par les inhibiteurs de la PKC : staurosporine et GO6983. L’immunoréactivité du recombinant NADK a augmenté de façon importante à la suite du traitement avec la PKC-delta. L’exposition au PMA ou avec la PKC semble induire la phosphorylation de NADK dans les neutrophiles et du recombinant de NADK, respectivement. L’analyse par LC MS/MS de la NADK a révélé qu’elle est partiellement phosphorylée sur S46, S48, S64 et T62 dans les neutrophiles au repos et que la phosphorylation de S46 et S64 augmente de façon significative à la suite de l’exposition des neutrophiles au PMA. De façon similaire, le PKC-delta induit la phosphorylation de la protéine recombinante NADK sur S46, S55 et S64 et

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augmente de façon importante son immunoréactivité avec l’anticorps monoclonal. Ces résultats suggèrent que l’immunoréactivité accrue de la NADK à la suite du traitement au PMA est due à la phosphorylation de S46 et/ou S64. Étant donné que la phosphorylation in vitro du recombinant NADK n’a pas affecté son activité enzymatique, nous émettons l’hypothèse que la PKC régularise l’activation de la NADK à travers les changements induits par la phosphorylation qui influence les interactions avec d’autres protéines. Nous avons obtenu des évidences préliminaires suggérant que dans les neutrophiles au repos, la NADK interagit avec la protéine 14-3-3 zeta et que cette interaction est réduite à la suite du traitement avec le PMA. Ceci suggère que la phosphorylation de la NADK induit un changement de conformation menant à une dissociation de la protéine 14- 3-3 ou d’une protéine connexe adaptatrice inhibitrice et a l’activation de la NADK. La phosphorylation de la NADK par la PKC pourrait donc être un mécanisme important de régulation pour fournir des niveaux suffisants de NADPH et ainsi supporter une importante explosion respiratoire dans les cellules phagocytaires.

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Contribution of authors

The following authors have contributed to the data presented in this manuscript based thesis:

Francois Graham performed some of the preliminary experiments on analysis of pyridine nucleotides in neutrophils and measurement of intracellular calcium. Chantal Cossette performed cloning of NADK in bacteria, Sylvie Gravel helped for neutrophils isolation and cell culture. Dr. William S. Powell designed the project and contributed to the writing.

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Chapter 1. Introduction and Literature review

1.1 Biology of neutrophils

1.1.1 Introduction Neutrophils are the most abundant cells of the immune system and comprise the first line of innate immunity. Upon pathogen invasion, neutrophils migrate to the site of infection, become activated and promote a well-tuned series of cascades resulting in production of reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hypochlorous acid, as well as they release of antimicrobial and proteolytic granule proteins to kill and degrade the pathogens. Moreover, cytokines and chemokines produced by neutrophils are involved in recruiting other effector cells including macrophages, T cells and additional neutrophils to the site inflammation. Finally, activated neutrophils undergo apoptosis, which is critical for resolution of inflammation because it prevents them from undergoing necrotic cell lysis and the associated tissue damage due to the release of cytotoxic effector proteins and ROS to the extracellular environment (1).

1.1.2 Neutrophil activation and migration

In the event of tissue damage, bacterial-derived lipopolysaccharide (LPS) and and/or both exogenous and endogenous inflammatory mediators are released, which in turn induce the expression of adhesion molecules, P-selectins, E-selectins and several members of the integrin superfamily on the luminal side of the endothelium (2). Neutrophils then interact with P-selectin and E-selectin via their two surface proteins, P-selectin glycoprotein ligand-1 (PSGL-1) and L- selectin, thereby promoting the tethering of neutrophils to the vessels wall followed by “rolling” of neutrophils along the endothelium (3). Interaction of neutrophils with selectins and inflammatory mediators results in the activation of β2 integrins and their expression on the cell surface (4). These β2 integrins then bind to their endothelial ligands to initiate the “firm adhesion” state, which facilitates the transmigration of neutrophils through endothelium junctions (5). Once neutrophils enter the interstitial space, they undergo the final step of activation in response to a variety of host-derived and pathogenic inflammatory mediators.

1.1.3 Phagocytosis Phagocytosis is a receptor-mediated process in which an invading pathogen is engulfed by the cell membrane into a vacuole called a phagosome. Internalization of the pathogen is dependent on the interactions between neutrophils and pathogen. Pathogen-derived molecules consist of pathogen-associated molecular patterns (PAMPs) that interact with the receptors on the surface of neutrophils including TLRs (toll-like receptor) and other receptors such as the peptidoglycan recognition protein (6). Ligation of the neutrophil pattern recognition receptors initiates the signaling pathways that trigger phagocytosis. Additionally, neutrophils interact with pathogens via an opsonin-dependent process by which pathogens are opsonized either with antibody or complement. When pathogens are opsonized with antibody, the Fc-region of the antibody will be recognized by various neutrophil receptors including FcγRI (CD64), FcγRIIa (CD32), FcγRIIIb (CD16), FcαR (CD89), and FcεRI (CD23). Activation of complement system leads to cleavage of C3, the most abundant protein of complement system into two major fragments: C3a (smaller fragment) and C3b (larger fragment). C3a is a chemoattractant and C3b initiate the complement opsonisation through binding to the surface of pathogens. Then, C3b is further degraded to iC3b, C3c and C3dg which interact with complement receptors including CD35, CD11b/CD18 and CD11c/CD18 on the surface of neutrophils. Eventually, ligation of opsonin receptors stimulates the cytoskeletal changes that result in the initiation of phagocytosis (7,8). In neutrophils, plasma membrane is the major source of membrane for phagosomes, whereas in macrophages fusion of endoplasmic reticulum to plasma membrane is an important process to supply membrane at various step of phagosome formation (9). Maturation of the neutrophilic nascent phagosomes requires the fusion of neutrophil granules to the phagosome. These fused granules, then, release anti-bacterial molecules into the phagosome which, together with ROS produced by activated NADPH oxidase, create a toxic environment for most pathogens (10). Characteristics of granules are discussed below in details.

1.1.4 Neutrophils granules Neutrophils contain three types of granules including azurophilic, specific, and gelatinase granules for storing their anti-microbial content. Azurophilic granules are called myeloperoxidase (MPO) positive, while specific and gelatinase granules are considered MPO negative granules. Other cargo of azurophilc granules include defensins, lysozyme, bactericidal/permeability-

increasing protein (BPI), neutrophil elastase, proteinase 3, and cathepsin G. Specific granules are characterized by the presence of lactoferrin. They also contain different antimicrobial compounds including NGAL (neutrophil gelatinase-associated lipocalin), hCAP-18 (human cationic antimicrobial protein 18), and lysozyme. Gelatinase granules are considered as a storage location for some metalloproteinases, such as gelatinase and leukolysin (10). There is another class of neutrophil granule called secretory vesicles that are formed through endocytosis during the late phase of neutrophil maturation rather than by budding from Golgi. Plasma-derived proteins such as albumin are the most abundant constituents of these vesicles. Moreover, the membranes of secretory vesicles serve as a reservoir for a number of main membrane-bound molecules involved in neutrophil migration (11).

1.1.5 Neutrophil extracellular traps (NETs)

Production of neutrophil extracellular traps (NETs) or NETosis is another feature of neutrophils to regulate the severity of infection, in which the nuclear contents of neutrophils are released into the extracellular space. NETs are fibrous structures containing de-condensed chromatin material, some granular (e.g. serine proteases) and cytoplasmic proteins. The NETs are able to trap and kill both gram-negative and gram-positive bacteria and eventually degrade pathogen-derived molecules due to their high serine protease content. NETs formation can be due to either release of chromatin from dying neutrophils or extrusion of chromatin material accompanied by serine proteases from intact neutrophils. The first mechanism occurs 2-3 h after exposure of neutrophils to PMA (phorbol myristate acetate), S. aureus, or C. albicans, whereas the second one is a very rapid response that happens within minutes upon recognition of LPS or pathogenic bacteria by platelets or neutrophils (12). It has been demonstrated that NET formation is linked to the activation of NADPH oxidase and production of ROS, as treatment of neutrophils with the NADPH oxidase inhibitor diphenylene iodonium (DPI) blocks PMA-induced NETosis (13). Furthermore, neutrophils isolated from chronic granulomatous disease (CGD) patients, that are defective in NADPH oxidase, were not able to form an effective NET (13,14). Although NETs enhance the capacity of the immune system to clear pathogens, dysregulation of NETosis and excessive formation of NETs may result in severe pathologic conditions as well as autoimmunity by exposing self-molecules to the extracellular environment. For example, systemic lupus

erythematosus (SLE) is an immune complex–mediated systemic autoimmune disease characterized by formation of antibodies against chromatin and neutrophil components. In this disease, antimicrobial peptide LL-37 that exists together with self-DNA in NETs activates plasmacytoid dendritic cells (pDCs). Activation of pDCs triggers production of type I interferon which in turn stimulates B cells to make antibodies against both the antimicrobial peptide and self- DNA. Moreover, interferon type I promotes activation of more neutrophils and consequently production of more NETs, thereby forming a positive feedback loop that contributes to the severity of SLE (15). NETs are also observed in the airway fluid of cystic fibrosis patients, possibly adversely affecting lung function by increasing the viscosity of sputum (16).

1.1.6 NOX family NOX is a family of enzymes that transport electrons across biological membranes leading to reduction of oxygen to superoxide at the expense of oxidation of NADPH to NADP+. So far 5 isoforms of NOX and two dual oxidase enzymes (i.e. DUOXA1 and DUOXA2) are identified that are expressed in most cells. Here we focus on NADPH oxidase NOX-2, the most studied member of this family, which is highly expressed in granulocytes and monocytes/macrophages. NOX-2 is a multi-component enzyme consists of cytosolic regulatory subunits (p47 phox, p67 phox and p40 phox) and two transmembrane subunits gp91 phox and p22 phox that form the flavocytochrome b558 in the membrane. Recruitment of the cytosolic subunits to the flavocytochrome b558 is required to yield an active enzyme complex. Phosphorylation, GTPase activation and protein-protein interactions are the major processes involved in the activation of NOX-2 (17). Assembly of the NOX complex2 is initiated upon phosphorylation of p47phox, releasing it from its autoinhibitory conformation. The p47phox consists of set of domains including an N-terminal phox homology (PX) domain that interacts with cell membrane phospholipids; two tandem Src homology 3 (SH3) domains forming a super-SH3 (sSH3) binding groove for interacting with the proline rich region (PRR) of p22phox; an autoinhibitory region (AIR); and a C- terminal PRR domain for interaction with other NOX-2 subunits. In the resting cells, masking of sSH33 groove by AIR, keeps the p47phox in an autoinhibitory conformation (18). It is also shown that Serine 379 (S379) on the C-terminal tail forms H-bonds with the hinge of sSH3 and the AIR, thereby connecting the sSH3 and AIR together and stabilizing the autoinhibitory state. Phosphorylation of S379 disrupts the link with the sSH3 and AIR which in turn destabilizes the

autoinhibtory conformation (19). Consequently, S303 and S304 within the AIR is phosphorylated which results in relieving the inhibitory intramolecular interactions. Then, p47phox translocates to the membrane where it binds to p22phox(18,19). Translocation of phosphorylated p47phox is accompanied with migration of activator subunit p67phox toward gp91phox (20) and further mediates the translocation of p40phox into the complex (17). To have an active NOX-2 complex, GTPase Rac is required to be activated by exchange of GDP with GTP first and then interact with gp91phox (21) and p67phox (22,23). Finally, the activated NOX-2 catalyzes the oxidation of NADPH to NADP+ that is accompanied with production of superoxide from oxygen (Figure 1.1). Superoxide anion is rapidly converted to hydrogen peroxide by superoxide dismutase. Moreover, superoxide reacts with nitric oxide, which is generated at high levels at the site of inflammation, forming peroxynitrite, a strong oxidant (24). In addition to antibacterial activity, ROS have the ability to affect the activities of certain host molecules by direct oxidation of cysteine residues and are involved in the regulation of several targets including phosphatases, metalloproteinases, and caspases (10).

Figure 1.1. Activation of NADPH oxidase. NADPH oxidase activation requires the assembly of the cytosolic subunits in the membrane. Upon stimulation, p47 phox is phosphorylated and subsequently translocated to the membrane to interact with p22 phox that also brings the activator subunit p67 phox as well as p40 phox toward gp91 phox. Interaction of GTPase Rac with gp91 phox is also required for full activation of NADPH oxidase (Adapted from (17) with modification).

1.1.7 Neutrophil apoptosis Aged neutrophils undergo apoptosis which is critical for the regulation of homeostasis in immune cells and preventing prolonged inflammation. Macrophages recognize and clear the apoptotic neutrophils, protecting the tissues from excessive damage. Although mechanisms that prolong neutrophil survival are beneficial to host defense against pathogens, induction of apoptosis is also critical for regulation of cell turnover and resolution of inflammation. Neutrophil apoptosis is influenced by a complex mixture of pathogen- and host-derived factors in the site of inflammation and it can be mediated by both intrinsic and extrinsic pathways (7). The intrinsic pathway of apoptosis is associated with the action of pro-apoptotic proteins and caspases. In neutrophils undergoing apoptosis, pro-apoptotic BCL2-associated X protein (BAX) is shifted from the cytosol toward the mitochondrial membrane and facilitates permeabilization of the outer mitochondrial membrane. Consequently, additional pro-apoptotic proteins such as cytochrome c and Smac/Diablo are released into cytosol. Cytochrome c interacts with apoptotic protease- activating factor 1 (APAF-1) to form the apoptosome complex, which recruits and activates caspase-9. Caspase activation is also regulated by the X-linked inhibitor of apoptosis (XIAP) which is quickly recruited to the apoptosome. Ultimately, Displacement of XIAP by Smac/Diablo potentiates caspase activation, including the apoptosis executioner caspase-3 (25).

The extrinsic pathway of apoptosis, on the other hand, is initiated upon ligation of death receptors such as the TNF-α receptor, FAS and TRAIL receptor that leads to the formation of a death-inducing signaling complex (DISC) which recruits and activates caspase-8. Activated caspase-8 directly activates other members of the caspase family and triggers cell apoptosis. In some cells, induction of the extrinsic pathway alone results in cell death, whereas in neutrophils the extrinsic pathway is required to be linked to the intrinsic pathway through the pro-apoptotic bcl-2 homologue Bid in order to trigger cell death. In such cases, caspase 8 truncates Bid, permitting it to translocate to the mitochondria, where it promotes the depolarization of the mitochondrial membrane, ultimately leading to activation of caspase 9 (26).

1.1.8 Neutrophils and adaptive immunity

The adaptive immune response is characterized by the activation of T and B cells against pathogenic organisms. Neutrophils not only are the major component of innate immune system,

but they also contribute to the adaptive immune response. Activated neutrophils release TNF-α, which in turn induces the maturation of dendritic cells (DCs), resulting in the production of IL-12 and activation of T cells (27,28). Furthermore, neutrophils can either act as antigen presenting cells as they express MHC-II when cultured in the presence of GM-CSF, IL-3, and IFN-γ (29) or present antigens to cytotoxic T cells through an alternative MHC class I antigen processing pathway (30)

1.1.9 Neutrophils and diseases

Failure to clear apoptotic remains results in accumulation of cytotoxic factors and chronic inflammation, which is associated with several disease including chronic obstructive pulmonary disease (COPD), rheumatoid arthritis (RA) and cystic fibrosis (CF). One of the key mediators in lung inflammation is leukotriene A4 hydrolase (LTA4H), which works in two opposing directions.

LTA4H hydrolyzes LTA4 into LTB4, which is a potent neutrophil chemoattractant and proinflammatory mediator. On the other hand, LTA4H has aminopeptidase activity that inactivates a specific neutrophil chemoattractant, the proline-glycine-proline tripeptide (PGP), thereby serving as an anti-inflammatory agent. In COPD patients, tobacco smoke (the most common cause of COPD) selectively inhibits only the aminopeptidase activity of LTA4H, raising both LTB4 and PGP levels which in turn enhances the recruitment of neutrophils and promotes chronic lung inflammation (31). IL-8 is a potent chemoattractant of neutrophils. Neutrophils themselves produce high level of IL-8 that promotes attraction of more neutrophils, thereby a self-perpetuating inflammatory state may be established (32). It is shown that the level of IL-8 is remarkably high in induced sputum of patients with COPD which is linked to the increased number of neutrophils (33). IL-8 level is also elevated in the bronchoalveolar lavage fluid of COPD patients which is correlated with the number of neutrophils (34). RA is another inflammatory disease characterized by significant elevation of neutrophils in synovial fluid of patients (35). Additionally, animal studies have shown that the synthesis of LTB4 by neutrophils in joints plays an important role in the development of RA. Interaction of LTB4 with its BLT-1 receptor mediates the recruitment of the neutrophils into joints through an autocrine process and also induces the production of IL-1 by neutrophils in the joints, promoting the synthesis of chemokines by synovial tissue cells, further augmenting neutrophil recruitment (36,37). In CF patients, neutrophils are resistant to TNF-α induced death, delaying spontaneous apoptosis and thereby contributing to chronic inflammation

in these patients (38). Moreover, it is shown that neutrophils from patients with intestinal inflammation produce high levels of proinflammatory mediators such as IL-1β and TNF-α compared to control neutrophils, suggesting that neutrophils may contribute to initiation and perpetuation of mucosal inflammation (39).

1.2 Pyridine nucleotides

1.2.1 Introduction Pyridine nucleotides including nicotinamide adenine dinucleotide (NAD+ and NADH) and nicotinamide adenine dinucleotide phosphate (NADP and NADPH) serve as coenzymes in many biological electron transfer processes. In addition to coenzymatic activity, they are involved in regulation of redox state, cell survival, ion channels and post translational modification of proteins (40). In this section, I provide some highlights on pyridine nucleotides biosynthesis (Figure 1.2) and their biological functions (Figure 1.3).

1.2.2 Biosynthesis of NAD+ NAD+ is necessary for production of NADH, NADP+ and NADPH, thus its biosynthesis is critical for several cellular functions. NAD+ is synthetized through two different pathways; the de novo and salvage pathways (41,42). In the de novo pathway, quinolinic acid, which is the precursor of NAD+, is generated either from L-tryptophan in animals and some bacteria or from aspartic acid in some bacteria and plants (43,44). Quinolinic acid is first converted to nicotinic acid mononucleotide (NaMN) by phosphoribosyltransferase (QPRT). Then, an adenylate moiety is transferred to NaMN to form nicotinic acid adenine dinucleotide (NaAD) which is subsequently is amidated to form NAD+ (45). In the salvage pathway, on the other hand, nicotinamide and nicotinic acid are the NAD+ precursors which are converted to nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), respectively, by phosphoribosyl transferases. Then, adenylyl transferases (NMNAT and NaMNAT) convert NMN and NaMN to NAD+ and NaAD, respectively. Finally, NaAD is converted to NAD+ by NAD+ synthase (NADS). It must be noted that the salvage pathway in mammals is different from that of yeast and invertebrates. In mammals, nicotinamide can be used directly as an NAD+ precursor, whereas in yeast and invertebrates it has to be converted to nicotinic acid by nicotinamidase prior to NAD+ synthesis (46,47).

Figure 1.2. Biosynthesis of pyridine nucleotides. NAD+ is synthetized via de novo and salvage pathways. In the de novo pathway, quinolinic acid, the precursor of NAD+ is converted to nicotinic acid mononucleotide (NaMN) by phosphoribosyltransferase (QPRT). Then, an adenylate moiety is transferred to NaMN to form nicotinic acid adenine dinucleotide (NaAD) which is subsequently is amidated to form NAD+ (45). In the salvage pathway, nicotinamide and nicotinic acid are the NAD+ precursors which are converted to nicotinamide mononucleotide (NMN) or nicotinic acid mononucleotide (NaMN), respectively, by phosphoribosyl transferases. Then, adenylyl transferase converts NMN and NaMN to NAD+ and NaAD, respectively. Finally, NAD+ synthase (NADS) produce NAD+ from NaAD. NAD+ can be reduced to NADH through TCA cycle or it can be utilized by NADK for synthesis of NADP+. Additionally, NADP+ can be converted to NAADP. NADPH is generataed form NADP+ by dehydorgenase enzymes and mitochondrial transhydrogenase. Finally, NADPH can be oxidized back to NADP+ through NADPH-dependent enzymes such as NOX (Adapted from (47) with modification).

Figure 1.3. Biological functions of pyridine nucleotides. Pyridine nucleotides act as electron carriers in metabolic pathways. They also serve as reducing factors in antioxidant system. Additionally, they are required as cofactors for the action of several enzymes (Adapted from (47– 49).

1.2.3 Production of NADP+ and NADPH The only pathway known for the de novo formation of NADP+ is phosphorylation of NAD+ by NAD kinase (50). NADP+ can also be produced from NADPH by NADPH-dependent enzymes such as NADPH oxidase and glutathione reductase (45). NADPH is principally generated from NADP+ through NADP+-dependent enzymes including glucose 6-phosphate dehydrogenase and 6-gluconate phosphate dehydrogenase (from the pentose phosphate pathway), cytosolic and mitochondrial isocitrate dehydrogenases (IDHc and IDHm), cytosolic and mitochondrial malic enzymes (MEc and MEm) and mitochondrial transhydrogenase (TH). Moreover, NADPH can be formed from NADH by mitochondrial transhydogenase (45,47). Pentose phosphate pathway (PPP) is one of the metabolic pathways of glucose, which consists of two phases; oxidative phase that leads to production of NADPH and non-oxidative phase that generates ribose which is required for the synthesis of nucleic acids (Figure 1.4). During the first phase, glucose 6-phosphate (G6P) is oxidized to 6-phosphogluconate (6PG) by glucose 6-

phosphate dehydrogenase (G6PDH) which is accompanied with reduction of NADP+ to NADPH. In the next step, 6PG is oxidized to ribulose 5-phosphate while NADP+ is reduced to NADPH. Then ribulose 5-phosphate initiates the non-oxidative phase to generate ribose (51). The reaction catalyzed by G6PDH is the rate-limiting step of PPP. G6PDH is regulated by NADP+/NADPH ratio as the increased level of NADP+ stimulates the G6PDH and elevation of NADPH level has an inhibitory effect through competing with NADP+ in binding to the enzyme (52). G6PDH is an essential factor for maintenance of the NADPH pool. It is found that exposure of cells to oxidative stress increased both expression level and activity of G6PDH (53). Additionally, inhibition of G6PDH results in more susceptibility in cells to oxidative damage (54). Taken together, increased level of glucose in cells results in activation of PPP pathway that is accompanied by decrease in NADP level and increase in NADPH level.

Figure 1.4. Scheme of pentose phosphate pathway (PPP). In the oxidative phase of the pathway, glucose 6-phosphate (G6P) is oxidized to 6-phosphogluconate (6PG) by glucose 6- phosphate dehydrogenase (G6PDH), which is accompanied with reduction of NADP+ to NADPH. In the next step, 6PG is oxidized to ribulose 5-phosphate while NADP+ is reduced to NADPH. Then ribulose 5-phosphate initiates the non-oxidative phase to generate ribose (51).

1.2.4 Pyridine nucleotides in metabolism and energy production NAD(P)+ and their reduced forms are involved in various metabolic processes. NAD+ is reduced through catabolic pathways including the glycolytic pathway and the tricarboxylic acid cycle (TCA), Generation of NADH will be coupled to production of ATP by entering to electron transport chain in mitochondria. Moreover, NADPH serves as an electron donor for reductive synthesis of fatty acids, steroids and DNA (47).

1.2.5 Pyridine nucleotides in protein modification NAD+ can serve as a substrates for different families of enzymes involved in protein modification, thereby playing important roles in the regulation of several biological processes. One of the common protein modifications is poly (ADP)-ribosylation of proteins catalyzed by poly (ADP)-ribosyl transferases (PARPs) in the nucleus. PARPs transfer an ADP- ribose moiety derived from NAD onto target proteins. Poly (ADP)-ribosylation is involved in various cell functions including apoptosis, cell-cycle regulation, DNA repair and transcription (55–57). Although PARP-1 activity is essential for DNA repair, its hyperactivation can lead to necrotic cell death due to massive NAD+ consumption. Hyperactivation of PARP-1 is observed in several pathological conditions that are accompanied by oxidative stress and DNA damage such as ischemia-reperfusion and inflammatory injuries (58). Mono (ADP)-ribosyl transferases (ARTs) are another family of enzymes that mediate mono (ADP)-ribosylation of proteins through consumption of NAD+. It has been shown that ART-2 is involved in induction of apoptosis in Treg cells, a subset of T cells that modulate the immune response through mono (ADP) ribosylation of

P2X7 (59,60). Furthermore, modification of α-defensin-1 (i.e. an antimicrobial protein secreted by immune cells) by ART-1 diminishes the antimicrobial effect of this protein (61). Another group of NAD+- dependent protein-modifying enzymes are histone deacetylases, also called Sir2 family or sirtuins. Sirtuins are mammalian homologues of the silent information regulator (Sir) proteins in yeast. There are 7 isoforms of SIR proteins in human; Sir4 serves as an ADP-ribosyl transferase and the other Sirs act as deacetylase enzymes that catalyze deacetylation of both histone and non-histone proteins through hydrolysis of NAD+ to O-acetyl-ADP-ribose and nicotinamide. Sirtuins are implicated in various cellular processes including transcriptional regulation, glucose and fatty acid metabolism, apoptosis, energy metabolism, and DNA repair (62,63).

1.2.6 Pyridine nucleotides in calcium signaling ADP-ribosyl cyclase converts NAD+ and NADP+ to cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP), respectively, which are both involved in calcium signaling. cADPR elevates cytosolic level of calcium by releasing calcium from endoplasmic/sarcoplasmic reticulum probably through ryanodine receptors, whereas NAADP is a potent calcium messenger that triggers release of calcium from lysosomal reservoir (48,64).

1.2.7 Pyridine nucleotides in regulation of redox state NADPH serve a dual function in regulation of cellular redox function; NADPH is implicated in reducing oxidized protein and ROS scavenging via the thioredoxin (Trx) and glutathione reductase systems (Figure 1.5). Intracellular glutathione is kept principally in the reduced state (GSH) to prevent the formation of non-native disulfide bonds, thereby protecting cells from oxidative damage. Under conditions of oxidative stress favoring generation of H2O2, GSH is oxidized to GSSG by glutathione peroxidase while hydrogen peroxide is converted to water and oxygen. GSSG will be reduced back to GSH by glutathione reductase, which is an NADPH-dependent enzyme. Thioredoxin also acts as a ROS scavenger and helps maintain proteins in their reduced form; with oxidized thioredoxin being converted to its reduced form by Trx reductase, which requires NADPH as cofactor. On the other hand, oxidation of NADPH by NADPH oxidase leads to production of superoxide, which augments oxidative stress (i.e. imbalance between production of ROS and the ability of the body to neutralize their harmful effects) (65–67). In addition to the glutathione and thioredoxin systems, cells have other antioxidant enzymes including catalase and superoxide dismutase (SOD), as well as non-enzymatic antioxidants such as ascorbate (vitamin C) and α-tocopherol (vitamin E). SOD converts superoxide to hydrogen peroxide, whereas catalase decomposes hydrogen peroxide to water and oxygen (68).

1.3 Eicosanoid Biosynthesis

1.3.1 Introduction As NADP serves as a cofactor for the synthesis of 5-oxo-ETE (5-oxo-6,8,11,14- eicosatetraenoic acid), a potent granulocyte chemoattractant, it is important to understand the metabolic pathway of eicosanoid synthesis and the biological functions of 5-oxo-ETE. Eicosanoids

are fatty acids that are produced through metabolism of arachidonic acid. Arachidonic acid (AA) is metabolized by a number of enzymes, the most important of which are cyclooxygenase and 5- lipoxygenase. Cyclooxygenases lead to the production of prostaglandins (PGs) and thromboxanes (TXs), while the 5-lipoxygenase pathway results in the generation of LTs and 5S-hydroxy-6, 8, 11, 14-eicosatetraenoic acid (5-HETE). Additionally, arachidonic acid can be metabolized through 8-, 12-, and 15-lipoxygenase and cytochrome P-450 pathways (49). Here we focus on production of 5-oxo-ETE via the 5-LO pathway.

Figure 1.5. Pyridine nucleotides in regulation of redox state. NADPH affects the redox state of the cell via two opposing ways. First, it serves as reducing factor in two antioxidant systems; glutathione and thioredoxin. Second, oxidation of NADPH by NOX-2 results in production of superoxide that promotes the oxidative stress (Adapted from (65) with modification).

1.3.2 5-Lipoxygenase pathway Enzymes involved in the 5-lypoxygense (5-LO) pathway metabolize free arachidonic acid released from membrane phospholipids by calcium-dependent phospholipase A2 (cPLA2), to produce leukotrienes (69) and 5-oxo-ETE (Figure 1.6). 5-Lipoxygenase catalyzes the stereospecific incorporation of molecular oxygen into the 5-position of AA to form the intermediate 5S-hydroperoxy-6, 8, 11, 14-eicosatetraenoic acid (5-HpETE) which is rapidly rearranged to produce LTA4. LTA4 is then converted to either LTB4 by LTA4 hydrolase or to LTC4 by LTC4 synthase. Then, γ-glutamyl transpeptidase converts LTC4 to LTD4. However, not all of the 5-HpETE is converted to LTA4, as a considerable amount is reduced to 5-HETE by peroxidases. 5-HETE can be oxidized to form 5-oxo-ETE by the enzyme 5-hydroxyeicosanoid dehydrogenase (5-HEDH), which is highly expressed in neutrophils (70) and requires NADP+ as a cofactor. The 5-LO pathway is regulated by calcium in two ways. First, release of AA from membrane phospholipids by cPLA2 is a calcium dependent process. Second, elevation of intracellular calcium is necessary for translocation of 5-LO to the nuclear membrane where it oxidizes AA bound to the nuclear membrane accessory protein 5-LO activating protein (FLAP). The majority of cellular 5-LO products are synthetized by inflammatory cells. Moreover, 5-LO pathway may contribute to resolution of inflammation through generation of lipoxins such as lipoxin A4 that acts as an anti-inflammatory lipid mediator (49).

1.3.3 5-oxo ETE formation and regulation As mentioned above formation of 5-oxo ETE by 5-HEDH is dependent on availability of NADP as cofactor. The majority of intracellular NADP+ is kept in its reduced form NADPH (71), hence the ability of resting cells to synthesize 5-oxo-ETE is limited. Stimulation of neutrophils with the protein kinase C (PKC) activator PMA, induces the formation of 5-oxo ETE. This effect could be blocked by the NADPH oxidase inhibitor DPI, indicating that it is dependent on the generation of NADP+ by this enzyme. Furthermore, nonenzymatic conversion of intracellular NADPH to NADP by phenazine meta sulphate (PMS) results in induction of 5-oxo ETE synthesis (72). Another process that can trigger the formation of 5-oxo ETE is oxidative stress, as exposure of inflammatory cells to H2O2 and other oxidants results in dramatic elevation of 5-oxo-ETE through a mechanism linked to the glutathione redox cycle (73). Glutathione peroxidase reduces the H2O2 at the expense of oxidation of GSH to GSSG. GSH is then regenerated by glutathione

reductase which is accompanied by the oxidation of NADPH to NADP+. Furthermore, glucose inhibits the formation of 5-oxo--ETE due to its metabolism by the pentose phosphate pathway, resulting in the conversion of NADP+ to NADPH (73).

1.3.4 Biological functions of 5-oxo-ETE in PMNs 5-oxo-ETE stimulates various cellular responses in neutrophils. For example, it promotes cell migration and calcium mobilization. However it is 10 times less potent than LTB4 in stimulating calcium mobilization (74). Moreover, it causes the elevation of intracellular F-actin level and also induces surface expression of the adhesion molecules CD11b and CD11c and neutrophil aggregation (75,76). Regarding the neutrophils degranulation, LTB4 is much more potent than 5- oxo- ETE. However, priming neutrophils with TNF-α, GM-CSF or G-CSF (granulocyte-colony stimulating factor) dramatically increased the potency of 5-oxo-ETE to induce neutrophils degranulation (77). It was shown by Norgauer et. al that, 5-oxo-ETE promotes the production of superoxide (78), however this effect was not observed by O’Flaherty and colleagues (76). Following priming with GM-CSF, the potency of 5-oxo ETE to induce the superoxide production is almost comparable to that of LTB4 (77). 5-Oxo ETE is a very potent stimulator of human eosinophil migration, in contrast to LTB4, which displays very little chemotactic activity for these cells (79). Other effects of 5-oxo ETE on eosinophils include stimulation of calcium mobilization, induction of expression of CD11-b, shedding of L-selectin, activation of the respiratory burst, actin polymerization (80,81). It also induced the transmigration of eosinophils across endothelial cell monolayers and artificial basement membranes (82) and elicits eosinophils infiltration into the skin when injected intradermally (83).

Figure 1.6. 5-Lypoxygenase (5-LO) pathway. Arachidonic aicd (AA) is metabolized through 5-LO pathway to produce leukotrienes and 5-HETE. cPLCA2 releases AA form membrane phospholipids. Then, 5-LO converts AA to the intermediate 5-HpETE which is rapidly rearranged to produce LTA4. LTA4 is then converted to either LTB4 by LTA4 hydrolase or to LTC4 by LTC4 synthase. Then, c-glutamyl transpeptidase converts LTC4 to LTD4. A considerable amount of 5-HpETE is converted to 5-HETE by peroxidases. 5-HETE can be oxidized to form 5-oxo-ETE by the enzyme 5-HEDH (Adapted from (49) with modification).

1.4 NAD kinase, a crucial enzyme for biosynthesis of NADP+

1.4.1 Introduction

In all organisms NAD kinase (NADK) is the only known enzyme that catalyzes the de novo synthesis of NADP+ from NAD+ by utilizing ATP or inorganic phosphate as phosphoryl donor (84) (Figure 1.7). NADK was discovered and partially purified from yeast Saccharomyces cerevisiae in 1950 (85). Later, some properties of NADK were characterized in different organisms including Candida utilis (86), Saccharomyces cerevisiae (87), Arabidopsis thaliana (88), pigeon liver (89), pigeon heart (90) and human neutrophils (91). The NAD kinase gene was identified for the first time in Micrococcus flavus and Mycobacterium tuberculosis (92). It has been reported that NADK is essential for the survival of Mycobacterium tuberculosis (93), Bacillus subtilis (94), Escherichia coli (95), and Salmonella enterica (96). Therefore, NADK plays a critical role in regulating the intracellular concentration of NADP+ and NADPH, two cofactors involved in many metabolic processes and cellular signal transduction mechanisms (97,98). Furthermore, dynamic regulation of NADK during embryogenesis in both Xenopus tropicalis and in sea urchins is essential for full embryonic growth (99). In spite of the importance of NADK in cellular functions, so far the majority of research on NADK has been done with lower organisms (bacteria and yeasts) and there is relatively limited information on mammalian NADK.

1.4.2 Structural properties of NADK NAD kinases in all organisms have a homo-oligomer structure consisting of two to eight identical subunits with molecular masses between 40 and 60 kDa (100). Analysis of known NADKs from different organisms revealed that there are 3 conserved sequences, including a GGDG motif, an NE/D short motif and a glycine rich motif (101) (Figure 1.8). The conserved catalytic domains are located within C-terminal of NADK, whereas N- terminal region is variable among different organisms. The GGDG domain, which mediates ATP binding, is a characteristic of a kinase superfamily that also includes diacylglycerol kinase, sphingosine kinase and 6-phosphofructokinase (102). Directed mutagenesis of GGDG in mycobacterial NAD kinase causes the loss of enzyme activity (103). Similarly, it was shown that the glycine rich motif, a specific motif present in all of the identified NADKs, is essential for NAD

binding (103). Conformational analysis of NADK in M. tuberculosis revealed that each subunit contains an N-terminal α/β domain and a C-terminal 12-stranded β sandwich domain, linked by swapped β strands. The N-terminal domain resembles a classical Rossmann fold and the structure of the C-terminal domain is similar to the human Ki67 fork-head associated domain (104,105).

Figure 1.7. Biosynthesis of NADP+ by NADK. NAD kinase catalyzes the transfer of phosphate group from ATP to NAD+ to produce NADP+. Activation of NADK is strictly dependent on divalent metal ions (84).

1.4.3 Subcellular location of NADK

1.4.3.1 Yeasts Three isoforms of NADK have been reported in S. cervisiae. ScNADK1 and ScNADK2 are localized in the cytosol (106), whereas ScNADK3 is a mitochondrial protein that prefers NADH as substrate rather than NAD+ (107). The expression level of ScNADK2 is considerably lower than that of ScNADK1 and ScNADK3 (108). This is consistent with another study showing that the majority of NADK activity in vivo is related to ScNADK1 and ScNADK3, as the disruption of these two isoforms are lethal, regardless if ScNADK is functional or disrupted (109), (110).

Figure 1.8. Partial multiple alignment of amino acid sequences of various NADKs using Clustal W. Sequence names, from top to bottom, are E. coli NADK (EcNADK), M. tuberculosis (MtNADK), S. cerevisiae NADK-1 (ScNADK-1/Utr1p), S. cerevisiae NADK-2 (ScNADK-2/Yef1p), S. cerevisiae NADK-3 (ScNADK-3/Pos5p), A. thaliana NADK-1 (AtNADK1), A. thaliana NADK-2 (AtNADK2), A. thaliana NADK-3 (AtNADK3), human cytosolic NADK (Human-Cyt) and human mitochondrial NADK (Human-Mit), respectively. The conserved motifs are shown in solid boxes (Adapted from (100) with modification).

Moreover, yeast cells that lack the mitochondrial isoform are very sensitive to oxidative stress indicating that ScNADK3 plays a critical role in supplying NADPH (107,111), whereas deletion of ScNADK1 and ScNADK2 do not result in hypersensitivity to oxidative stress (107). Furthermore, it has been shown that ScNADK1 is required for NADP production to support cytosolic NADP-dependent dehydrogenases and detoxifying systems, whereas the specific role of ScNADK2 is not identified yet (109).

1.4.3.2 Plants Three NADK isoforms that have been originally characterized in plants are localized in cytoplasm (112), mitochondria (113) and chloroplasts (114,115). For example, a study of NADK isoforms in Arabidopsis thaliana have shown that AtNADK1 and AtNADK3 are expressed in most tissues, whereas AtNADK2 is only expressed in leaves (88). Fluorescence microscopy studies have revealed that AtNADK2 is localized in chloroplasts (116) whereas AtNADK1 and AtNADK3 are located in the cytoplasm (117). In agreement with the protective role of yeast NADKs in oxidative stress, treatment of plant cells with hydrogen peroxide or irradiation leads to up-regulation of AtNADK-1 at both mRNA and protein levels. Additionally, cells that lack the AtNADK1 gene are more sensitive to oxidative stress (118). Moreover, mutation of AtNADK2 results in delayed growth and development, which in turn decreases leaf size and seed production. AtNADK2 mutation also results in lower chlorophyll content, indicating the important role that AtNADK2 plays in chlorophyll synthesis (116).

1.4.3.3 Human For a long time, it was thought that only one isoform of NADK exists in human, localized in the cytosol (50,97). However, a mitochondrial protein that exhibits NADK activity has recently been identified (119,120). The characteristics of both cytosolic and mitochondrial NADK in human will be discussed in detail later.

1.5 Enzymatic properties of NADK

1.5.1 Substrate specificity Although, all NADKs contain the conserved domains described above, they exhibit some differences in enzymatic properties. For example, substrate specificity for both the phosphoryl donor and phosphoryl acceptor varies among different organisms. NADKs from gram-positive bacteria and eukaryotes can phosphorylate both NAD and NADH, whereas NADK from gram- negative bacteria phosphorylate NAD but not NADH. It is has been reported that an arginine residue in a conserved domain of NADKs plays a critical role in specificity toward NAD (121). Replacing this arginine by glycine or a polar amino acid residue in NADK from E.coli (gram negative) results in the loss of the NADK substrate specificity, so that it accepts both NAD and NADH, although the rate of phosphorylation of NADH is still considerably lower than that of NAD+. On the other hand, replacement of glycine by arginine (inverse of the replacement in gram- negatives, mentioned above) in Micrococcus flavus (gram positive) doesn’t convert the non- selective NADK to the selective enzyme, but it causes a remarkable decrease in NADK activity regardless of substrate type. Based on these results, it can be concluded that the arginine residue affects the catalytic properties of NADK, but it is not the only factor for substrate specificity determination. Regarding the phosphoryl donor, NADKs from both prokaryotes and eukaryotes use ATP as phosphoryl donor. However, NAD kinase from gram-positive bacteria and archaea utilizes both ATP and poly(P) (84). Poly(P), an ancient energy carrier, is a polymer of inorganic orthophosphate residues linked by high-energy phosphoanhydride bonds as in ATP (122).

1.5.2 Modulation of NADK activity Besides differences in substrate specificity, NADKs from different organisms are subject to different regulatory mechanisms. Divalent metal ions regulate NADK activity in a strict fashion. It has been shown that Mg2+ and Mn2+ are the most efficient metal cofactors for NADK in eubacteria, whereas Ca2+ and Zn2+ are as efficient as Mg2+ in promoting NADK activity in B. subtilis and E.coli, respectively. In human, Zn2+, Mn2+ and Mg2+ are the most potent activators of NADK (123).

Various studies have demonstrated that purified NADK from different sources is regulated by pyridine nucleotides. For example, NADH and NADPH are potent negative allosteric regulators of NADK in gram-negative bacteria like E. coli (124) and S. enterica (96), whereas NADK in Sphingomonas sp. A1 is negatively regulated by NADP+ and NADPH. In gram positive bacteria, NADK activity from both B. licheniformis (125) and B. subtilis (126) is negatively modulated by NADP+ . Activity of NADK from M. tuberculosis is also inhibited by NADP+ (103). NADKs from S. cervisiae are also regulated by pyridine nucleotides, as Utr1p is inhibited by NADP+ , NADH and more potently by NADPH (106) and Yef1p is slightly inhibited by NADH and NADPH (127). Additionally, it has been reported that quinolinic acid, the precursor of NAD+ biosynthesis, regulates NADK activity in bacteria. Quinolinic acid blocks the activity of NADK in S. enterica serotype Typhimurium (128) whereas it stimulates NADK activity from B. subtilis (126).

It has also been shown that NADK activity is regulated by calmodulin (CaM), a key regulatory protein in some organisms. CaM acts as a ubiquitous intracellular receptor for Ca2+ and the formation of a Ca2+/CaM complex triggers several cellular signaling cascades through the regulation of different enzymes including calmodulin dependent protein kinases I, II, IV and myosin light chain kinase as well as calmodulin dependent protein phosphatase, calcineurin (129). In plants, NADK can be either Ca2+/CaM-dependent or Ca2+/CaM-independent based on its subcellular localization (112,130). Surprisingly, none of three recombinant isoforms of NADK from Arabidopsis thaliana are activated by Ca2+/CaM, even though isoform2 contains a CaM binding domain in its N-terminal domain, which binds to CaM in a Ca2+ dependent manner (88). It is well known that NADK in sea urchin egg is activated by Ca2+/CaM after fertilization (131), resulting in elevation of NADPH level, which is required for DNA and protein synthesis (132),(133). This increase in NADPH also triggers the activation of NADPH oxidase and subsequently the production of hydrogen peroxide (134) which is involved in hardening of fertilization membrane (135). It has also been demonstrated that the activity of partially purified NADK from human neutrophils is up-regulated by addition of calcium in the presence of calmodulin, but not by calcium alone (91). Similar to recombinant NADKs from A.thaliana, human recombinant NADK is not activated by Ca2+/CaM (136).

1.6 Human cytosolic NADK

1.6.1 Properties of human NADK According to Genbank, human NADK gene (gene Id: 65220) encodes a 49 kDa protein. Very little was known about human NADK until Williams et al. characterized some kinetic properties of partially purified NADK from human neutrophils (91). In this study, NADK from neutrophil extracts was purified using red agarose, ion exchange and gel filtration chromatography sequentially. The calculated Km values were 0.6 and 0.9 mM for NAD and ATP, respectively. The molecular mass of NADK was estimated to be approximately 169 kDa by gel filtration chromatography. Several years later in 2001, human NADK from fibroblasts was overexpressed in E.coli for the first time and the molecular and kinetic properties of the recombinant enzyme (molecular mass 49 KDa) were compared with those of partially purified NADK from bovine liver (136). Recombinant NADK displayed an apparent molecular mass of about 190 kDa (determined by size exclusion chromatography), suggesting that it is catalytically active as a tetramer complex. Similarly, the apparent molecular mass of bovine NADK was calculated to be 210 kDa. The recombinant human enzyme had a Km value of 3.3 mM and 0.54 mM for ATP and NAD, respectively, which is consistent with those reported for bovine liver and other animal tissues (137). Regarding the substrate specificity, human NADK is very selective for NAD and ATP and replacement of ATP with GTP reduces the enzyme activity more than ten times. Analysis of tissue specific expression of NADK mRNA revealed that it is expressed in most tissues (136). However, it is not expressed in skeletal muscle and its expression in intestinal tissue is very low. The lack of NADK expression in skeletal muscle can be explained by its metabolic specificity, as neither the pentose phosphate nor the fatty acid synthetic pathways exist in this tissue (136). However, there isn’t any clear reason for the low expression of NADK in intestinal tissue. Later, Ohashi et al. introduced an improved method to express and purify recombinant NADK and studied the kinetic properties of NADK (138). They succeeded to purify the recombinant NADK by two steps of purification including heat treatment and TALON column chromatography, whereas the recombinant NADK produced by Lerner’s group was purified in a three-step process (Heat treatment, DEAE and nickel–nitrilotriacetic acid column chromatography). Based on their study, NADK showed sigmoidal kinetic behavior toward ATP

while saturation curve toward NAD+ and its catalytic activity was negatively regulated by NADH and NADPH but not by NADP+. Furthermore, they subjected the purified NADK to SDS-PAGE and found that its subunit molecular mass was 43 kDa, which was smaller than the predicted mass (49 kDa). They analyzed the N-terminal amino acid sequences of the recombinant NADK and found that N-terminal sequences consisting of 60, 63, and 74 amino acid residues of human NADK were missed in the purified enzyme, explaining its lower molecular mass. The purified enzyme yielded a peak on size exclusion chromatography corresponding to 172 kDa, indicating a tetramer structure.

1.6.2 Regulation of human NADK by divalent cations The study of NADK in neutrophils (91) revealed that the enzymatic activity of NADK was strictly dependent on Mg2+ at the optimum pH of 7.0-9.5. Furthermore, the enzyme was sensitive to micromolar levels of free calcium, as at the concentration below micromolar range, very low level of NADK activity was observed. Increasing calcium concentration resulted in elevation of NADK activity. The enzyme exhibited half maximal level of activity at 0.4 µM of free calcium. In agreement with this, it was found that the catalytic activity of both recombinant and bovine NADK were strictly dependent on the availability of divalent cations, which form complexes with the substrate nucleotides. The optimum concentration of Mg2+ required to activate NADK is 10 mM (Intracellular concentrations of magnesium range from 5-20 mM and 1-5% of that is ionized (139)) . However other metal ions such as Zn2+, Mn2+, Co2+ and to some extent Ca2+ can also activate the enzyme (136).

1.6.3 Role of human NADK in regulating intracellular NADPH In 2007, Pollak et al. (97) conducted a study that revealed more physiological aspects of NADK. They confirmed that human NADK is localized in cytosol and prefers NAD+ as substrate rather than NADH. They established a cell line stably overexpressing human NADK that resulted in a 200-fold increase in NADK in both mRNA and proteins levels. Subsequently, the catalytic activity of NADK was increased comparably. Overexpression of NADK elevated the intracellular NADPH level without any significant effect on NADP+ concentration. This effect may be due to rapid conversion of the produced NADP+ to NADPH through dehydrogenase pathways. However, NADPH level was increased by only 4-5 fold, which is not comparable to a 180-fold increase in

NADK activity. The authors suggested that inhibitory effect of NADPH on NADK prevents further production of NADP+. Moreover, they down regulated the expression of NADK by a small hairpin RNA targeting NADK that resulted in a 3-fold decrease in both the level of mRNA and NADK activity accompanied by a 3-fold decrease in NADPH levels. Unexpectedly, they found that strong overexpression of NADK only slightly increased protection against oxidative stress. They explained this by the possibility that under oxidative stress, the cells are mostly relying on their existing pool of NADP+ rather than on NADP+ synthesis. The cells provide more protection by increasing the rate of regeneration of NADPH from NADP+ through activation of NADP+- dependent dehydrogenases. Apparently, only a significant decrease in the NADP+ pool can be a limiting factor, as knock down of NADK resulted in more sensitivity of the cells toward oxidative stress.

1.6.4 Regulation of human NADK by Ca2+/ calmodulin˛ It is shown that partially purified NADK from human neutrophils is activated by Ca2+/calmodulin (91). The enzyme activity was not affected significantly by 1 mM Ca2+alone, whereas adding calmodulin in the presence of Ca2+ caused 3.5 fold increase in the NADK activity. Another study demonstrated that stimulation of neutrophils with calcium and A23187, a calcium ionophore, as well as stimulation with zymosan A increased the total intracellular pool of NADP+ and NADPH. Pre-treatment of neutrophils with calmodulin antagonist trifluoperazine or with the intracellular calcium antagonist, TMB-8 (3,4,5-trimethoxybenzoic acid 8-[diethylamino]octyl ester), blocked the zymosan-induce increase in the concentration NADP+ and NADPH, suggesting that calcium may be involved in regulation of NADK (140). In contrast to partially purified NADK from human neutrophils, recombinant human NADK was not stimulated with Ca2+/CaM, suggesting that Ca2+/CaM regulates NADK in neutrophils indirectly; possibly through cytosolic components (136). Similar to human NADK, it was previously shown that Ca2+/CaM activates NADK in sea urchin (131), but the molecular mechanism of its activation was not elucidated. Another study was done recently by Love et al. (99) to further investigation of NADK regulation by Ca2+/CaM in human and sea urchin (Strongylocentrotus purpuratus). They produced recombinant NADK from human (HsNADK) and sea urchin (SpNADK-1 and Sp-NADK-2) and showed that SpNADK2

which has an N-terminal CaM binding domain is activated directly by Ca2+/CaM, in contrast the activity of SpNADK-1 and HsNADK which lack such a domain is not affected by Ca2+/CaM. Since Ca2+/CaM can activate Calmodulin-dependent protein kinase II (CaMKII), they hypothesized that regulation of NADK by Ca2+/CaM can be phosphorylation-dependent. To investigate this, they looked at in vitro phosphorylation of NADK by CaMKII. They showed that CaMKII is able to phosphorylate Sp-NADK-1 and HsNADK but not Sp-NADK-2. Additionally, they found that the phospho-sensitive region of human NADK is located within amino acids 30- 64 by analysis of different N-terminally truncated HsNADKs. Phospho-site mapping of HsNADK by site-directed mutagenesis revealed that serine 64 (S64) is the most sensitive amino acid for phosphorylation. To study whether the detected phosphorylation is involved in elevation of NADK catalytic activity, they substituted the S64 with either a non-phosphorylatable aminoacid (alanine) or a phosphomimetic amino acid (aspartic acid). Although neither of these alterations affected NADK enzymatic activity, the authors suggested that NADK phosphorylation may modulate NADK trough regulation of association of NADK with other factors. A recent study on mouse liver phosphoproteomes showed that the analogous S64 residue of NADK in mouse liver is phosphorylated (141). It is noted that ∼ 70 amino acids surrounding the phosphorylated residue are identical in mouse and human NADKs, supporting the biological relevance of phosphorylation of human NADK on S64 (99).

1.6.5 Activation of NADK by PMA In 1985, Williams et al. (91) found that total pool of NADP+ + NADPH was increased in neutrophils stimulated with PMA, indicating that PMA induced activation of NADK. However, they didn’t investigate the mechanism of NADK activation. Another study from the same year (140), showed that total concentration of NADP+ + NADPH is increased in neutrophils stimulated by PMA. They also showed that pre-treatment of neutrophils with either trifluoperazine or W-7 (two calmodulin antagonists) blocked the PMA-induced increase of NADP+ + NADPH in neutrophils. As trifluoperazine and W-7 are also inhibitors of PKC (142), the authors suggested that PMA may cause the activation NADK by PKC that consequently results in the elevation of NADP+ + NADPH pool. However, they didn’t investigate this possibility. On a final note, recently Cheng and colleague observed the stimulation of NADK activity by PMA while studying PMA-

induced net formation in neutrophils (143). They found that stimulation of neutrophils with PMA resulted in increase in the intracellular levels of NAD+ and NADP+ which was in parallel with increase in the activity of NADK measured in neutrophil lysates before and after 1h stimulation with PMA. They connected the increase in the NADK activity to the induction of NADK expression, as the intensity of protein band observed with western blotting using anti-NADK antibody was augmented after PMA stimulation for one hour or longer.

1.6.6 Potential binding partner of NADK Since many cellular transductions are carried out by protein-protein interactions, identifying proteins that can potentially interact with NADK may be helpful to better understanding the NADK regulation. We searched the BioGRID database of molecular interactions to find the NADK interacting partners. List of proteins that possibly interact with NADK and the interaction detection methods are summarized in Table 1.1.

1.6.7 Alternatively spliced isoforms of NADK We used the Unipro UGENE program to find different alternatively spliced transcripts of human NADK. Among several potential spliced transcripts, those that have molecular mass of higher than 30 kDa are summarized in Table 1.2. Different spliced transcripts have variable N- terminals, while their C-terminals are identical. In Figure 1.9 sequence of the selected spliced transcripts are aligned using Clustal W program. The cell type and tissue-specific expression of these isoforms is not studied yet.

Protein name MW. role Interaction detection Reference kDa method

14-3-3 protein gamma 28.3 Bait Affinity capture-MS (144) (YWHAG)

14-3-3 protein zeta (YWHAZ) 27.7 Bait Affinity capture-MS (145)

14-3-3 protein beta (YWHAB) 28 Bait Affinity capture-MS (146)

14-3-3 protein epsilon (YWHAE) 29.2 Bait Affinity capture-MS (147)

14-3-3 protein theta (YWHAQ) 27.8 Bait Reconstituted complex (148)

Syndecan-binding protein 1 33 Bait two hybrid pooling (149) (SDCBP) approach

Glutaredoxin 3 (GLRX3) 40 Bait two hybrid pooling (150) approach

growth factor receptor-bound 25 Bait two hybrid pooling (151) protein 2 (GRB2) approach

nudix-type motif 18 (NUDT18) 35 Bait two hybrid pooling (150) approach

ligand of numb-protein X 1, E3 80 Hit two hybrid pooling (150) ubiquitin protein ligase (LNX1) approach

eukaryotic translation elongation 50 Hit two hybrid pooling (152) factor 1 gamma (EEF1G) approach

Mov10 RISC complex RNA 113 Bait Affinity capture-MS (153) helicase (MOV10)

Table 1.1. Potential binding partner of human NADK based on the BioGRID (database of molecular interactions)

Table 1.2. Alternatively spliced transcripts of NADK. DNA sequence of human NADK were analyzed to find different alternatively spliced transcripts using Unipro UGENE program.

1.7 Human mitochondrial NAD kinase As mentioned above, it was originally thought that only a single NAD kinase exists in human cells. Although NADPH is an essential factor in mitochondria to protect cells against oxidative stress, the source of mitochondrial NADP+ was unknown. As the mitochondrial membrane is not permeable to NADP+, the exchange of cytosolic NADP is impossible. A recent study identified a non-characterized human gene C5ORF33 that encodes a protein which is homologous to NADK3 of A.thaliana. and was introduced as a novel mitochondrial NADK (MNADK) (119). C5ORF33 was expressed in E.coli and the purified recombinant protein was shown to have NAD kinase activity with either ATP or poly (P) as phosphoryl donor. In + comparison to cytosolic human NADK, C5ORF33 had lower Km (for NAD ) and Vmax values As C5ORF33 carries mitochondrial-targeting sequence, it was expected to be localized to mitochondria. To verify this, HEK293A cell were transfected with C5ORF33, which was found to be localized to mitochondria using fluorescence microscopy. In agreement with this, C5ORF33 was detected by western blotting only in the mitochondrial fraction of HEK293A cells but not in the cytosolic fraction. Moreover, they showed that knock-down of C5ORF33 resulted in higher production of ROS in mitochondria, indicating that C5ORF33 is involved in regulation of mitochondrial ROS generation.

Figure 1.9. Multiple alignment of alternatively spliced transcripts of human NADK. Among several potential alternatively spliced transcripts of NADK, those with molecular mass of higher than 30 kDa were aligned using Clustal W program. The part of C-terminal sequence that was identical in all the transcripts is not shown. The transcripts are named based on the number of their aminoacids and their molecular mass.

Simultaneously, another group identified a mouse orthologue of C5ORF33 named MNADK (mouse homologue 1110020G09Rik) as a novel mitochondrial NADK (120). They reported that MNADK is evolutionarily conserved and contains both NAD and NADH kinase domains. Human MNADK is 89% identical to mouse MNADK and interestingly doesn’t share significant homology with human cytosolic NADK. As predicted from its sequence, recombinant MNADK was shown to have NAD kinase activity using an in vitro NADK assay. However, MNADK produced NADPH from NADH, the amount of produced NADPH was minimal compared to the amount of produced NADP+ utilizing NAD+ as substrate. MNADK was found to be localized in mitochondria in Hep G2 cells, a human liver cell line, by fluorescence imaging. MNADK is nutritionally regulated in mice, as fasting increased its expression in both liver and white adipose tissue. Furthermore, MNADK expression was dramatically lower in human liver tumor compared to normal human liver. It was shown that mitochondrial functions play an important role in tumor progression. Some of the mitochondrial gene mutations in cancer are associated with production of more ROS, thereby perturbing the redox state of transcription factors and finally favoring tumorogenesis (154). MNADK may contribute to regeneration of antioxidant defense system in mitochondira through production of NADP+ which is turn converted to NADPH by mitochondrial malic enzyme, mitochondrial NADP+-dependent isocitrate dehydrogenase and aldehyde dehydogenase

1.8 Protein kinase C

1.8.1 Introduction The protein kinase C (PKC) family is a large group of serine-threonine kinases belonging to the AGC superfamily of protein kinases. They consist of a highly conserved catalytic domain involved in ATP/substrate binding and catalysis as well as a regulatory domain that keeps the enzyme in an inactive state. The PKC regulatory domain is located within the NH2-terminus of the protein and comprises an autoinhibitory pseudosubstrate region and two distinct membrane targeting modules; C1 and C2 (155). There are several isoforms of PKC that are distinguished based on their structure, cell/tissue distribution and abundance This diversity enables them to play a role in numerous cellular transduction processes including development, differentiation, proliferation, carcinogenesis (156). Furthermore, several studies have shown that PKC is involved

in induction of inflammatory response. For example, PKCδ stimulated pro-inflammatory chemokines expression through NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway in vascular smooth muscle cells (157). PKC activation induces gene expression related to NF-κB and TNF-α pathways in human bronchial epithelial cells (158). Additionally, PKC activation is associated with proliferation and differentiation of T-cells through induction of transcription factors such as NF-κB, NFAT (nuclear factor of activated T-cells), c-Jun, c-Fos and AP-1 (activator protein 1) (159).

1.8.2 Classification of PKC enzymes PKC isoforms are subcategorized into conventional PKCs (α, β1, β2 and γ), novel PKCs (δ, θ, ε, η and μ) and atypical PKCs (ζ, ι and λ) (160) based on differences in their regulatory domain structure. In conventional PKC isoforms, the C1 domain contains two cysteine-rich motifs involved in DAG/PMA binding, while C2 domain is capable of calcium-dependent binding to anionic phospholipids. Therefore, this class requires both DAG (diacyl-glycerol) and calcium to become activated. The regulatory domain of novel PKCs is very similar to conventional isoforms with the exception that its C2 domain lacks the putative calcium-binding residues, thus novel isoforms require only DAG for the activation. Atypical PKCs require neither DAG nor Ca++ to be activated, as they lack the calcium-sensitive C2 domain and their C1 domain contains only one cysteine-rich motif that binds to PIP3 (phosphatidylinositol 3,4,5-trisphosphate) or ceramide, but not to DAG or PMA (155,160). They also contain a protein-protein interaction PB-1 domain (Phox and Bem1 domain) that facilitates the interaction with other PB-1 containing proteins (161). Although, most PKC isoforms are expressed ubiquitously, some isoforms are expressed in a cell/tissue specific manner. Many cells co-express multiple isoforms. For example neutrophils express 4 isoforms: PKC-α, PKC-β, PKC-δ and PKC-ζ (162–164).

1.8.3 Regulation of PKCs PKC is activated through the dissociation of pseudosubstrate from its internal binding site caused by PKC’s interactions with DAG and PS (155,160). DAG is a hydrophobic anchor that recruits PKC to the membrane and also increases the affinity of cPKCs and nPKCs to acidic lipids. Translocation of PKC to the membrane occurs through binding of its C1 and C2 domains to DAG and acidic lipids such as phosphatidylserine (PS), respectively. DAG together with IP3 are products

of PIP2 hydrolysis by phospholipase C (PLC). DAG remains in the membrane while IP3 goes to the cytosol and then interacts with its receptor to trigger the release of calcium from intracellular stores. (155,160). Interaction of acidic lipids with the C2 domain in cPKCs is calcium-dependent, as calcium induces a conformational change in the C2 domain that increases its affinity to acidic lipids. PKCs do not require calcium for activation, as their C2 domains already possess the required structure for interaction with lipids Post translational modifications are also involved in modulation of PKCs. It has been demonstrated that PKC isoforms undergo translational modifications that are essential for their function. Three phosphorylated sites have been identified in PKCs that are called activation-loop (A loop), turn motif (TM) and hydrophobic motif (HM). All three sites are conserved in all PKCs with the exception of aPKCs, in which Serine/Threonine residue in HM motif is replaced by glutamic acid, perhaps mimicking a constitutively phosphorylated state. (165). Phosphorylation of the A-loop in many PKC isoforms is critical for the catalytic activity, since it is stabilizes the active conformation of the kinases by enabling ionic interactions with positively charged residues located close to the kinase domain (166,167). However, in the case of PKC-δ, phosphorylation of the A- loop is not necessary for kinase activation, but instead modulates the specificity of the enzyme toward some substrates (168). Phosphorylation of TM and HM contributes to the stability of the kinase core of the enzyme, and is required for the catalytic activity of certain PKC isoforms. For example, phosphorylation of TM is required for the activation of PKC-β, PKC-δ and PKC-ζ and activation of PKC-α and PKC-θ is dependent on HM phosphorylation (165).

The activity of PKC isozymes can also be controlled by their localization within cells. It has been shown that specific anchoring proteins such as Receptor for activated C-kinase (RACK), annexin and other cytoskeletal proteins are implicated in the localization of PKCs (169). Subcellular distribution of eight PKC isoforms was studied by performing immunocytochemical analyses. The results showed that the majority of PKCs are located in the cytoplasm in resting cells. Upon activation, each isoform specifically translocated to a distinct cell compartment such as plasma membrane, Golgi, mitochondria, nuclear membrane and endoplasmic reticulum that apparently contains the substrate for that isoform (170).

1.8.4 ATP-competitive Inhibitors of PKCs

The vast majority of small molecule kinase inhibitors act by competing with ATP for its binding site on PKC. An important point that should be taken into account regarding the inhibitor is its selectivity for the kinase of interest. Several small molecule PKC inhibitors are commercially available, with the most commonly used ones being ATP-competitive derivatives of bisinolylmalemides or indocarbazoles. Two derivatives of bisinolylmalemides, Gö6983 and G06850, inhibit isoforms from all three classes of PKCs in vitro with greater selectivity toward conventional and novel PKCs compared to atypical PKCs. These inhibitors do not inhibit closely related PKA and PKD (protein kinase A and D). On the other hand, Gö6976 and staurosporine are indocarbazole-based inhibitors. Gö6976 is an inhibitor of conventional PKC isoforms, an inefficient inhibitor of novel and atypical isoforms and a highly promiscuous inhibitor of other kinases. However, staurosporine is the least selective of commonly-used PKC inhibitors. It is a potent inhibitor of most PKC isoforms but is even more promiscuous than Gö6976 in inhibiting other non-PKC protein kinases (171). Since the ATP-binding site is conserved among all ser/thr kinases, it is impossible to avoid at least some degree of promiscuity among active-site inhibitors. Therefore, a non-catalytic domain or a less-conserved domain of the kinases should be targeted for synthesis of more selective inhibitors.

1.9 14-3-3 proteins

1.9.1 General properties 14-3-3 proteins form a family of small proteins (25-30 kDa) that are relatively abundant and are ubiquitously expressed in all eukaryotic cells. Mammalian cells possess 7 isoforms of 14- 3-3 proteins that are mainly located in the cytoplasm, but can move easily from cytosol to nucleus and vice-versa. The hallmark of 14-3-3 proteins is their ability to bind to numerous proteins thereby regulating a wide variety of signaling pathways involved in cell cycle control, survival, cell adhesion and neuronal plasticity. Binding of 14-3-3 proteins to target proteins may alter enzymatic activity, promote or inhibit protein-protein interactions or induce post translational modifications. However, in most known cases, 14-3-3 proteins inhibit the function of the target protein. (172).

1.9.2 Interaction of 14-3-3 proteins with other proteins

1.9.2.1 Phosphorylation-dependent interactions 14-3-3 proteins interact with target proteins mostly in a phosphorylation-dependent manner, through binding to the consensus motifs RSXpSXP and RXY/ FXpSXP where pS is phosphoserine and X represents any amino acid (173). Although, it has been well established that phosphorylation of the target protein regulates its binding to 14-3-3 protein, there is evidence that phosphorylation of 14-3-3 proteins also modulates their interaction with their binding partners. 14- 3-3 proteins contain several residues that can be phosphorylated by different protein kinases including certain isoforms of PKC, AKT, JNK (c-Jun N-terminal protein kinase), CkI (cyclin- dependent kinase inhibitor) and Bcr (174). Regulation of the pro-apoptotic molecule BAD (Bcl-2- associated death promoter) by 14-3-3 is an example of phosphorylation-dependent interactions (Figure 1.10). Survival signals promote Akt activation that result in phosphorylation of BAD, which in turn promotes association of 14-3-3 with BAD. Binding of 14-3-3 to BAD causes a conformational change that exposes S155 of BAD to PKA. Phosphorylation of S155 results in dissociation of the anti-apoptotic factor Bcl2 from 14-3-3, resulting in inhibition of apoptosis (175). On the other hand, stress and DNA damage induce activation of JNK and phosphorylation of 14-3-3 that result in dissociation of BAD from 14-3-3. As a result, BAD is dephosphorylated and translocates to the mitochondria, where it binds to and inhibits Bcl2, thereby inducing apoptosis (174). Furthermore, 14-3-3 is involved in regulation of cell growth through modulation of the Raf-1/ERK pathway. Raf-1 contains two 14-3-3 binding sites. In unstimulated cells, only one site is phosphorylated, preventing the formation of a stable complex with 14-3-3. This enables Raf-1 to translocate to membranes where its activator Ras is located. Activation of Raf-1 stimulates MEK1 and MEK2, activators of ERK. Eventually, ERK is activated and induces latent transcription factors and alters patterns of gene expression involved in cell growth and differentiation. Inhibition of Raf-1/ERK signaling occurs when both 14-3-3 binding sites on Raf- 1 are phosphorylated, in which case Raf-1 forms a complex with 14-3-3 that resides in the cytoplasm, where it is dissociated from its activator Ras (176).

Figure 1.10. Regulation of BAD with 14-3-3. In non-stimulated cells, pro-apoptotic molecule BAD is sequestered in the cytosol through interaction with 14-3-3 protein. Upon cellular stress and DNA damage, JNK-mediated phosphorylation of 14-3-3 leads to dissociation of BAD from 14-3-3. Consequently, BAD is dephosphorylated and translocated to mitochondria where it binds to the anti-apoptotic molecule, Bcl-2/XL, which in turn triggers apoptosis (Adapted from (174) with modification).

1.9.2.2 Interaction with non-phosphorylated proteins Although most of regulatory effects of 14-3-3 proteins are mediated by a phosphorylation- dependent process, it has been reported that 14-3-3 also interacts with non-phosphorylated proteins. For example, the proapototic protein Bax binds 14-3-3 without being phosphorylated. Bax remains in the cytoplasm while it is complexed with 14-3-3. During apoptosis, Bax undergoes a conformational change that results in dissociation of 14-3-3. This permits Bax to become integrated into the mitochondrial membrane, triggering the release of apoptotic factors such as cytochrome c. The release of Bax from 14-3-3 occurs either via a caspase-independent pathway or through direct cleavage of 14-3-3 by caspases (177).

Chapter 2. Aim of study

2.1 Activation of NADK by PKC in neutrophils

Despite to critical role of NAD kinase (NADK) in maintaining the redox state of cells and supporting respiratory burst, relatively little information is available on mammalian NADK. For example, although it has been shown that PMA activates NADK in neutrophils, the mechanism for this effect is not yet understood. Stimulation of neutrophils with PMA, which activates PKC, activates NOX-2 which results in a robust respiratory burst in which superoxide is generated at the expense of oxidation of NADPH to NADP+. Our preliminary measurements of pyridine nucleotides in neutrophils following stimulation with PMA showed an expected rapid rise in NADP+ levels, but, contrary to expectations, this was not accompanied by the loss of NADPH, which initially also increased, but rather by the depletion of NAD+. The time course for NADP+ formation was precisely mirrored by a comparable decline in NAD+ levels, suggesting that NADK had been activated. The major aim of this study was to elucidate the mechanism of NADK activation in neutrophils by PMA. We first wanted to determine whether the effects of PMA are mediated by PKC and, if so, whether PKC directly modifies NADK or whether it acts indirectly, through its well-known activating effect on NADPH oxidase, for example. If found to act directly on NADK, we wanted to determine the mechanism for this effect, and in particular, to determine whether PKC could phosphorylate NADK, resulting in its activation. We hoped to achieve these objectives using a variety of techniques, including measurement of pyridine nucleotides by HPLC, analysis of NADK activity under a variety of conditions, purification of NADK and examination of potential post-translational modifications using mass spectrometry. In this way we hoped to elucidate novel regulatory mechanisms in neutrophils that could be important for maintaining sufficient intracellular levels of NADP+ and NADPH, which serve very important roles in phagocytic cells, including initiation of the respiratory burst, which is critical in host defense.

Chapter 3. Material and Methods

3.1 Materials Adenosine trinuvleotide phosphate (ATP), 3-[(3-cholamidopropyl)-dimethylammonio]1- propanesulfonate (CHAPS), dextran 500 (from Leuconostoc), dimethyl sulfoxide (DMSO), diphenyleneiodonium chloride (DPI), ethylenediaminetetraacetic acid (EDTA), glucose 6- phosphate (G6P), glucose 6-phosphate dehydrogenase (G6PDH), reduced glutathione (GSH), thiazolyl blue tetrazolium blue (MTT), nicotinamide adenine dinucleotide (NAD), nicotinamide hypoxanthine dinucleotide (IS), phorbol-12 myristate-13-acetate (PMA), phenazine methosulfate (PMS) and anti-NADK (polyclonal A) were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Complete mini EDTA-free protease inhibitor cocktail tablets, NADP+ and NADPH were purchased from Roche Diagnostics (Laval, QC). Ficoll-Paque, gluthatione-sepharose beads and prescission protease were obtained from GE Healthcare Bio-Sciences, Uppsala, Sweden. RPMI 1640 and other cell culture materials were obtained from Invitrogen (Burlington, Ontario, Canada). Phenylmethylsulfonyl flouride (PMSF) were purchased from ICN Biomedicals Inc. (Aurora, OH). HPLC solvents were from Fisher Scientific, Nepean, ON. Acrylamide/bisacrylamide solution, ammonium persulfate (APS), N, N,N”,N’-tetramethylethylenediamine (TEMED), pre-cast acrylamide gel, CHT hydroxyapatite (HA), Macro-Prep t-butyl-HIC media and gel filtration protein standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Monoclonal anti NADK, anti-14-3-3 zeta protein, anti-p47 antibodies were obtained from Santa Cruz Biotechnology, Inc. (Dallas, Texas, U.S.A). Anti-phospho (Ser) PKC substrate antibody and anti- NADK (Polyclonal B) were obtained from Cell Signaling technology (Danvers, Massachusetts, U.S.A) and Atlas Antibodies (Stockholm, Sweden), respectively. Immunoprecipitation kit was from Pierce Biotechnology (Rockford, IL, U.S.A). Recombinant human PKC-δ and its activator were from SignalChem (Richmond, BC, Canada).

3.2 Isolation of neutrophils Isolation of neutrophils from healthy subjects blood was carried out by dextran sedimentation by removing red blood cells followed by centrifugation over Ficoll-Paque to separate mononuclear cells and hypotonic lysis of remaining red blood cells (70). Performing this method results in purification of polymorphonuclear leukocytes (more than 95% neutrophils and

less than 5% eosinophils) without any contamination with mononuclear leukocytes. Purified neutrophils were resuspended in PBS+ followed with stimulation with PMA for various time at 37º C.

3.3 Quantitation of NAD+ and NADP+ NAD+ and NADP+ were converted to fluorescent naphthyridine derivatives with alkaline acetophenon derivitization, followed with RP-HPLC (reversed phase high-performance liquid chromatography) analysis (178). 2 x 106 neutrophil in 250µl of PBS+ were stimulated with 50nM PMA. To stop the stimulation, 250 ul of an ice cold mixture of 50 mM acetophenone in Methanol + and 3 M KOH (1:1 ratio) along with 30 ng of deamino-NAD (as an internal standard) was added to the neutrophils suspension. The mixture was kept on ice for 15min, followed by adding 62.5 µl of formic acid. Then the mixture was extracted with ethyl acetate and the aqueous phases were incubated with 100 µM PMS. 50 µl aliquots were analyzed by RT-HPLC on an Ultracarb ODS column (31% carbon loading; 5 μm particle size; 150 mm×4.6 mm; Phenomenex) using a Waters model 2475 Multiwavelength Fluorescence Detector (λex, 371 nm; λem, 438 nm). The mobile phase was a gradient between solvent A (100 mM citric acid containing 4 mM Tetrabutylammonium hydroxide (TBAH)) and solvent B (acetonitrile) with 1-25% B over 12min and flow rate of 1.25ml/min.

3.4 Measurement of NADPH NADPH was measured by RP-HPLC by taking advantage of its native fluorescence (178). 6 Neutrophils (1.5×10 cells in 100 μl PBS+) were incubated with 50nM PMA for various times. The incubations were terminated by addition of 50 μl of 200 mM Na PO . The mixture was sonicated 3 4 for 10 sec at medium level, followed with centrifugation for 10 min at 13500 rpm. pH of the supernatant was adjusted to 7.00 by addition of NaH2PO4. 100 μl aliquots from each sample were analysed by RP-HPLC using a SupelcosilTM LC-18-T column (5um particle size; 250×4.6mm; Sigma). The mobile phase was a gradient between solvents A (100 mM potassium phosphate pH: 6 containing 6 mM TBAH) and B (100 mM potassium phosphate pH: 5.5 containing 6 mM TBAH and 35% methanol) with 0-100% B over 10min, hold for 10min at 100%B. NADPH was detected

using a fluorescence detector (λex, 325 nm; λem, 450 nm). The amounts of NADPH were calculated from a standard curve.

3.5 Measurement of cytosolic calcium levels in PMA stimulated neutrophils Neutrophils were incubated with the acetoxymethyl ester form of the fluorescent dye indo- 1 for 30 min in PBS- (179). The cells were then washed in PBS- and resuspended in PBS containing Ca2+ /Mg2+ before the addition of PMA. Following stabilization of the baseline fluorescence, calcium measurements were performed in temperature-controlled cuvette at 37C using a Photon Technology International (PTI) Deltascan 4000 spectrofluorometer with a magnetic stirrer. Fmax was determined by adding ionomycin.

3.6 Differentiation of PLB-985 cells into neutrophil like cells Human myeloid leukaemia cell line PLB-985 was cultured in RPMI-1640–glutamine medium supplemented with serum and antibiotics. To Differentiate PLB-985 cells into neutrophilic granulocytes, cells were cultured in the presence of 1.25% endotoxin-free DMSO for 5 days (180). Differentiation was controlled based on morphological characterization.

3.7 Preparation of cell lysates Neutrophils were resuspended in PBS+ and incubated with either vehicle or PMA. To lyse the cells, one volume of 2X lysis buffer containing 20mM Tris-HCl, 150mM NaCl, 6mM CHAPS, 1mM EDTA, 1mM NaF, 1mM β-glycerophosphate, 1mM PNPP, 1mM PMSF and protease inhibitors cocktail were added and vortexed well. The mixture was incubated on ice for 10min followed with centrifugation at 13000 rpm for 10min.

3.8 Protein quantitation Protein concentrations for all purification steps were measured using the Bio-Rad Bradford protein assay (181). Protein levels were calculated using an external standard curve of bovine serum albumin (BSA).

3.9 Measurement of NAD kinase activity NAD kinase activity was measured in cell lysates performing a two-step procedure, as described previously, by minor modification (182). 20 µg of cell lysate was added to a reaction solution containing 50mM Tris-HCl pH: 8, 10mM MgCl2, 1mM NAD+ and 5mM ATP at final volume of 100 µl, incubated at 37º C for 20 min. Incubation was terminated by boiling the samples for 2min. The amount of produced NADP+ was measured doing a cycling assay. The mixture was added to 400 µl of solution containing 50mM Tris-HCl, 5Mm MgCl2, 5mM glucose 6-phophate, 1.5mM PMS and 0.5mM MTT. One unit of glucose 6 phosphate dehydrogenase was added to the reaction mixture and reduction of MTT was immediately monitored at 600 nm for 2min using spectrophotometer. NADP+ level was quantified by comparison to known concentrations of NADP+ standards.

3.10 Western Blotting Whole cell extracts were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus (Bio-Rad) at 4 °C for 80 min. After incubation with 5% non-fat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary antibodies at 4 °C overnight. Membranes were washed three times for 10 min and incubated with a 1: 10000 dilution of DyLight fluorochrome-conjugated anti-mouse or anti-rabbit antibodies for 1 h at room temperature. Blots were washed with TBST three times and scanned to acquire images.

3.11 Immunoprecipitation

Immunoprecipitation was performed using PierceTM Crosslink IP kit to minimize co- elution of heavy and light chains of IgG with the purified proteins. In this method, the IP antibody is covalently crosslinked to Protein A/G Agarose resin with disuccinmidyl suberate (DSS). The antibody resin is then incubated with cell lysates at 4 °C overnight, allowing the antibody-antigen interaction. After washing to remove non-bound proteins, the protein of interest is recovered by dissociation from the antibody with elution buffer.

3.12 Production of recombinant NADK

3.12.1 Cloning of cDNA of NADK Total RNA was extracted from human neutrophils using Trizol reagents followed with RT- PCR to synthesize cDNA. Bacterial expression of NADK was achieved by cloning the cDNA into a pGEX-6P vector that has two tags including GST (N-terminal) and PreScission cleavage site.

3.12.2 Expression of NADK in E.Coli The construct was transformed to E.Coli Rosetta and overnight culture was done in LB medium with ampicillin (100 µg/ml) and chloramphenicol (34 µg/ml) at 25°C. The expression of NADK was induced by testing different concentrations of IPTG (Isopropyl β-D-1- thiogalactopyranoside) at an optical cell density of 0.6 (600 nm). After 4 h of expression, the cells were harvested and lysed in lysis buffer (50 mM Tris–HCl, pH: 8, 1mM EDTA, 0.5% Triton, 100mM NaCl, 5% glycerol, 1mM PMSF, 100 µg/ml lysozyme), followed with 15 min incubation on ice; vortexing every 5 min. Then 3 U/ml DNase and 2mM MgCL2 were added to the mixture. After 15 min incubation on ice, the mixture were sonicated for 8 sec, 3 times, followed with centrifugation at 8000 rpm for 10 min at 4 °C. An aliquot of the bacterial extract was subjected to SDS-PAGE electrophoresis, followed with coomassie blue staining to evaluate the NADK expression.

3.12.3 Purification of GST-NADK from bacterial extracts The recombinant NADK was purified from the bacterial extract using gluthatione- sepharose beads (GE healthcare). 1ml of the bacterial extract was incubated with 20 µl of glutathione-sepharose beads for 2h in cold room while rotating. Then the mixture was centrifuged and the pellet was washed 3 times with PBS (phosphate buffered saline). To elute the recombinant NADK, the beads were incubated with 100 mM reduced glutathione in 50 mM Tris-HCl, pH: 8 for 10 min at room temperature while rotating, followed with spinning down and saving the supernatant. The elution process was repeated one more time to elute the remaining NADK.

3.12.4 Cleavage of GST using PreScission protease To cleave the GST-tag, the GST-NADK was treated with PreScission protease (GE healthcare) overnight at 4°C in a buffer containing 50 mM Tris-HCl pH: 7, 150 mM NaCl, 1mM EDTA and 1mM DTT (dithiothreitol). To test if the cleavage was done efficiently, the sample was subjected to SDS-PAGE followed with Coomassie Blue staining.

3.13 In vitro phosphorylation of GST-NADK by PKC-δ Recombinant GST-NADK was treated with PKC-δ (SignalChem) according to manufacturer’s instructions. Briefly, 10 mM ATP stock solution (5x) was prepared in 5x kinase assay buffer (25mM MOPS, pH 7.2, 12.5mM, β-Gylcerol phosphate, 25mM MgCl2, 2mM EDTA). Then recombinant NADK, active PKC-δ, PKC lipid activator (phosphatidyl serine and diacylglycerol) and ATP were mixed in a tube and incubated for 15 min at 30°C. An aliquot of the mixture was tested for NADK activity and the rest was subjected to SDS-PAGE followed with immunoblotting with either monoclonal anti-NADK or anti-phospho serine PKC substrate antibody.

3.14 Partial purification of NADK

3.14.1 Size exclusion chromatography Proteins from neutrophil lysates (0.5-1 mg) were concentrated to 20 μl using 10 kDa Amicon centrifugal filter (Millipore) and size separated using a 7.8 x 300 mm TSKgel G3000SWxl size exclusion column (Tosoho bioscience) with 5 μm particle size. The column was connected to the Waters 2695 Alliance system and chromatography was performed at room temperature using isocratic conditions of 100 mM phosphate buffer pH 7 containing 100 mM sodium sulfate at a flow rate of 0.5 ml/min. A mixture of molecular mass markers (Bio-Rad), including bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa) and vitamin B12 (1.3 kDa) was also injected to determine NADK molecular mass. The protein levels were monitored at 280 nm and fractions (0.5 ml) were collected every minute to test for NADK activity.

3.14.2 Hydroxyapatite chromatography Hydroxyapatite (HA) is a “mixed-mode” ion exchanger in which positively charged calcium ions interact with carboxyl residues of proteins and negatively charged phosphate groups interact with amino groups on proteins. CHT hydroxyapatite medium (Bio-Rad) was packed into a 10*100 mm column according to the manufacturer’s instructions and attached to the Waters 2695 Alliance system. Proteins from neutrophil lysates (3-5 mg) were concentrated to 100 μl using 10 kDa Amicon centrifugal filter and diluted in 5mM phosphate buffer pH 7.0 to have a final volume of 0.5 ml to load onto the column. The chromatography started with a solvent containing 5mM phosphate buffer pH: 7.0 (solvent A) and the concentration of phosphate was gradually increased to 400 mM (solvent B) as follows: 4 min isocratic solvent A, 0-100% B over 20 min, 4min isocratic solvent B, at a flow of 1ml/min. 2ml fractions were collected and aliquots were tested for NADK activity and protein quantitation.

3.14.3 Hydrophobic interaction chromatography A 10*100 mm column was packed with 50 µm Macro-Prep t-butyl-HIC media (Bio-Rad) according to manufacturer’s instructions. T-Butyl is mildly hydrophobic and interacts with hydrophobic sites of proteins. 0.5 ml of neutrophil lysates (~2 mg) were loaded on the column with 5mM phosphate buffer, pH 7.00 containing 1M sodium sulfate at a linear flow rate of 0.5 ml/min for 10min, followed with decreasing the sulfate concentration to zero over 30min and maintaining at this condition for 10 min. Fractions were collected every 3min, 0.5 ml of each fraction was analyzed for NADK activity and protein quantity.

3.14.4 Ion exchange chromatography To separate the proteins based on their charge, ion exchange chromatography was performed using an anion exchange column (Rainin Company). Neutrophil lysates were concentrated to 50 µl and then diluted with 20 mM phosphate buffer, pH 7.4 to final volume of 200 µl. The mobile phase was a gradient between solvent A (20mM phosphate buffer pH: 7.4) and B (1 M ammonium acetate) as follows: 0-4 min: 100% A, 34 min: 70% B, flow rate: 0.5 ml/min. 1ml fractions were collected and assayed for NADK activity and protein concentration.

3.15 LC-MS-MS analysis

3.15.1 Preparation of samples To prepare neutrophil lysates, isolated neutrophils from 4-5 healthy donors’ blood were lysed and the lysates were pooled together. Then NADK was purified from the lysates either through conventional partial purification or IP. The purified NADK was subjected to SDS-PAGE using precast 10% acrylamide gel (Bio-Rad). The gel was fixed overnight in a buffer containing 50% methanol and 10% acetic acid followed with silver staining. After staining, the bands were excised and stored at -20 °C until processing for in-gel digestion of proteins.

3.15.2 In-gel digestion of proteins Trypsin digestion of proteins (183) in excised bands (silver stained) and the LC-MS/MS analysis were performed by Dr. Denis Faubert’s group at Institut De Recherches Cliniques De Montreal, QC. After digesting the proteins, tryptic peptides were re-solubilized in 2% ACN / 1% formic acid for 15 min under agitation. Then, the peptides were subjected to liquid chromatography (LC).

3.15.3 LC-MS/MS To perform LC, a 15 cm, 75 µm i.d. Self-Pack PicoFrit fused silia capillary column (New Objective, Woburn,MA) was packed with the C18 Jupiter 5 µm 300 Å reverse-phase material (Phenomenex, Torrance, CA). The mobile phases were 0.2 % formic acid (solvent A) and 100 % acetonitrile / 0.2 % formic acid (solvent B). The LC column was installed on the Easy-nLC II system (Proxeon Biosystems, Odense, Denmark) and coupled to the LTQ Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) equipped with a Proxeon nanoelectrospray ion source. LC-MS/MS data acquisition was done using an eleven scan event cycle included of a full scan MS for scan event 1 acquired in the Orbitrap. The mass resolution for MS was set to 60,000 (at m/z 400) and used to trigger the ten additional MS/MS events acquired in parallel in the linear ion trap for the top ten most intense ions. Mass over charge ratio range was from 360 to 2000 for MS scanning with a target value of 1,000,000 charges and from ~1/3 of parent m/z ratio to 2000 for MS/MS scanning with a target value of 10,000 charges. A dynamic exclusion of 15 sec was used to limit resampling of previously selected ions. Nanospray and S-lens voltages were set to

1.5 kV and 50 V, respectively, and the temperature of capillary was set to 225ºC. Following conditions were applied for MS/MS: normalized collision energy, 35 V; activation q, 0.25; and activation time, 10 ms.

3.15.4 Protein identification Protein database searches were performed with Mascot 2.3 (Matrix Science) using carbamidomethyl (CAM) as a fixed modification and methionine oxidation as a variable modification. Proteins were identified from peptide sequences available for all species.

3.16 Data analysis Data are presented as Mean ± SEM. Statistical analysis were performed using GraphPad Prism 6. Paired T-test were performed comparing two groups with one variable. Analysis of the experiments with more than two groups or two groups with two variables were performed by either one-way or two-way ANOVA (analysis of variance), as were appropriate. Sidak’s post-test was used for multiple comparison. P < 0.05 was considered statistically significant. Western blots were quantified using ImageJ program.

Chapter 4. Results

4.1. PMA activates NADK in intact neutrophils

4.1.1 PMA has a dramatic effect on pyridine nucleotide levels in intact neutrophils

Activation of NOX-2 in neutrophils by stimulators of PKC, such as PMA, results in a robust respiratory burst, in which superoxide is generated from oxygen at the expense of oxidation of NADPH to NADP+. We measured the levels of pyridine nucleotides by fluorescence HPLC following addition of PMA to neutrophils (Figure 4.1.A to D). The expected rapid rise in NADP+ levels was observed, but, contrary to expectations, this was not accompanied by the loss of NADPH, which initially also increased, but rather by the depletion of NAD+. The time course for NADP+ formation was precisely mirrored by a comparable decline in NAD+ levels (Figure 4.1.E), suggesting that NADK had been activated.

Figure 4.1. Effect of PMA on pyridine nucleotide levels. NAD+, NADP+ (A, B) and NADPH (C, D) were measured by fluorescence-HPLC in neutrophils. The time courses for the changes in the levels of NAD+ (○; n=7), NADP+ (□; n=6) and NADPH (Δ; n=6) following addition of PMA (50 nM) to neutrophils are shown in panel E. For NAD+ and NADP+ all points after addition of PMA and for NADPH points 2 and 4 are significantly different from time zero (NAD+: p< 0.001 and NADP+: p <0.01, NADPH: p< 0.05).

4.1.2 PMA-induced activation of NADK is independent of NADPH oxidase and calcium mobilization

As mentioned in chapter 1, because human NADK is inhibited by NADPH but not by NADP+ (138), depletion of NADPH could result in activation of NADK. Since NADPH levels initially increased in PMA-stimulated neutrophils, PMA-induced activation of NADK could not be explained by the loss of NADPH due to NOX-2 activation. To confirm this, neutrophils were treated with the NOX inhibitor DPI prior to stimulation with PMA. The results clearly show that DPI did not block the PMA-induced depletion of NAD+ (Figure 4.2A). Although DPI blocked the increase in NADP+ induced by PMA (Figure 4.2B), this was presumably due to its rapid conversion to NADPH via the pentose phosphate pathway, as we observed significantly increased levels of NADPH following PMA stimulation of DPI-treated neutrophils (Figure 4.2C). These results demonstrate that the activation of NADK by PMA is independent of its effects on NOX-2.

Another potential mechanism for the activation of NADK in our experiments could be related to changes in intracellular calcium levels, as NADK has been shown to be activated by 2+ Ca /calmodulin (91). We, therefore measured intracellular calcium levels in neutrophils following addition of PMA and found that it has no effect (Figure 4.3).

Figure 4.2. Effects of the NADPH oxidase inhibitor DPI on PMA-induced changes in pyridine nucleotide levels in neutrophils. NAD+ (A), NADP+ (B) and NADPH (C) quantified fluorescence-HPLC in neutrophils preincubated for 10 min with either vehicle (○) or 10µM DPI (●) prior to stimulation with 50 nM PMA for different periods of time. Data are represent as means ± SEM (n=5). ns, not significant **, p< 0.01 and *, p< 0.05.

Figure 4.3. Lack of effect of PMA on intracellular calcium levels in neutrophils. Neutrophils were incubated with the acetoxymethyl ester form of the fluorescent dye indo-1 for 30 min in PBS-, then washed and stimulated with 50 nM PMA. Intracellular calcium was measured using a Photon Technology International (PTI) Deltascan 4000 spectrofluorometer with a magnetic stirrer. Ionomycin was added to determine the maximal fluorescence.

4.1.3 PKC is required for the activation of NADK by PMA

To determine whether PMA activates NADK through a PKC-dependent process, neutrophils were treated with the PKC inhibitors staurosporine (non-selective inhibitor of PKCs as well as some other protein kinases) and Gö6983 (inhibitor of PKCα, β, γ, δ, and ζ). Both staurosporine (0.5 µM) and Gö6983 (1 µM) blocked both the depletion of NAD+ (Fig. 4.4A) and the increase in NADP+ (Fig. 4.4B) induced by PMA (50 nM; 5 min) without significantly affecting NADPH levels (Fig. 4.4C). Concentration-response experiments were conducted to determine the + + IC50 values of staurosporine and Gö6983 for NAD depletion (Fig. 4.5A) and NADP generation + + (Fig. 4.5B). The IC50 values for the effects of staurosporine on NAD depletion and NADP generation were 37.90 ± 12.41 and <5 nM respectively, whereas the corresponding values for Gö6983 were 261.38 ± 25.47 and 47.63 ± 5.99 nM.

Figure 4. 4. Effects of PKC inhibitors on PMA-induced changes in pyridine nucleotide levels in neutrophils. Inhibition of changes in NAD+ (A) and NADP+ (B) and NADPH (C) in neutrophils treated with vehicle (Δ), 1µM Gö6983 (○) or 0.5 µM staurosporine (□) before 50 nM PMA stimulation for different time points. n=3, Data are mean ± SEM. **, p< 0.01 and *, p< 0.05.

Figure 4.5. Concentration-response curves for PKC inhibitors. Neutrophils were incubated for 10 min with different concentrations of staurosporine (●) or Gö6983 (○) and then stimulated with 50 nM PMA. The inhibition of NAD+ decrease (A) or NADP+ increase (B) following incubation with PMA for 5 min was quantified comparing their levels at time 0 and time 5 min. n=4. Data are mean ± SEM.

4.2 NADK activity in cell lines As neutrophils have a very short life span, it is not feasible to use techniques that require transfection, such as RNA interference, to reveal the mechanism of NADK activation by PKC. Therefore, we sought to identify a cell line in which PMA induced a rapid decline in intracellular NAD+ levels, indicative of activation of NADK. To do so, PLB-985 cells were differentiated into neutrophil like cells with 1.25% DMSO. Although differentiated PLB-985 cells morphologically looked similar to neutrophils and they expressed the p47 subunit of NOX-2 (Figure 4.6A), we were not able to replicate the neutrophils response to PMA in these cells (Fig. 4.6B). Interestingly, monocytes isolated form human blood showed a response to PMA (Figure 4.7A), but a number of monocytic cell lines, including THP-1, U937 and MD (monocyte/macrophage) cells (Figure 4.7B- D) were not affected significantly in response to PMA. We also tried other cell lines such as Ramos (B lymphocyte) and Cess (lymphoblast) cells (Figure 4.8 A, B), but none of them responded to PMA.

Figure 4.6. Effects of PMA on NAD(P)+ levels in PLB-985 cell. PLB-985 cells were differentiated to neutrophil-like cells by treatment with 1.25% DMSO for 5 days. Their differentiation was validated by assessing expression of p47phox, a subunit of NADPH oxidase, by western blotting (A). Changes in NAD+ (○) and NADP+ (●) levels in response to 50 nM PMA were analyzed in differentiated PLB-985 cells (B). Values are presented as means ± SEM.

Figure 4.7. Effects of PMA on NAD(P)+ levels in monocytes and monocytic cell lines. Changes in NAD+ (○) and NADP+ (●) levels in response to 50 nM PMA were analyzed in monocytes (A), THP-1 (B), U937 (C) and MD (D) cells.

Figure 4.8. Effects of PMA on NAD(P)+ levels in lymphocytic cell lines. Changes in the levels of NAD+ (○) and NADP+ (●) following addition of PMA (50 nM) to Ramos (A) and Cess (B) cells.

4.3 NADK activity is elevated in lysates from PMA-stimulated neutrophils We also measured NADK activity in neutrophil lysates following PMA stimulation over 15 min using a spectrophotometric assay in which increasing absorbance at 600 nm caused by the reduction of NADP+ to NADPH is measured (Figure 4.9.A, B). In agreement with our initial results, we observed that NADK activity is elevated in lysates from PMA-stimulated neutrophils compared to vehicle-treated neutrophils with the maximal response being reached by 2 min (Figure 4.8.C). The stimulatory effect of PMA on NADK activity was blocked by pretreatment of neutrophils with Gö6983 (Figure 4.9.D).

However, based on the rapid decline in intracellular NAD+ measured by HPLC, we expected to observe a more pronounced increase in NADK activity, which was elevated by only about 60% compared to lysates from control cells. In these experiments we assayed NADK activity by measuring the amounts of NADP+ that were produced from exogenous NAD+ and ATP. To ensure that endogenous NADP+ and NADPH did not interfere with the assay, we performed the assay on lysates of vehicle and PMA treated neutrophils in the absence of the exogenous NADK substrates NAD+ and ATP. The values obtained were comparable to those of measured in blanks (Figure 4.10) showing that endogenous levels of NADP+ and NADPH are too low to interfere with the assay.

One explanation for the rather modest increase of NADK activity in lysates in response to PMA could be that under physiologic conditions, NADK activity is inhibited by some intracellular factors such as NADH and NADPH. As a consequence of lysing the cells, these inhibitors would be diluted, which in turn could increase the basal level of NADK activity. To investigate this, we lysed the vehicle/PMA treated neutrophils in lysis buffer containing 150 µM of NADH. The results showed that NADH inhibited NADK activity in both vehicle and PMA treated neutrophils similarly (Figure 4.11). Therefore, it is unlikely that the modest increase in NADK activity observed in lysates from PMA-treated neutrophils is due to dilution of endogenous NAD(P)H in the lysates.

Figure 4.9. NADK activity in neutrophil lysates. A: Schematic diagram of assay used to measure NADK activity. B: NADP+ generated by NADK in neutrophil lysates was measured using a spectrophotometric assay in which amount of NADP+ was reflected by the slope of the line indicating formazan blue formation. C: Time course showing NADK activity in lysates (20 µg protein) from neutrophils treated with either vehicle (○) or PMA (100 nM;●) for different times (n=9; ***, p<0.001). D: NADK activity in neutrophils preincubated for 10 min with either vehicle (left) or Gö6983 (right) followed by incubation with either vehicle (open bars) of PMA (100 nM; black bars) for 2 min (n = 3; ** p<0.01). Data are presented as means ± SEM.

Figure 4.10. Effect of endogenous NADP and NADPH on NADK activity assay: 20 µg protein of vehicle and PMA treated neutrophil lysates was tested for NADK activity either in the presence of exogenous NAD and ATP (right bars) or in the absence of them (middle bars), n=1. The values obtained were compared to that of blank sample (left bar).

Furthermore, because the activity of NADK is dependent on the availability of certain cations (91,136), we assayed NADK activity from vehicle and PMA treated neutrophils at three different concentrations of MgCl2, 0.1 µM, 0.5 mM and 10 mM (concentration of magnesium in the regular assay). At very low concentration of magnesium, very little NADK activity was detected. Increasing the concentration of magnesium, increased the enzymatic activity in both vehicle and PMA treated neutrophil (Figure 4.12 A). As it was previously reported that NADK is sensitive to micromolar concentrations of calcium (91), we performed the NADK activity assay in 2+ the presence of 10 µM Ca Cl2 and different concentrations of MgCl2. As shown in figure 4.12B, changing the concentrations of ions affected NADK from vehicle and PMA treated neutrophils, similarly.

Figure 4.11. Effect of NADH on NADK activity. Vehicle (white bars) and PMA (100nM, 2min, dark bars) treated neutrophils were lysed either in regular lysis busffer (vehicle) or in abuufer containing 150µM of NADH, followed with measurement of NADK activity. n=5, * p<0.05. Data are presented as mean ± SEM.

Figure 4.12. Effect of cationic ions on NADK activity. Activity of NADK in lysates of vehicle (white bars) and PMA (100nM, 2min, dark bars) treated neutrophils were measured in the presence of A. different concentrations of magnesium and B. different concentrations of magnesium in the presence of 10 µM calcium.

4.4 Detection and immunoprecipitation of NADK using various antibodies To further investigate PMA-induced changes in NADK activity we tested a series of commercially available antibodies, including one monoclonal antibody (MC-aNADK Ab) and 4 polyclonal antibodies.

4.4.1 Monoclonal anti-NADK antibody The first commercially available NADK antibody was NADK monoclonal antibody M01, clone 5F4 from Abnova. It was raised against full-length NADK containing a GST tag. This monoclonal NADK antibody revealed a rather weak band at 49 KDa when used to analyze a lysate from unstimulated neutrophils. However, lysates from neutrophils that had been treated with PMA (100 nM) gave a much stronger band, which reached maximal intensity by 2 min (Figure 14.13. A,B).

Figure 4.13. Time course for the generation of immunoreactivity to MC-aNADK in neutrophils treated with PMA. A: Western blots of NADK in lysates (50 µg protein) from neutrophils incubated with PMA (100 nM) for various times using the monoclonal antibody MC- aNADK. B: Quantification of NADK protein in the western blots shown in A using the ImageJ program.The densities of the NADK bands were normalized to those of -actin (Data are presented as mean ± SEM.; n = 2).

We also performed an immunoprecipitation (IP) experiment in which we analyzed NADK immunoreactivity (western blots) and enzymatic activity in both the immunoprecipitate and supernatant fractions from lysates of vehicle- and PMA- treated neutrophils. The percent of total lysate NADK activity that was immunoprecipitated by MC-aNADK from lysates of PMA-treated neutrophils was significantly higher than that from lysates of vehicle-treated neutrophils (Figure 4.14A). Only a small proportion of NADK enzyme activity was precipitated from vehicle-treated neutrophils, with most of the activity remaining in the supernatant fraction. In contrast, a much larger amount of NADK activity was precipitated from PMA-treated neutrophils, with an approximately equal amount remaining in the supernatant (Fig. 4.14B). However, MC-aNADK precipitated virtually all of the immunoreactivity in lysates from both vehicle- and PMA- treated neutrophils (Figure 4.14C, D). These results suggest that there are at least two enzymatically active forms of NADK, presumably differing from one another by one or more post-translational modifications.

To determine whether the increase in immunoreactivity of NADK toward MC-aNADK Ab is mediated by PKC, we treated neutrophils with the PKC inhibitors staurosporine and Gö6983 prior to activation with PMA. Both staurosporine (Figure 4.15A, B) and Gö6983 (Figure 4.15 C, D) strongly inhibited the emergence of NADK immunoreactivity in response to PMA, suggesting that this effect is meditated by PKC.

Figure 4.14. Immunoprecipitation of NADK using MC-aNADK. A. Percent of total lysate NADK activity that was immunoprecipitated by MC-aNADK from lysates of neutrophils treated either with vehicle or 100 nM PMA (n=3; ** p<0.01). B. NADK activity in immunoprecipitate and supernatant fractions following IP of NADK from lysates of neutrophils treated with vehicle or PMA (100 nM) for 2 min (n = 4). C. Western blots of immunoreactive NADK in whole lysates, immunoprecipitates and supernatants after removal of immunoprecipitated material from neutrophils treated with vehicle or PMA (100 nM) for 2 min. (The bands are from the same blot, but their order is changed). D: Quantitation of the blots shown in panel C using ImageJ (n = 3). Values are presented as presented as mean ± SEM.

Figure 4.15. Effects of PKC inhibitors on PMA-induced immunoreactivity of NADK. Neutrophils were pretreated with 100 nM staurosporine (A) or 500 nM Gö6983 (C) prior to stimulation with 100 nM PMA for 2 min. Western blot analyses were performed using MC- aNADK Ab. Blots were quantified using ImageJ. B. The intensities of the NADK bands after treatment with staurosporine (Sts) or Gö6983 (GO) are shown in panels B and D, respectively (n = 3). Values are means ± SEM (n = 3). *** p<0.001.

4.4.2 Polyclonal (H300) anti-NADK antibody Because MC-aNADKAb only interacted appreciably with a modified form of NADK, we sought to identify additional antibodies that would recognize all forms of NADK to a similar extent. We tested a rabbit polyclonal antibody from Santa Cruz Biotechnology called (H300) anti- NADK Ab which is raised against amino acids 8-300 mapping near the N-terminal of human NADK Figure (4.16A). This antibody didn’t detect any band at 49 kDa in the lysates of either vehicle- or PMA- treated neutrophils. Instead, two bands were detected with the apparent molecular masses of 20 and 30 kDa which are unlikely to correspond to NADK (Figure 4.16B).

Figure 4.16. Western blotting of NADK using polyclonal H300 anti NADK antibody. A. Full length human NADK with immunogen corresponding to amino acids 8-300 highlighted. B. 50 µg of protein lysates from neutrophils incubated with vehicle/100 nM PMA for 2min were analyzed by western blotting using polyclonal H300 anti-NADK Ab.

4.4.3 Polyclonal (A) anti-NADK antibody Another polyclonal antibody which will be called poly (A) aNADK Ab is a rabbit polyclonal antibody raised against full length NADK. The band detected by this antibody was slightly lower than that detected using the monoclonal antibody (apparent KDa calculated 43). Unlike the monoclonal antibody, however, there wasn’t a significant difference in the intensity of this band between control and PMA-treated neutrophils (Figure 4.17.A, B). Similarly, measurement of immunoprecipitated NADK activity showed that the efficiency of poly (A) antibody toward NADK from PMA-treated neutrophils is comparable NADK from vehicle-treated cells (Figure 4.17. C). However, the total NADK proportion of NADK that was immunoprecipitated by the poly (A) was less than that by the monoclonal Ab.

Figure 4.17. Immunoprecipitation of NADK using polyclonal antibody (poly (A) aNADK Ab). A: Western blots of NADK in lysates (50 µg protein) from neutrophils incubated with vehicle or PMA (100 nM) for 2 min. B: Quantitation of the bands for NADK shown in panel A using ImageJ (n = 2). C: Percent of total lysate NADK activity that was immunoprecipitated by PC1-aNADK from lysates of neutrophils treated either with vehicle or 100 nM PMA (n = 3). Data are presented as mean ± SEM.

4.4.4 Polyclonal (B) anti-NADK antibody We also used another polyclonal Ab (Poly (B) aNADK) which recently became available. This antibody was raised against amino acids 223-312 of NADK sequences (Figure 4.18A). It gave two bands at 43 and 49 kDa and the intensity of the bands were not affected by PMA significantly (Figure 4.18B). We speculated that 43 and 49 kDa bands detected with this Ab are similar to the 43 kDa and 49 kDa bands recognized by the poly (A) aNADK and the MC aNADK antibodies, respectively.

Figure 4.18. Western blotting of NADK using polyclonal antibody (poly (B) aNADK Ab). A. Full length human NADK with immunogen corresponding to amino acids 223-312 highlitghed. B. 50 µg of protein lysates from neutrophils incubated with vehicle/100 nM PMA for 2min were analyzed by western blotting using polyclonal H300 anti-NADK Ab.

4.4.5 Polyclonal (C) anti-NADK antibody

Another rabbit polyclonal antibody (Poly (C) aNADK) from GeneTex became available last year. This antibody was raised against a recombinant protein containing a proprietary sequence within the central region of human NADK. Western blotting using this antibody resulted in

detection of several bands with apparent molecular masses of 70, 40, 30 and 25 kDa, suggesting that crossreacts with other unrelated proteins in the neutrophil lysates (Figure 4.19).

Figure 4.19. Western blotting of NADK using polyclonal antibody (poly (C) aNADK Ab). Western blots of NADK in lysates (50 µg protein) from neutrophils incubated with vehicle or PMA (100 nM) for 2 min.

4.5 Post-translational modification of NADK Because of the PKC-dependent changes in NADK activity and immunoreactivity we postulated that PKC could induce the phosphorylation of this enzyme. We employed various approaches, including the use of a phospho-(Ser) PKC substrate antibody to detect PKC-induced phosphorylation of NADK in neutrophils, modification of recombinant NADK by PKC, and mass spectrometric analysis.

4.5.1 Detection of NADK phosphorylation in neutrophils with a phospho-(Ser) PKC substrate antibody To determine whether PMA induces the phosphorylation of NADK we immunoprecipitated NADK using either the MC-aNADK or poly (A) aNADK antibodies followed with immunoblotting with a phospho-(Ser) PKC substrate antibody. This antibody recognizes proteins phosphorylated at serine residues surrounded by Arg or Lys in the -2 and +2 positions and a hydrophobic residue in the +1 position. The results show that this Ab only recognizes NADK to an appreciable extent when neutrophils have been stimulated with PMA (Figure 4.20 A-C), providing evidence for PKC-mediated phosphorylation of NADK.

Figure 4.20. Detection of phosphorylated NADK with anti-phospho (Ser) PKC substrates antibody. Neutrophils were stimulated with vehicle or 100 nM PMA for 2 min. NADK from the lysates were immunoprecipitated using either MC-aNADK (A) or Poly (A) aNADK (B), followed with immunoblotting with anti-phospho (Ser) PKC substrates antibody. C: Blot quantification of polyclonal Ab experiment, n=2.

4.5.2 PKC-induced modification of recombinant NADK

4.5.2.1 Production of recombinant NADK in E.coli The full-length human NADK cDNA from neutrophils was cloned into E. coli DH5-α using pGEX-6P-1 vector which contains an open reading frame encoding GST (Figure 4.21A,B). Sequencing of the cloned cDNA revealed that it codes for one additional glutamic acid at the c- terminal. The purified construct was transformed into E.coli Rosetta and NADK expression was induced by adding IPTG to the culture. Consequently, the expression of NADK was evaluated by subjecting the bacterial extract to SDS-PAGE, followed with Coomassie Blue staining. Protein bands with the expected molecular mass of GST-NADK (75 kDa) were observed in the bacteria cultured in the presence of IPTG (Figure 4.21C). GST-NADK was then purified using glutathione- Sepharose beads, followed by cleavage of GST by prescission protease (Figure 4.21.D).

Figure 4.21. Production of recombinant NADK. A. Schematics of pGEX-6P-1 vector B. Schematic diagram showing cloning and expression of NADK in bacteria. B: Induction of NADK expression in E.coli Rosetta by treatment with different concentration of IPTG at 25°C for 4 h, followed by staining with Coomassie Brilliant Blue. C: GST-NADK was purified from bacterial extracts using glutathione-Sepharose beads (Lane 1) and the GST tag was removed by PreScission protease treatment (1U/200 ml of bacterial lysate) overnight at 4°C (Lane 2).

4.5.2.2 Characterization of recombinant NADK The activity of purified recombinant NADK was measured before and after cleavage of GST. Recombinant NADK and GST-NADK both exhibited high levels of NADK activity, which was not affected by the presence of GST (Figure 4.22A). Western blotting of recombinant NADK using MC-aNADK Ab revealed that this antibody reacts only very weakly with recombinant NADK. The enzymatic activity and immunoreactivity of recombinant NADK were compared to those of neutrophils by titration of recombinant NADK activity against NADK activity from neutrophil lysates, followed by western blotting. As shown in Figure 4.22B, 100 ng of recombinant NADK is not recognized by the monoclonal Ab, although it exhibits the same level of enzyme activity as NADK from PMA-stimulated neutrophils (Figure 4.22C), which reacts very well with the Ab. Furthermore, increasing the amount of recombinant NADK by 3 times didn’t lead to its recognition by MC-aNADK Ab, confirming that similar to NADK from non-stimulated neutrophils, recombinant NADK does not interact with the MC-aNADK Ab efficiently. Additionally, we subjected the recombinant NADK to size exclusion chromatography to determine its apparent molecular mass. The apparent molecular mass of recombinant NADK was estimated to be about 213 kDa by comparing the retention time of NADK enzyme activity with those of standard proteins (Figure 4.23A), in agreement with its existence as a homotetramer (136). Figure 4.23B shows a chromatogram of a mixture of standards along with the elution position of NADK activity. In figure 4.23C the retention times of standards are plotted against log molecular masses. Arrow shows the time that NADK activity was eluted.

Figure 4.22. Characterization of recombinant NADK. A. 100 ng of purified recombinant NADK was tested for NADK activity before and after removal of GST. B. Interaction of recombinant NADK with the monoclonal NADK antibody was compared to NADK from neutrophils. Recombinant NADK (100 ng) with a comparable level of activity to NADK from lysates (60 µg protein) from vehicle- and PMA-treated neutrophils was subjected to western blotting using MC-aNADK. C. The intensities of the bands for NADK in the western blots shown in panel B were compared to the NADK activity in neutrophils and two concentrations of recombinant NADK.

Figure 4.23. Size exclusion chromatography of recombinant NADK. A.Purified NADK was chromatographed on a TSKgel G3000SWxl size exclusion column (7.8 x 300 mm; Tosoh Bioscience) with 100 mM sodium sulfate, pH: 7.4 (flow rate 0.5 ml/min) as the mobile phase. Fractions (1 ml) were collected and an aliquot of each fraction was tested for NADK activity. B. A representative chromatogram showing the profile of a mixture of standards eluted from the column as detected by UV absorbance at 280 nm. C. Retention times of standards were plotted against their molecular masses and it was used to calculate the native molecular mass of NADK. Arrow shows point in which NADK activity was eluted. Values are presented as means ± SEM (n = 4).

4.5.2.3 In vitro phosphorylation of recombinant NADK Our results with NADK from neutrophil lysates suggested that PKC mediates phosphorylation of NADK, resulting in increased immunoreactivity toward the MC-aNADK Ab. To further investigate this, we performed an in vitro phosphorylation assay in which recombinant GST-NADK was incubated with recombinant PKC-δ in the presence of ATP for 15 min in the presence of DAG and PS. Western blotting using MC-aNADK Ab revealed that PKC-δ induced a dramatic increase in immunoreactivity (Figure 4.24A-C). Furthermore, treatment of GST-NADK with PKC-δ resulted in its recognition with anti phospho (Ser) PKC substrate antibody (Figure 4.24 D, E). This suggests that PKC-δ phosphorylates NADK and that MC-aNADK Ab interacts strongly with NADK only after it is phosphorylated. We also measured NADK enzyme activity before and after incubation with PKC-δ, but did not detect any significant changes (Figure 4.24F).

4.6 Partial purification of NADK from human neutrophils To further investigate NADK phosphorylation, we decided to do mass spectrometric analysis of NADK before and after stimulation with NADK. Because MC-aNADK Ab did not react effectively with NADK from non-stimulated neutrophils and because, until recently, there was no efficient polyclonal antibody available for immunoprecipitation of NADK, we attempted to partially purify NADK through conventional chromatographic methods. Because our experiments suggested the existence of different forms of NADK in neutrophils, we hoped that one of the chromatographic approaches employed might be useful for the separation of these forms.

Figure 4.24. In vitro phosphorylation of recombinant GST-NADK. Recombinant GST- NADK (1.5 μg) was treated for 15 min at 30 ºC with PKC-δ (0.5 μg), ATP (2 mM) and the PKC activators DAG and PS. A. Western blotting of recombinant GST-NADK before and after PKC-δ treatment using MC-aNADK. B. Quantification of blots shown in panel Awith ImageJ. C: Coomassie Brilliant Blue staining of the membrane after western blotting. D. Western blotting of recombinant GST-NADK before and after PKC-δ treatment using anti-phospho (Ser) PKC substrate Ab. E. Quantification of blots shown in panel E with ImageJ (n=2). F. Measurement of the activity of recombinant GST-NADK before and after PKC-δ treatment. Values are presented as means ± SEM (n = 3).

4.6.1 Size exclusion chromatography Size exclusion chromatography (SEC) was carried out using a TSKgel G3000SWxl HPLC column to separate NADK from other proteins in vehicle- and PMA- treated neutrophil lysates and to evaluate the apparent molecular mass of native NADK. Lysates from neutrophils treated with either vehicle or PMA were concentrated and analyzed by SEC. NADK enzyme activity was measured in aliquots of column fractions as shown in Fig. 4.25A. A mixture of molecular mass markers (Bio-Rad) including bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), horse myoglobin (17 kDa) and vitamin B12 (1.3 kDa) was also injected to be used for determination of the apparent molecular mass of NADK (Figure 4.25 B, C). The majority of NADK activity was detected in fractions that should contain proteins with molecular masses between 220 and 300 kDa. We estimated the molecular mass of NADK to be about 266 and 268 kDa in vehicle and PMA treated neutrophils, which is higher than what we observed for recombinant NADK (213 kDa; Section 4.5.3) and higher than the mass of tetrameric NADK (4 x 49 = 196 kDa). This discrepancy could possibly be explained by the interaction of NADK with another protein(s). We didn’t observe any apparent difference in the retention time of NADK activity between samples from PMA-treated and vehicle-treated neutrophils. The enzymatic activity of NADK in column fractions from PMA-treated neutrophils was considerably higher than that from vehicle treated neutrophils, while the amount of total protein loaded onto the column was the same for both. Furthermore, we performed western blot on the fractions and the result was in agreement with the activity measurement (Figure 4.25D, E).

Figure 4.25. Purification of NADK performing size exclusion chromatography (SEC). Neutrophils were treated with vehicle or 100 nM PMA, lysed and 0.5-1 mg of protein from the lysates were injected on an HPLC SEC column (7.8 x 300 mm TSKgel G3000SWxl column from Tosoh Bioscience) using 100 mM sodium sulfate, pH: 7.4 as mobile phase at a flow rate of 0.5 ml/min. 1 ml fractions were collected and an aliquot of each fraction was tested for NADK activity. A. NADK activity in fractions of vehicle (○) and PMA (○) treated neutrophils (Data are presented as means ± SEM, n = 5); ***p<0.001. B. A representative chromatogram showing the profile of proteins eluted from the column as detected by UV absorbance at 280 nm. C. Retention times of

standards were plotted against their molecular masses and it was used to calculate the apparent native molecular mass of NADK. Arrow shows point in which NADK activity was eluted. D. Western blots of the fractions with NADK activity using the MC-aNADK. E: Quantitation of NADK bands shown in panel C. Data are presented as means ± SEM (n = 2).

4.6.2 Hydroxyapatite chromatography CHT-hydroxyapatite (Bio-Rad) was packed into a 10 x100 mm column and the lysates from vehicle and PMA treated neutrophils were loaded onto the column. The initial buffer was 5 mM phosphate, pH: 7.4 and the proteins were eluted by increasing the buffer concentration up to 400 mM over 30 min at the flow rate of 1 ml/min. Aliquots of column fractions were analyzed for NADK enzyme activity and protein content. As shown in figure 18A NADK from both vehicle and PMA stimulated neutrophils is well retained by the column and NADK activity is detected in two fractions eluted between 320 and 350 mM phosphate. The elution positions of NADK activity appeared to be similar between vehicle-treated and PMA-treated neutrophils (Figure 4.26A). However, the enzyme activity in the earlier fraction (19 min) from PMA-treated neutrophils is about double that from vehicle-treated neutrophils (P < 0.05). In contrast, the enzyme activity in the second fraction (21 min) was unaffected by treatment with PMA. Figure 4.26B shows a representative profile of protein elution from a lysate from vehicle-treated neutrophils. NADK was separated from about 75% of proteins eluted from the column However, it appears that some proteins were not eluted from the column under these conditions, and these would also be separated from NADK.

4.6.3 Hydrophobic interaction chromatography In hydrophobic interaction chromatography (HIC), hydrophobic groups in the protein interact with a hydrophobic stationary phase, which is enhanced by a high ionic strength buffer. Proteins are eluted from the column by gradually decreasing the ionic strength of the mobile phase. A 10 x 100 mm column with was packed with butyl-HIC media (Bio-Rad) and examined for purification of NADK. The mobile phase was a linear gradient between 1 and 0 M sodium sulphate pH: 7.4 over 30 min. Similar patterns of two peaks of NADK enzyme activity were observed for both vehicle- and PMA- treated neutrophils (Figure 4.27A).The enzymatic activity of NADK from

PMA-treated neutrophils was consistently higher than that from vehicle-treated neutrophils in both peaks. However, when the material in the first peak was rechromatographed under the same conditions, two peaks of enzyme activity with retention volumes similar to the initial two peaks were again obtained, whereas rechromatography of the second peak resulted in a single peak with a retention volume similar to that of the second peak from the first chromatography (Figure 4.27B). Figure 4.27C shows a protein profile from a lysate of vehicle-treated neutrophils.

Figure 4.26 Purification of NADK by hydroxyapatite chromatography (HA). Lysates (3-5 mg protein) from neutrophils treated with either vehicle or PMA (100 nM) for 2min were chromatographed at atmospheric pressure on a column ((10 x 100 mm) packed with CHT- hydroxyapatite resin. The mobile phase was a gradient between 0.05-0.4 M sodium phosphate at a flow rate of 1ml/min. A: NADK activity in fractions from vehicle (○) and PMA (●) treated neutrophils. Data are means ± SEM (n = 7); * p <0.05. B: A representative profile of protein elution from a lysate from vehicle-treated neutrophils.

Figure 4.27 Purification of NADK doing hydrophobic interaction chromatography (HIC). A column (10 x 100 mm) packed with Macro-Prep t-butyl-HIC media (50 µm particle size) was used for HIC. Neutrophils were treated with vehicle or 100 nM PMA for 2 min, lysed and the lysate chromatographed as described in the Materials and Methods section. A. NADK activity in lysates from neutrophils treated with vehicle (○) or PMA (○). B: Fractions corresponding to peak 1 (○) and peak 2 (○) from lysates of PMA-treated neutrophils were rechromatographed using the same conditions as for panel A. C. Representative chromatogram showing the amounts of protein in HIC column fractions from a lysate of vehicle-treated neutrophils.

4.6.4 Ion exchange chromatography Ion exchange chromatography is a widely used technique for purification of polar molecules based on their charge. A strong anion exchange HPLC column (SAX from Rainin Instrument Comapny) was used to test its capability to separate NADK from other proteins. In anion exchange chromatography, negatively charged molecules are attracted to a positively charged solid support. Proteins are negatively charged at pHs above their pI and are able to interact with the positively charged anion exchanger. The pI of NADK is 6.2, so we used 20 mM phosphate buffer, pH: 7.4 as an initial solvent. A gradient of 0 to 70% ammonium acetate was used to elute the proteins. NADK enzyme activity and immunoreactivity were evaluated in column fractions.

Chromatography of lysates from vehicle-treated neutrophil gave a single peak of NADK activity (Figure 4.28A). In contrast, two peaks of NADK activity were observed after ion exchange chromatography of lysates from PMA-treated neutrophils. The later peak had the same retention volume and total activity as the peak detected for vehicle-treated neutrophils. In contrast, the earlier peak, which eluted with the majority of total protein (Figure 4.28B), was not observed in the case of vehicle-treated neutrophils and contained substantially more enzyme activity than the second peak. Western blotting of the material in the two peaks showed that only the earlier one was recognized by the MC-aNADK Ab (Figure 4.28C, D).

Pretreatment of neutrophils with staurosporine (300 nM) prior to PMA stimulation completely suppressed the immunoreactivity of this peak but reduced enzyme activity by only 40% (Figure 4.29A, B). However, increasing the concentration of staurosporine to 1 µM resulted in 100% inhibition of both enzyme activity and immunoreactivity (Figure 4.29C, D).

Figure 4.28. Purification of NADK by anion exchange chromatography (SAX). Lysates from vehicle and PMA (100 nM for 2 min) treated neutrophils were chromatographed on an HPLC anionic exchange HPLC column using a gradient between 0 and 0.7 M ammonium acetate in 20 mM phosphate buffer (pH: 7.4) over 30 min and a flow rate of 0.5 ml/min. A: NADK activity in fractions from the vehicle-treated (○) and PMA-treated (●) neutrophils. Data are means ± SEM (n = 6); **** p<0.0001. B: Representative chromatograms showing the total protein elution profile. Arrows show points that NADK was eluted C: Fractions with NADK activity correspond to peak 1 and peak 2 were selected for western blotting using the MC-aNADK Ab. D: Quantitation of western blots shown in panel C (Data are means ± SEM, n = 2).

Figure 4.29. Effect of staurosporine on activity of NADK partially purified by SAX. Neutrophils pretreated with vehicle or A. 300nM, B. 1µM stauroporine followed with 2 min stimulation with 100nM PMA (in A) and with vehicle or PMA stimulation in panel B. C. Western blot analysis of fractions correspond to peak 1 and peak 2 shown in panel A using MC-aNADK Ab. D. Western blot analysis of peak1 fraction from PMA-stimulated neutrophils of panel B.

We intended to test more specific inhibitors of PKC, such as Gö6983 on the elution profile of NADK activity in SAX-HPLC, but unfortunately our column suddenly developed very high back pressure and could no longer be used. We tried many approaches to rejuvenate the column but none were successful. We were unable to replace the column by a new one, as the Rainin Instrument Company no longer manufactures HPLC columns. Therefore we purchased a new SAX column from Tosoh Bioscience LLC (TSKgel Q-STAT (100 x 4.6 mm, particle size 7µm). We chromatographed lysates from vehicle and PMA treated neutrophils under the same chromatographic conditions used with the Rainin column (0 to 0.7M ammonium acetate in 20 mM phosphate buffer, pH: 7.4). However, we were not able to detect NADK activity in any of the column fractions after chromatography of lysates from either vehicle or PMA treated neutrophils, suggesting that the enzymatic activity of NADK was destroyed. Then we attempted to replicate the results obtained with the Rainin column by testing different buffers and salts. We were able to detect NADK activity in column fractions when ammonium acetate was replaced by sodium sulfate (Figure 4.30 A) or ammonium sulfate (Figure 4.30B), but the enzymatic activity of NADK appeared as a single peak. Moreover, NADK activity was not detected in any of the column fractions when chromatography was performed using sodium acetate as elution solvent. A pH gradient of 7 to 5.8 using 10mM phosphate buffer as mobile phase also did not result in detection of NADK in any of column fractions.

Figure 4.30 Purification of NADK doing anion exchange chromatography using Q- STAT column. Lysate from PMA- treated neutrophils chromatographed on an HPLC anionic column called TSKgel Q-STAT (10 cm, particle size 7µm) using a gradient between 0 and 0.7 M sodium sulfate (A) and amoonium sulfate (B) in 20 mM phosphate buffer (pH: 7.4) over 30 min and a flow rate of 0.5 ml/min, followed with measurement of NADK in column fractions.

4.7 Analysis of post-translational modification of NADK by LC-MS/MS Because until recently, there was no anti-NADK antibody available to detect NADK from non-stimulated neutrophils, we partially purified NADK from non-treated neutrophils through conventional chromatographic methods. NADK from PMA-treated neutrophils was purified by performing SEC followed with IP using MC-aNADK Ab. When the poly (B) aNADK Ab became available, we used that to purify NADK from both vehicle and PMA-treated neutrophils. Purified NADKs were subjected to SDS-PAGE followed by silver staining. Regardless of the type of purification, two prominent protein bands were observed with apparent molecular masses of 43kDa and 49kDa. Both bands were excised and subjected to trypsin digestion. The sequenced digested peptides were analyzed by MASCOT, a search engine that matches mass spectrometry data to available primary sequence databases to identify proteins. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm (184). Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet Algorithm (185). NADK was detected in both 43k and 49k bands from all the samples analyzed by LC-MS/MS. The peptide sequence coverage and post translational modifications of each sample are discussed in the following sections.

4.7.1 Analysis of NADK purified from PMA- treated neutrophils by IP (MC-aNADK Ab) NADK was partially purified from PMA treated neutrophils by performing SEC, followed with IP using MC-aNADK Ab. Two bands at 43 and 49kDa were visualized with silver staining (Figure 4.31 A,B) and analyzed by LC-MS/MS. NADK was the most abundant protein identified in both 43k and 49k bands with peptide sequence coverage of 28% and 35% respectively. Analysis of post translational modifications revealed that Serine 64 (S64) is phosphorylated on NADK from both bands. Additionally, S46 was phosphorylated on 49k NADK, but the corresponding peptide was not detected in 43k NADK (Figure 4.31 C, D).

Figure 4.31 LC-MS/MS analysis of NADK from PMA-treated neutrophils purified by SEC and IP (MC-aNADK). A. Schematics of preparation of NADK from PMA-treated neutrophils. B. Silver stained protein bands indicated in blue boxes were excised for LC-MS/MS. Full length sequence of human NADK is showed with peptides identified in C. 43k band and D. 49k band. Phosphorylated serine residues are shown in red.

4.7.2 Analysis of NADK purified from vehicle-treated neutrophils by conventional chromatographic methods NADK from vehicle-treated neutrophils was partially purified by hydroxyapatite chromatography, SAX chromatography, HIC and SEC, sequentially. Similar to NADK purified from PMA-treated neutrophils, two prominent bands were observed with silver staining (Figure 4.32 A, B). Although, NADK was identified in both 43 and 49 kDa bands, it appeared that the samples were highly contaminated with keratin. Additionally there were a few other proteins identified that were present in higher abundance than NADK, including protein disulfide isomerase (mostly in the 49k band with a smaller amount in the 43k band), and β-actin (mostly in the 43k band). These contaminating proteins interfered with the mass spectrometry and as a result the identified peptides accounted for only 6% and 11% of the total NADK sequence (i.e. 6 and 11% peptide coverage) in the 43k and 49k bands, respectively (Figure 4.32 C,D). Although the peptide corresponding to S46 was sequenced, this serine was not phosphorylated in either the 43k or the 49k isoforms of NADK.

Figure 4.32 LC-MS/MS analysis of NADK from vehicle-treated neutrophils purified by conventional methods. A. Different chromatographic methods used for purification of NADK from vehicle treated neutrophils. SAX was performed using Rainin column. B. Silver stained gel to evaluate the purification. Selected bands for LC-MS/MS analysis are shown in boxes. NADK full length sequence is shown with peptides (blue text) sequenced in C. 43k band and D. 49k.

4.7.3 Analysis of NADK purified from vehicle treated neutrophils by a combination of chromatography and IP (Poly (B) aNADK) When poly (B) aNADK became available, we decided to purify NADK from vehicle- treated neutrophils by IP using this Ab. Initially, we thought that one step of partial purification of NADK prior to IP would be useful to increase the purity of the NADK. Therefore, a combined lysate from vehicle-treated neutrophils was first chromatographed on a hydroxyapatite column, followed by IP using the poly (B) aNADK Ab (Figure 4.33A,B). The 43 and 49 kDa NADK isoforms were detected by mass spectrometry with peptide coverages of 26% and 13%, respectively (Figure 4.33 C, D). The S46 residue that was phosphorylated in NADK from PMA- treated neutrophils (Section 4.7.1), did not appear to be phosphorylated in either 43k or 49k NADK from vehicle-treated neutrophils. The peptide encompassing S64 was not detected in the either of 43k and 49k NADK isoforms. To improve the peptide coverage of NADK, we purified NADK from vehicle-treated neutrophils by performing SAX chromatography (Rainin), which showed higher resolution than HA to separate NADK from other proteins, followed by IP using poly (B) aNADK Ab (Figure 4.34A,B). This resulted on 26% and 35% peptide sequence coverage following mass spectrometric analysis for the 43k and 49k NADK isoforms, respectively. Similar to the previous sample, S46 was not phosphorylated in either of the two bands. Furthermore, NADK from the 49k band was phosphorylated on S64, whereas the corresponding peptide was not detected in the 43k NADK isoform (Figure 4.34C, D).

Figure 4.33 Analysis of NADK from vehicle-treated neutrophils purified by HA-IP (poly(B) aNADK). A. Purification of NADK from vehicle treated neutrophils. B. The bands excised from the silver stained gel are shown in boxes. The protein sequence of NADK is shown with peptides (blue text) from the C. 43 kDa band and D. 49k band identified by LC-MS/MS spectra.

Figure 4.34. LC-MS/MS analysis of NADK from vehicle-treated neutrophils purified by SAX (Rainin)-IP (poly(B) aNADK). A. Schematics of NADK purification from vehicle treated neutrophils. NADK protein sequence is shown with peptides (blue text) identified in B. Silver stained protein bands indicated in blue boxes were excised for LC-MS/MS. NADK full length sequence is shown with peptides (blue text) sequenced in C. 43k band and D. 49k.

4.7.4 Analysis of NADK purified by IP (poly (B) aNADK Ab) Because the peptide sequence coverage obtained in the previous samples was not as high as we would have liked, we eliminated the initial step of chromatographic purification in an attempt to improve the yield of NADK. Therefore, NADK from vehicle and PMA treated neutrophils was purified by performing IP of NADK directly on the cell lysates (Figure 4.35A). As a result about 50% and 45% peptide coverage were obtained for NADK from vehicle-treated (Figure 4.35B, C) and PMA-treated (Figure 4.35D, E) neutrophils, respectively. Analysis of post-translational modification of NADK revealed multiple phosphorylation sites on NADK immunoprecipitated from both vehicle and PMA treated neutrophils. In the case of the 43k band, S46 was only phosphorylated in NADK from PMA-treated neutrophils, whereas S48 and S64 were phosphorylated in NADK from both vehicle and PMA treated neutrophils. On the other hand, NADK in the 49k band was phosphorylated on S46, S48, S64 and T62 in both vehicle and PMA treated neutrophils. Furthermore, another serine residue, S44, was phosphorylated in 49k NADK of vehicle treated neutrophils.

Figure 4.35. Analysis of NADK from vehicle and PMA-treated neutrophils purified by IP (poly(B) aNADK). A.NADK was purified form lysates of vehicle and PMA treated neutrophils by IP using poly(B) aNADK Ab. The protein sequence of NADK is shown with peptides (blue text) from B. 43 kDa band from vehicle , C. 49k from vehicle, D. 43k band from PMA and E. 49k band from PMA treate neutrophils identified by LC-MS/MS spectra. Phosphorylated residues are shown in red text.

Because, in the previous samples analyzed from vehicle-treated neutrophils, we only detected phosphorylation of S64, we speculated that the level of phosphorylation of S46 and S48 in the present sample may be different between vehicle-treated and PMA-treated neutrophils. Therefore, we compared the degree of phosphorylation in the two samples on the basis of the intensities of the ion signals for the peptides carrying each of the phosphorylated residues. The values were normalized to correct for differences in total protein between the samples by dividing the phosphopeptide ion intensity by the sum of the ion intensities of a series of nonphosphorylated reference peptides, which included the non-phosphorylated counterparts of the phosphorylated peptides. The ion intensities of the peptides bearing phospho-S46 (Table 4.1), phospho-S48 (Table 4.2), and phospho-S64 (Table 4.3) are shown in the relevant tables. The major ions bore 2 charges, whereas ions of less intensity with 3 charges were also detected in many cases and were included in the calculations. The ion intensities of the reference peptides that were included in the calculations are shown in Table 4.4. Table 4.5 shows the ratios of the normalized ion intensities for the phosphopeptides from PMA-treated neutrophils divided by the normalized intensities of the phosphopeptides from vehicle-treated neutrophils. The ion intensity of the peptide bearing phospho-S46 was over 3 times higher in the sample from PMA-treated neutrophils compared to that from vehicle-treated neutrophils for both the 43 and 49 kDa isoforms of NADK, whereas there were relatively small differences for the peptide bearing phosphor-S48. Overall, the peptide bearing S48 was only phosphorylated in this position about 20% of the time in all cases (Table 4.2). The ratios obtained for the peptide bearing phospho-S64 were intermediate, with a relatively small difference for the 43 kDa NADK isoform and a larger difference (2.3 times higher for PMA- treated neutrophils) for the 49 kDa isoform. We only observed phosphorylation of S44 in NADK from vehicle-treated neutrophils. However, only about 20% of the relevant peptide was phosphorylated in this position in the cases of both the 43 and 49 kDa isoforms. Taken together, these results suggest that NADK in resting neutrophils is phosphorylated to some extent on T62, S48, S64 and S46. Stimulation of neutrophils with PMA results in phosphorylation of S46 and possibly, to a lesser extent, S64.

Table 4.1. The ion intensities of the peptides bearing phospho-S46

Table 4.2. The ion intensities of the peptides bearing phospho-S48

Table 4.3. The ion intensities of the peptides bearing phospho-S64

Table 4.4. The ion intensity of the reference peptides

Table 4.5. The ratios of the normalized ion intensities for the phosphopeptides from PMA- treated neutrophils divided by the normalized intensities of the phosphopeptides from vehicle- treated neutrophils.

4.7.5 Analysis of GST-NADK before and after treatment with PKC-δ 57% peptide sequence coverage was obtained from analysis of GST-NADK before and after treatment with PKC-δ. Analysis of post translational modification showed S15 and T127 are phosphorylated in GST-NADK before treatment with PKC-δ. After PKC-δ treatment, four phosphorylated residues including S15, S46, S55 and S64 were identified on GST-NADK. (Figure 4.36A,B). These results strongly support phosphorylation of NADK by PKC-δ. All the LC-MS/MS results are summarized in table 4.6 and MS/MS spectra correspond to all identified phospho peptides are shown in supplementary figure (S.1-S.5) in appendix.

Figure 4.36. LC-MS/MS analysis of GST-NADK before and after PKC-δ. The protein sequence of NADK is shown with peptides (blue text) from GST-NADK A. before and B. after PKC-δ treatment identified by LC-MS/MS spectra. .

Table 4.6. Summary of LC-MS/MS analysis. ND: Corresponding peptide was not detected * The intensity of phospho peptides in NADK from vehicle-treated neutrophils was 3 times less than that in NADK form PMA-treated neutrophils.

4.8 Interaction of NADK with other proteins

4.8.1 14-3-3 protein, a candidate for regulation of NADK As shown in figure 4.24F, in vitro phosphorylation of recombinant NADK with PKC-δ did not significantly alter the activity of NADK. Therefore, we hypothesised that phosphorylation of NADK may regulate its activation by affecting its interaction with other proteins. As mentioned in the introduction section, high throughput studies of protein interactions illustrated that NADK can physically interact with a number of other proteins, including 14-3-3 ζ, β, γ, θ and ε isoforms, eukaryotic translation elongation factor 1 gamma, Syndecan-binding protein 1, nudix-type motif 18, ligand of numb-protein X 1, E3 ubiquitin protein ligase, Glutaredoxin3 and growth factor receptor-bound protein 2. We searched for these proteins among the proteins that were pulled down along with NADK as a result of the immunoprecipitation procedure and analyzed by LC-MS/MS. Interestingly, we detected both the zeta and beta isoforms of 14-3-3 in the 43k band following - immunoprecipitation (Poly (B) aNADK Ab) of NADK from untreated neutrophils, whereas these proteins were not detected when NADK was immunoprecipitated using the same antibody from neutrophils stimulated with PMA. The sequence coverages of identified 14-3-3 zeta and beta were 19% and 16%, respectively (Figure 4.37A, B). Since the primary goal of performing LC-MS/MS analysis was detection of PTMs of NADK, rather than the identification interacting partners, only two selected bands in each lane were excised for LC-MS/MS analysis that contained the proteins with molecular mass of 40-60 kDa. Therefore, it was unexpected to see the proteins smaller than 40 kDa such as 14-3-3 protein (30 kDa) in the bands. To further investigate the presence of interacting partners of NADK, additional IP experiments should be done followed with identifying all of the co-immunoprecipitated proteins.

4.8.2 PMA changes the interaction of NADK with 14-3-3 zeta protein

To further investigate a potential interaction of NADK with 14-3-3 protein, a coimmunoprecipitation experiment was done in which NADK from vehicle and PMA-treated neutrophil lysates was immunoprecipitated using poly (A) aNADK Ab, followed by western blotting with anti-14-3-3 zeta antibody. In a preliminary experiment, we were able to detect 14- 3-3 zeta pulled down along with NADK in the lysates from non-treated neutrophils, while the

intensity of the band was lower in the lysates of PMA-stimulated neutrophils (Figure 4.38 A, B). This suggests that NADK could interact with 14-3-3 zeta in resting neutrophils and that PMA stimulation may lead to its dissociation from NADK.

Figure 4.37. 14-3-3 proteins as interacting partners of NADK. 14-3-3 beta and zeta were identified as proteins co eluted along with NADK immunoprecipitated from vehicle treated neutrophils using poly(B) aNADK. The protein sequences of A. 14-3-3 beta and B. 14-3-3 zeta are shown with peptides (blue text) identified by LC-MS/MS spectra.

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Figure 4.38. PMA-induced change in interaction of 14-3-3zeta with NADK PMA. A. coimmunoprecipitation of NADK and 14-3-3 zeta. NADK was immunoprecipitated from vehicle and PMA treated neutrophils using poly (A) aNADK, followed with western blotting using 14-3- 3 zeta antibody. B. Blots shown in panel A were quantified using ImageJ (n=2).

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Chapter 5. Discussion

5.1. PMA activates NADK in intact neutrophils

Despite the important roles that NADK plays in various biochemical pathways, there is surprisingly little information available on NADK in mammalian cells. Although previous studies have shown that PMA increases the activity of NADK in neutrophils, this was not investigated in much detail and the mechanism was unclear. One study showed that stimulation of neutrophils with PMA for 5 minutes causes a more than 2 -fold increase in the total concentration of NADP+ and NADPH, accompanied by the activation of NADPH oxidase (91). Another study from the same year further showed that elevation of the NADP+/NADPH pool by PMA in neutrophils was blocked by calmodulin antagonists, trifluoperazine and W-7 (140), which appeared to suggest a role for calcium/calmodulin in this process. Furthermore, in a recent study, where neutrophils were treated with PMA for longer time points (30-120 min), NADP+ level was increased with treatment time, whereas NADPH levels transiently decreased within the ﬁrst 60 min, and returned to basal levels by 120 min. A transient drop in NAD+ levels were also observed, followed by a gradual increase, which they interpreted to imply that the increase in NADP+ was due to activation of NADPH oxidase and NADK, both. Furthermore, they suggested that PMA increases NADK activity in neutrophils through induction of NADK expression (143).

The present study stems from a prior finding in our laboratory during an investigation of the role of NADP+ in regulating the synthesis of 5-oxo-ETE by neutrophils. In that study it was found that stimulation of neutrophils with PMA led to a very rapid increase in NADP+ levels followed by an increase in 5-oxo-ETE synthesis. The objective of this study was to determine the mechanism for effect of PMA on intracellular NADP+ concentrations. We initially expected that PMA-induced NADP+ synthesis was due to activation of the respiratory burst, resulting in the rapid oxidation of NADPH by NADPH oxidase. However, when we measured the intracellular levels of pyridine nucleotides we found that PMA did not deplete NADPH, the levels of which initially tended to rise, but instead induced a rapid fall in NAD+ levels that mirrored the increase in NADP+, suggesting activation of NAD kinase. Although the depletion of NAD+ occurred within the first 4-8 min after addition of PMA, NADP+ generation continued up to 12 min, which could

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possibly be explained by additional mechanisms such as increased de novo synthesis of NAD+ due to the initial decrease in its levels or to oxidation of NADPH. It has been reported that NADPH allosterically inhibits human NADK (138), which raised the possibility that activation of NADPH oxidase in neutrophils could relieve the this inhibitory effect, resulting in and activation of NADK. However, this explanation cannot explain our results clearly demonstrated that NADPH was not depleted in response to PMA. To clearly rule out a role for NADPH oxidase we showed that treatment of neutrophils with the NOX inhibitor DPI prior to addition of PMA had no effect on PMA-induced depletion of NAD+, despite blocking the increase in NADP+ levels. This is presumably due to the rapid reduction of NADK-derived NADP+ by the pentose phosphate pathway (PPP), resulting in the accumulation of NADPH (Figure 5.1).

The above experiments ruled out roles for NOX-2 and NADPH depletion in inducing NADK activity. Another potential mechanism for the activation of NADK by PMA could have been an elevation of the intracellular concentration of calcium, as it has been reported that NADK activity in neutrophils is increased by Ca/CAM (91,140). However, we showed that PMA has no effect on intracellular calcium levels, confirming that activation of NADK by PMA in neutrophils is not related to the calcium mobilization.

Figure 5.1. Regulation of pyridine nucleotides by NADK. Activation of NADK results in production of NADP+ which is rapidly converted to NADPH through PPP. NADPH is then oxidized to NADP+ by the action of NOX-2. Inhibition of NOX-2 by DPI favors the production of NADPH.

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We hypothesized that a novel isoform of PKC, which requires DAG but not calcium for its activation, mediates the PMA-induced activation of NADK by PMA. PKC isoforms that are known to be expressed in neutrophils are α and β (conventional isoforms), δ (novel isoform) and ζ (atypical isoform) (162–164). We treated neutrophils with 0.5 µM staurosporine (non-selective inhibitor of PKCs as well as some other protein kinases) and 1 µM Gö6983 (inhibitor of PKCα, β, γ, δ, and ζ) followed by stimulation with PMA. Both inhibitors blocked the PMA-induced decrease in NAD+ as well as the increase in NADP+ levels, suggesting that these effects were mediated by PKC. The initial transitory increase in NADPH levels following PMA stimulation was also + attenuated with both inhibitors. The IC50 for inhibition of NAD depletion is higher than the IC50 for inhibition of NADP+ synthesis for both inhibitors. This could possibly be due to additional effects of PMA on pathways involved in the synthesis of NAD+ (e.g. de novo synthesis) and NADP+ (e.g. oxidation of NADPH).

Further investigation of the role of specific PKC isoform in activation of NADK would be possible by using siRNA against PKC-δ or other PKC isoforms. Since it is not feasible to transfect neutrophils due to their short life span, we attempted to replicate the PMA response observed in neutrophils, in a granulocytic cell line. To do so, we differentiated PLB-985 cells to neutrophil- like cells by treatment with DMSO, however they did not respond to PMA at all. We also investigated several other cell lines including Ramos (B lymphocyte), U937 (lymphocyte), MD (monocyte/macrophage), THP-1 (monocyte) and Cess (lymphoblast) cells. None of these cells showed response to PMA as neutrophils did. However, we were able to replicate the PMA response in monocytes isolated from human blood, suggesting that such a response to PMA is unique to the cells with high respiratory burst. Because of the lack of an appropriate cell line we were unable to conduct experiments in which the expression of specific PKC isoforms was up- or down- regulated.

5.2 NADK activity is elevated in lysates from PMA-stimulated neutrophils

To look more closely at NADK we set up an assay to measure its activity in cell-free systems. NADK activity was assayed by measuring the increasing absorbance at 600 nm caused by the reduction of NADP+ (generated by NADK using exogenous NAD+ and ATP) to NADPH We validated the accuracy of the assay by showing that endogenous levels of NADP+ and NADPH

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didn’t interfere with the assay, as the values obtained from the lysates without exogenous NAD+ and ATP were very close to the value obtained with blank. Possibly, using high concentration of NAD+ in the assay makes the impact of endogenous NADP+ and NADPH insignificant.

Only limited information is available in the literature about NADK activity in neutrophils. One study reported a 2-fold increase in NADK activity following stimulation of neutrophils with PMA for 5min (91), whereas another showed that one hour stimulation of neutrophils with PMA resulted in more than 5-fold increase in activity (143). We similarly found that NADK activity is elevated in the lysates of neutrophils following PMA stimulation for between 2 and 15 min the with maximal response occurring at 2min. Consistent with our results on pyridine nucleotide levels in intact neutrophils, we were able to block the PMA-induced increase in NADK activity in neutrophil lysates by prior treatment of neutrophils with the PKC inhibitor, Gö6983, suggesting that PMA activates NADK through a PKC-dependent process.

The ~60% increase in NADK activity in lysates of PMA-treated neutrophils seemed rather modest in view of the dramatic rapid decline in intracellular NADP+ levels that we observed in intact cells. We considered the possibility that this could have been due to elevated basal activity in lysates from vehicle-treated neutrophils due to the dilution of endogenous NADH and NADPH, which are known to inhibit NADK and thereby regulate its activity. In an attempt to circumvent this, we added exogenous NADH to either the incubation medium or the medium used for cell lysis in an attempt to mimic the intracellular environment. We found that although NADH reduced NADK activity in lysates from vehicle-treated neutrophils, it had a similar effect on NADK activity in PMA-treated neutrophils so that the percent increase in NADK activity due to PMA was unaltered by NADH. We were not able to do such experiments with NADPH, as would have interfered with the NADK activity assay. However the effects of NADH and NADPH on NADK activity are similar (138).

Because cationic metal ions are required for NADK activity (50,91) we also considered the possibility that reduced Ca2+ or Mg2+ concentrations in the neutrophil lysates could have contributed to the modest increase in NADK activity that we observed following treatment of neutrophils with PMA. We therefore measured NADK activity in lysates of vehicle- and PMA- treated neutrophils in the presence of various concentration of Mg2+ and Ca2+ or combination of

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the two ions. However, none of the combinations of metal ions that we employed affected the magnitude of the response to PMA.

5.3 Post translational modification of NADK

The results we obtained using western blotting of NADK with the first commercially available antibody to NADK (the monoclonal antibody MC-aNADK Ab) were initially difficult to interpret. This antibody revealed an intense band with lysates from PMA-treated neutrophils but only a weak band with vehicle-treated neutrophils. Measurement of immunoprecipitated NADK activity confirmed that this antibody is more reactive to NADK from PMA-stimulated neutrophils compared to vehicle-treated cells. Cheng et al. also reported that treatment of neutrophils with PMA for 1 h or longer increased NADK immunoreactivity and interpreted this to mean that PMA induced NADK expression (143).

In our experiments, the incubation time with PMA was too short (2 min) to induce increased protein expression. The reason for the selective recognition of NADK from PMA-treated neutrophils is difficult to explain. The MC-aNADK Ab was raised against full length GST-NADK. One possibility is that the recombinant NADK expressed in bacteria was somehow modified by the bacteria and that this modification is reproduced in human neutrophils NADK following stimulation with PMA. However, it would seem unlikely that bacterial enzymes would modify a eukaryotic protein in this manner. Another more likely possibility is that the antigen was being modified while processing in the immunized mouse. We were able to immunoprecipitate almost 100% of the immunoreactive form of NADK with the MC-aNADK Ab based on Western blotting. However, about 30% of the initial NADK activity remained in the supernatant. This suggests that at least one additional form of NADK, which possesses enzyme activity but little immunoreactivity, exists in neutrophils. It is possible that the degree of enzymatic activity of the two immunoreactive differ from one another, but we were unable to address this directly.

When we used a polyclonal anti-NADK antibody (Poly (A)-aNADK Ab), the band we detected was lower (~ 43kDa) than the one we detected with the MC-aNADK Ab, and its intensity was not affected by PMA. We were able to immunoprecipitate NADK activity with the poly (A)- aNADK Ab from lysates of both vehicle and PMA-stimulated neutrophils, indicating that the band

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observed with Poly (A)-aNADK Ab corresponds to NADK. The efficiency of poly (A)-aNADK Ab toward NADK from PMA- and vehicle- treated neutrophils were comparable, but the total recovery of NADK activity with the poly (A)-aNADK Ab was less than with the MC-aNADK Ab. Based on these results, it seems that both bands detected at 43 and 49 kDa correspond to NADK, providing more evidence for the existence of more than one form of NADK in neutrophils. Furthermore, using another polyclonal anti-NADK antibody (Poly (B)-aNADK) that became available only recently, we detected two bands, one at 43 kDa and the other at 49 kDa. Incubation of neutrophils with PMA did not affect the intensity of either band, suggesting that this Ab recognizes both forms of NADK.

We demonstrated that PMA increased the immunoreactivty of NADK toward the MC- aNADK Ab in a PKC-mediated manner, as treatment of neutrophils with PKC the inhibitors staurosporine and Gö6983 prior to PMA stimulation blocked the emergence of the immunoreactivity. The most obvious explanation for this would be that PMA induced the PKC- dependent phosphorylation of NADK, resulting in generation of the epitope recognized by MC- aNADK. We sought evidence for PKC-induced phosphorylation of NADK using an anti-phospho (Ser) PKC substrate Ab. NADK from vehicle and PMA treated neutrophils was immunoprecipitated using either MC-aNADK or poly (A) aNADK, followed by western blotting using the anti-phospho (Ser) PKC substrate antibody. The results suggested that PMA induced the phosphorylation of NADK, as the antibody recognized NADK only when neutrophils were treated with PMA. However, the band detected from NADK immunoprecpitated using poly (A) aNADK Ab has an apparent molecular mass of 49kDa similar to the band obtained from NADK immunoprecipitated using MC-aNADK Ab which is not in agreement with results obtained with western blotting using poly(B) aNADK Ab.

To further investigate the post-translational modification of NADK we produced GST- tagged recombinant NADK. Although this protein should theoretically be identical to the immunogen used to raise MC-aNADK, it was barely recognized by the antibody, even when very large amounts were used. In spite of its lack of immunoreactivity, the recombinant protein displayed high NADK activity, which was unaffected by the presence of the GST tag. Analysis by size exclusion chromatography showed that apparent native molecular mass of the recombinant

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NADK (after GST cleavage) is 213 kDa, which is in agreement with its reported tetrameric structure (50).

Interestingly, in vitro phosphorylation of the GST-NADK by PKC-δ dramatically increased its immunoreactivity toward the monoclonal Ab, indicating that phosphorylation by PKC permits its recognition by the antibody. The phosphorylation of GST-NADK by PKC-δ was confirmed by western blotting using the anti-phospho (Ser) PKC substrate Ab.

5.4 Partial purification of human NADK from neutrophils

To further investigate post-translational modifications of NADK, we decided to do mass spectrometric analysis of NADK purified from vehicle and PMA treated neutrophils. Because MC- aNADK Ab did not recognize NADK from non-stimulated neutrophils and because, until recently, there was no efficient polyclonal antibody available for immunoprecipitation of NADK, we attempted to partially purify NADK through conventional chromatographic methods. Since we had evidence for multiple forms of NADK in neutrophils we hoped to be able to separate them by one of the chromatographic approaches employed.

We performed size exclusion chromatography of vehicle and PMA treated lysates. The results revealed that apparent native molecular masses of NADK is 266 and 268 kDa in vehicle and PMA treated neutrophils, respectively. These masses are higher than the theoretical native molecular mass of NADK (~200 kDa) and the molecular mass of our recombinant NADK (213 kDa), suggesting that NADK in neutrophils exists in a complex with other protein(s). Although, treatment of neutrophils with PMA did not change the apparent molecular mass of the NADK complex, it is possible that a change involving a small protein occurred but was not detectable because of the limited resolution of our column in this molecular mass range. Moreover, the protein standards that we used unfortunately lacked proteins with molecular masses between 200 and 300 kDa, which would have been useful in characterizing the NADK complex. The mixture we used contained bovine thyroglobulin (670 kDa), bovine γ-globulin (158 kDa) and three other proteins with molecular masses of less than 50 kDa. In term of separation of NADK from other proteins, SEC was a very effective method, as, based on UV absorbance, the majority of proteins in the lysate were eluted in the molecular mass range below 50 kDa. Interestingly, we consistently found

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that the total NADK activity eluted from the column was considerably higher for lysates from PMA-treated neutrophils compared to those of vehicle-treated neutrophils.

There was some evidence for multiple forms of NADK using other chromatographic approaches. In the case of hydroxyapatite chromatography NADK activity appeared to be eluted slightly earlier from lysates of PMA-treated neutrophils compared to vehicle-treated neutrophils. As with SEC, the total NADK activity eluted using hydroxyapatite chromatography was consistently greater with lysates from PMA-treated neutrophils. This technique removed about three-quarters of lysate proteins from the NADK fraction.

Another chromatographic method we employed was hydrophobic interaction chromatography (HIC). In this case, two peaks of NADK activity of similar magnitude were obtained with lysates from both PMA- and vehicle- treated neutrophils, with the total activity again being considerably higher with PMA-treated neutrophils. We wondered whether the two peaks could have been due to two forms of NADK that were initially present in the neutrophil lysates or whether they could have been formed artifactually during the purification procedure. Rechromatography of the material in the two peaks under the same conditions revealed that the material in the first peaks was transformed to the material in the second peak, whereas the material in the second peak remained unaltered. This could possibly be explained by an effect of the high (1 M) salt concentration used in the initial stage of the chromatography, which could promote the dissociation of the native tetrameric form of NADK into a dimer, for example, which could appear as the later eluting peak. This process could be repeated when the earlier peak was rechromatographed, resulting in the formation of the second dimeric peak. In contrast, rechromatography of the material in the second peak, which may already be the dimer, would not result in any further changes. If this is true, this would mean that both dimeric and tetrameric forms of NADK have similar enzymatic activities. Since NADK activity was spread over several fractions, only about 50% of total proteins were separated from NADK. Thus, HIC may not be an efficient method for single step purification of NADK, but it could be performed after hydroxyapatite or ion exchange chromatography, as it is compatible with high salt in the eluted fractions from these methods.

Finally, we explored the use of anion exchange chromatography using a strong anion exchange (SAX) HPLC column that had been purchased some time ago from Rainin, who no

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longer manufacture these columns. SAX chromatography gave a single peak for NADK activity from lysates of non-treated neutrophils. In contrast, two peaks of enzymatic activity were observed after SAX chromatography of lysates from PMA-treated neutrophils. The earlier peak was only observed with PMA-treated neutrophils and had markedly higher enzymatic activity. Interestingly, western blotting of the two peaks showed that only the material in the earlier peak was recognized by MC-aNADK, providing further evidence that PMA transforms NADK into a second distinct form in neutrophils. Immunoreactivity of this peak was suppressed with low concentration of staurosporine, whereas higher concentration of staurosporine was required to inhibit the activity. This may suggest that the earlier peak may contain at least two forms of NADK, one of which is enzymatically active but not immunoreactive, and the second of which is immunoreactive and possibly also enzymatically active, but this requires further investigation.

Our next step would have been to treat neutrophils with more specific PKC inhibitors such as Gö6983, prior to PMA stimulation, to determine their effects on the immunoreactivity and enzymatic activity of the materials in the first peak obtained after SAX chromatography. We had also planned to purify NADK from the first peak by immunoprecipitation using MC-aNADK Ab. However, unfortunately the SAX column suddenly developed a very high back pressure and became unusable, and many attempts to rejuvenate it using a variety of approaches failed. Since the Rainin Instrument Company, the provider of our 30 cm SAX column, no longer manufactures HPLC columns, we were unable to replace it with an identical column. After evaluating several possibilities, we replaced the Rainin column with an anion exchange column from Tosoh Bioscience (TSKgel Q-STAT) that we hoped would behave similarly to the Rainin column. However, attempts to replicate our previous results with the TSKgel Q-STAT column using an identical gradient (0-0.7M) ammonium acetate, pH 7.4 over 30 min) failed, and were unable to detect any NADK activity in the column eluate, including the flow-through fraction, even after further increasing the concentration of ammonium acetate. We initially thought that the NADK was not eluted from the column, but it is also possible that its activity was lost during chromatography. After testing a variety of salts, buffers and pHs we found that NADK activity could be eluted as a single peak using either sodium or ammonium sulfate. However, we were never able to obtain two peaks of NADK activity as we had done reproducibly with the Rainin SAX column, presumably due to differences in the stationary phases. It may be possible to

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replicate our previous results with another SAX HPLC columns, but unfortunately these columns are quite expensive (>USD 1000) and there is no guarantee that they will work.

Overall, we were successful in setting up chromatographic methods including SEC, hydroxyapatite, HIC and SAX for partial purification of NADK and we have used these methods for the preparation of samples for mass spectrometry as discussed below. Additionally, the results of SAX chromatography suggests that PMA stimulation of neutrophils may result in the formation of second form of NADK that was eluted earlier than the form present in unstimulated neutrophils. However, the nature of the material in this peak would require further investigation using another anion exchange column.

5.5 LC-MS/MS analysis of NADK

Treatment of neutrophils with PKC inhibitors provided clear evidence that PKC mediates the PMA-induced increases in NADK activity and immunoreactivity towards the MC-aNADK Ab in neutrophils. The dramatic increase in immunoreactivity of the GST-NADK after treatment with PKC-δ further supported a role for PKC in the recognition of NADK by the MC-aNADK Ab MC- Ab. Furthermore, detection of phosphoserine on NADK from PMA-stimulated neutrophils and PKC-δ treated GST-NADK using the anti-phospho (Ser) PKC substrate Ab demonstrated that PKC-δ phosphorylates NADK. To identify the sites on NADK that are phosphorylated, LC- MS/MS analysis was performed.

When this part of the study was initiated the only antibody recognizing NADK that was commercially available was MC-aNADK, which only appreciably interacts with NADK from neutrophils treated with PMA. Therefore NADK was initially purified from PMA-treated neutrophils using MC-aNADK and from and unstimulated neutrophils using conventional chromatographic methods of purification. However, the results obtained with purely chromatographic methods were not satisfactory, as many contaminating proteins were present, and very poor peptide coverage (~11%) was obtained, which made it difficult to draw any conclusions about phosphorylation of NADK from unstimulated neutrophils. Although we started with about 100 mg protein from 2 billion neutrophils, the final yield was quite low, largely because of the multiple steps in the purification procedure. Purification of NADK from PMA-treated neutrophil

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lysates gave much better results (about 35% coverage) for the modified protein, but we were not able to compare it to NADK from unstimulated cells. To accomplish this we required an antibody that could equally recognize NADK from stimulated and unstimulated cells, but no such antibody was available when this part of the study was initiated.

Although the poly (A) aNADK Ab later became available, it only recognized a band at 43 kDa in both stimulated and unstimulated neutrophil lysates, but as it did not recognize the 49 kDa band,ö we were reluctant to include it in our purification scheme. More recently the poly(B) aNADK Ab became available and could detect both the 43 and 49 kDa bands attributable to NADK from both vehicle- and PMA- treated neutrophils and, thus, this antibody was used for all subsequent IP experiments used for the purification of NADK.

SDS-PAGE of the purified NADKs followed by silver staining resulted in two prominent bands (43 and 49 kDa), regardless of the purification method used. Both bands were excised for trypsin digestion and LC-MS/MS analysis. Analysis of the mass spectra using the Mascot search algorithm revealed that both the 43 and 49 kDa bands contained NADK. However, the peptide sequence coverage varied among different samples, depending on the type of purification. In the case of poly (B) a-NADK Ab we expected to immunoprecitate both the 43 and 49 kDa forms of NADK, yielding two bands with silver staining, because of our prior western blot analyses with this antibody. In contrast, we didn’t expect to observe two bands with the MC- aNADK Ab, as it only gave one band at 49 kDa with western blotting. This suggests that NADK might exist as a complex of 43 and 49 kDa subunits and that the 43 kDa subunit is immunoprecipated as part of this complex, rather than by direct interaction with MC-aNADK.

Initially, we thought that performing one step of partial purification prior to IP would help to reduce the amount of contaminating proteins at the final step. Therefore, NADK from non- treated neutrophils was purified first by either HA or SAX chromatography followed by IP using the poly (B) aNADK. These experiments gave 26% and 35% protein coverage, respectively. In an attempt to increase sequence coverage, NADK was immunoprecipitated using the poly (B) aNADK Ab from vehicle and PMA treated neutrophils without any initial purification to avoid protein loss. This approach resulted in improved results, with 50% and 45% sequence coverage for vehicle and PMA, respectively.

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It would seem likely that the 43 and 49 kDa NADK isoforms are alternatively spliced transcripts of the protein. The amino and carboxyl termini of the two isoforms were identical, so that the portion of NADK that is absent from the 43 kDa isoform would have to be within the inner region of the molecule. We did not find any amino acids in the 43 kDa isoform that were not present in the 49 kDa isoform, so it seems probable that the 43 kDa isoform lacks an internal sequence that is present within the 49kDa isoform, but was not sequenced in the mass spectrometric analysis. Since we achieved only about 50% coverage of the sequence of the 49 kDa isoform there were unsequenced regions within the molecule, in particular between amino acids 159 and 251 and between amino acids 291 and 367 that could potentially account for the missing sequence in the 43 kDa isoform. The Unipro UGENE program was used to find the theoretical alternatively spliced transcripts of NADK. We found 12 transcripts with MW of between 34 and 63 kDa, which are listed in Table 1.2. One of the spliced forms (401 amino acid, 44 kDa) almost matches the NADK identified in 43k band with regard to molecular mass, but their sequences do not match. This transcript lacks 45 amino acids from its N-terminal compared to sequence of the 49 kDa transcript, but the results of LC-MS/MS experiments showed that the first 10 amino acids from the N-terminal of the 43 kDa protein were the same as those of the 49 kDa protein (Figure 5.2). However, it is possible that this amino terminal peptide could be a contaminant from the 49 kDa band because it was detected in only 2 out of 6 experiments. If so, then it is possible that the 43 kDa isoform could correspond to the 44 kDa alternatively-spliced isoform. Otherwise, it is not clear what causes the difference between the molecular masses of the two NADK isoforms. This could be further investigated by PCR analysis of neutrophils using specific primers designed for each theoretical transcript.

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Figure 5.2. Alignment of 44k Da and 49kDa alternatively spliced transcripts of human NADK using Clustal W program

Analysis of the phosphorylated peptides by LC-MS-MS revealed multiple phosphorylation sites on NADK. The 49 kDa isoform of NADK from vehicle- and PMA- treated neutrophils contained 5 and 4 phosphorylated sites between residues 44 and 64, respectively. Serine 44 (S44) was phosphorylated in NADK from vehicle-treated neutrophils prepared by IP using poly(B) aNADK Ab, whereas the corresponding peptide was not detected in other samples from vehicle- treated neutrophils (prepared by conventional purification methods, and combinations of hydroxyapatite and IP-poly(B) aNADK Ab or SAX- IP-poly (B) aNADK Ab). S44 was not

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phosphorylated in any of the samples purified from PMA-treated neutrophils, although in 43k NADK purified by MC-aNADK, the corresponding peptide was not detected.

S46 was phosphorylated in both samples prepared from PMA-treated neutrophils by IP- MC aNADK Ab and IP-poly (B) aNADK Ab, while it was not phosphorylated in 3 out of 4 independent samples prepared from vehicle-treated neutrophils. Although, phosphorylation of this residue was detected in one of the samples from vehicle-treated neutrophils (purified by IP-poly (B) aNADK Ab), the ion signal intensity of the S46 phosphopeptide was about 3 times higher in the sample prepared from PMA-treated neutrophils (purified by IP-poly (B) aNADK Ab). This suggest that S46 of NADK is phosphorylated to some extent in resting neutrophils and that activation of neutrophils with PMA markedly increases its phosphorylation.

S48 was also phosphorylated in NADK purified from PMA-treated neutrophils by IP-poly (B) aNADK Ab, but not in NADK purified by IP-MC aNADK Ab). It was also phosphorylated in one out of the 4 samples prepared from vehicle-treated neutrophils (IP-poly (B) aNADK Ab). In contrast to P-S46, the intensities of the ion corresponding to the peptide bearing P-S48 in vehicle- treated neutrophils were comparable to those the PMA-treated neutrophils. Additionally, the intensity of the unmodified S48-peptide was much higher than that of the P-S48-peptide in both vehicle and PMA treated neutrophils (i.e., 5 times more in vehicle and 4 times more in PMA- treated neutrophils), indicating that in both cases only a minor degree of phosphorylation occurs at S48.

NADK was also phosphorylated on S64 in both samples prepared from PMA treated neutrophils and in two of the samples prepared from non-treated neutrophils (SAX-IP-poly (B) and IP-poly (B) aNADK Ab). Based on the relative ion intensities of this peptide, S64 appeared to be phosphorylated to a greater extent in NADK from PMA-treated neutrophils, but the difference was not as great as with S46. Finally, phosphorylation of Threonine 62 (T62) was identified in NADK purified by IP-poly (B) aNADK Ab from both vehicle and PMA treated neutrophils.

A pattern of phosphorylation sites similar to that described above was detected on the 43 kDa isoform of NADK. The peptide bearing either S44 or P-S44 was not detected in any of the mass spectra of the 43 kDa isoform. S46 was not phosphorylated in any of the samples from vehicle-treated neutrophils but was phosphorylated on NADK purified by IP-Poly (B) aNADK in

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PMA-treated neutrophils. However, no peptides bearing S46 were detected in the sample prepared by IP-MC aNADK Ab from PMA-treated neutrophils.

S48 was phosphorylated in NADK from both vehicle and PMA treated neutrophils purified by poly (B) aNADK Ab, whereas it was not phosphorylated in 3 other samples form vehicle treated neutrophils. In case of sample from PMA-treated neutrophils prepared by MC-aNADK, the corresponding peptide was not detected.

S64 was phosphorylated in both samples from PMA-treated neutrophils and one sample from vehicle treated neutrophils (IP-poly (B) aNADK Ab) and the corresponding peptide was not sequenced in 3 other samples from vehicle-treated neutrophils. Finally, T62 was not phosphorylated in any of the samples of the 43 kDa isoform of NADK

Taken together, these data suggests that a small portion of NADK in non-treated neutrophils is phosphorylated on S46, S48, S64 and T62. Treatment of neutrophils with PMA increases the phosphorylation of S46, and possibly to a lesser extent S64, of NADK without affecting phosphorylation of S48 and T62. A recent phosphoproteome study has revealed that the murine ortholog of NADK is phosphorylated at T62 and S64 in the liver (141). As human NADK and mouse NADK share more than 92% homology, phosphorylation of these residues may have some as yet unknown biological relevance.

LC-MS/MS analysis of the recombinant NADK revealed that GST-NADK was phosphorylated on S15 before and after treatment with PKC-δ, whereas T127 was phosphorylated only on GST-NADK before PKC-δ treatment. Treatment of the GST-NADK with PKC-δ resulted in phosphorylation of S46, S55 and S64. As PKC-δ treatment dramatically increased the immunoreactivity of the GST-NADK toward the MC-Ab, we propose that phosphorylation of at least one of these residues is associated with recognition of the protein by the MC-Ab. Comparison of the phosphorylated residues in NADK from neutrophils with the PKC-treated recombinant GST-NADK suggests that phosphorylation of S46 is likely to play the most important role in recognition of NADK by the monoclonal antibody, whereas phosphorylation of S64 could possibly also be involved. Since S55 was not phosphorylated in any of the neutrophil samples that we analyzed it is unlikely to be involved in recognition of recpombinant NADK-GST by the monoclonal antibody.

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5.6 PMA changes the interaction of NADK with 14-3-3 zeta protein

Although, our results provide clear evidence that NADK is phosphorylated by PKC (most likely by PKC-δ), it is not clear how phosphorylation of NADK regulates its enzymatic activity, as in vitro phosphorylation of GST-NADK by PKC-δ did not increase its enzymatic activity significantly. This is consistent with the study conducted by Love et al. (99) on the mechanism of regulation of NADK by Ca2+/CAM. They showed that calmodulin kinase phosphorylates S64 in recombinant NADK in vitro without affecting its activity, in contrast to a previous report that partially purified NADK from human neutrophils was activated by Ca2+/CAM (91). We postulate that phosphorylation of NADK regulates its catalytic activity indirectly, possibly through release of NADK from an inhibitory complex. Searching the BioGRID, biological database of protein- protein interactions resulted in identification of NADK as an interacting partner of 12 proteins, as shown in table 1.1. These interactions were all found in high throughput studies of protein-protein interactions.

Our data on LC-MS/MS analysis of immunoprecipitated NADK showed that several other proteins were co-immunoprecipitated along with NADK. In our search for NADK interacting partners among the proteins identified by LC-MS/MS, we interestingly found that 14-3-3 beta and zeta were co-immunoprecipitated with NADK in the 43 kDa band from vehicle-treated neutrophils using the poly (B) anti-NADK Ab, while these proteins were not detected when NADK was immunoprecipitated from PMA-treated neutrophils. Members of the 14-3-3 protein family are adaptor proteins involved in regulation of many intracellular signal transduction pathways. In most known cases, they function as an inhibitory binding partner, thereby negatively regulating the function of the target protein (172). It is possible that NADK forms a complex with 14-3-3 proteins and that treatment of neutrophils with PMA results in dissociation of 14-3-3 proteins. Unfortunately, as our initial goal for LC-MS/MS analysis was to identify post-translational modifications of NADK, we didn’t analyze the entire lane of the acrylamide gel from the SDS- PAGE of immunoprecipitated NADK. We did, however, analyze the region containing proteins with molecular masses in the range between about 40 and 55 kDa. Thus the detection of 14-3-3 proteins, which have molecular masses around 30 kDa, may be due to incomplete separation of the proteins on the acrylamide gel. Although our current LC-MS/MS results are not conclusive with regard to the identification of NADK interaction partners, they nevertheless provide us with

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a potential candidate protein for further investigation. To examine a possible role for14-3-3 in regulating NADK activity we immunoprecipitated NADK from vehicle- and PMA- treated neutrophils followed by western blotting using an anti-14-3-3 zeta antibody. In preliminary experiments, we were able to detect 14-3-3 zeta co-immunoprecipitated with NADK from vehicle treated neutrophils, whereas the intensity of the 14-3-3 zeta band was decreased in PMA-treated neutrophils, suggesting that PMA modified the interaction of NADK with 14-3-3 zeta. This, however, needs further investigation and it will be important to determine whether 14-3-3 can inhibit the enzyme activity of recombinant NADK

Taken together, we hypothesize that formation of a complex between 14-3-3 proteins and NADK results in inhibition of enzyme activity. Phosphorylation of NADK, possibly at S46, could induce conformational changes that result in the release of 14-3-3 and activation of NADK (Figure 5.3).

Figure 5.3. Schematics mechanism of NADK activation by PKC

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5.7 Conclusion and implications in diseases

In this study we investigated, for the first time, the mechanism of activation of NADK by PMA in neutrophils and discovered a novel PKC-dependent regulatory mechanism for this enzyme. We showed that the effect of PMA is not indirectly mediated by activation of NOX2 or calcium mobilization, but rather by direct activation of NADK by PKC. To further investigate NADK activation we produced recombinant GST-tagged NADK and showed by mass spectrometric analysis that it is phosphorylated at serines 46, 55 and 64 following treatment with PKC-. We also showed that PMA stimulates the phosphorylation of NADK in neutrophils at serine-46 and to a lesser extent, at serine-64. However phosphorylation of GST-NADK did not increase its catalytic activity, suggesting that PKC regulates NADK indirectly, possibly by causing changes in its interaction with other proteins. Finally, we provided preliminary evidence that NADK forms a complex with 14-3-3 zeta and that PKC causes dissociation this complex. Taken together, we hypothesize that phosphorylation of NADK by PKC causes a conformational change that results in release of 14-3-3 zeta and activation of the NADK.

It is of a great importance to elucidate the mechanisms governing NADK regulation, as this enzyme is the only biosynthetic route for the formation of NADP+ in mammalian cells. NADP+ in turn is required for NADPH formation through its reduction by the pentose phosphate pathway. The respiratory burst, which is characterized by rapid generation of superoxide at the expense of oxidation of NADPH to NADP+, plays a critical role in the bactericidal function of neutrophils, the first line of defense against infection. Thus, activation of NADK could play an important role in this process by providing sufficient levels of NADPH to permit neutrophils to mount a robust respiratory burst. In patients with chronic granulomatous disease (CGD), the respiratory bust is defective due to a NADPH oxidase disorder (186) and as a result, CGD patients suffer from recurrent infections with Staphylococcus aureus, Pseudomonas, Nocardia, as well as fungi such as Aspergillus and Candida albicans (186). Furthermore, NADK is an essential enzyme in the regulation of redox homeostasis of cells by providing NADPH, which in turn provides the reducing power for both the glutathione and thioredoxin systems that scavenge ROS and protect cells against oxidative damage (66). Although mechanisms that prolong neutrophil survival are beneficial to host defense against pathogens, induction of apoptosis is also critical for resolution of inflammation. NADK may also be involved in enhancing granulocyte infiltration sites of

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inflammation by providing NADP+, which is required for the synthesis of the potent granulocyte chemoattractant 5-oxo-ETE. For example, it is shown that neutrophils are significantly elevated in synovial fluid of rheumatoid arthritis patients (35), therefore, relative inhibition of NADK may contribute to modulate the neutrophils infiltration via reducing the production of 5-oxo-ETE.

6. Prospects for future studies The present study has demonstrated that PMA activates NADK in neutrophils by PKC- mediated phosphorylation. However, this does not appear to directly activate the enzyme, suggesting that the role of phosphorylation is to regulate the interaction of NADK with a binding partner. Future research should be directed at clarifying the potential role of 14-3-3 in regulating NADK binding activity as well as at identifying additional regulatory proteins that complex with NADK to modulate its activity.

6.1 To further investigate the interaction of NADK with 14-3-3 isoforms as well as other proteins, recombinant GST-NADK bound to glutathione-Sepharose beads could be treated with vehicle and PKC-δ, followed by incubation with a lysate from vehicle-treated neutrophils. The beads could then be washed, and GST-NADK eluted with reduced GSH, followed by LC- MS/MS analysis to identify the proteins captured by GST-NADK from the neutrophil lysate. The experiment could then be repeated after PKC--induced phosphorylation of GST-NADK to look for changes in the profile of binding partners.

6.2 A more rigorous search should be made for NADK binding partners by analyzing more comprehensively the proteins that are co-immunoprecipitated with NADK from resting and PMA-stimulated neutrophils. The protocol should be similar to what we have used previously except that additional bands from the gel should be analyzed.

6.3 In the case of 14-3-3 isoforms, further co-immunoprecipitation experiments are required to provide stronger evidence that PKC inhibits the interaction of NADK with these proteins. To do so, NADK from vehicle and PMA treated neutrophils could be immunoprecipitated using poly (B) anti-NADK Ab, followed with western blotting using anti-14-3-3 beta and zeta

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antibodies. Such experiments should also be done in the reverse direction, in which 14-3-3 zeta and beta are immunnoprecipitated, followed by western blotting using poly (B) anti-NADK Ab.

6.4 To investigate whether binding of 14-3-3 proteins to NADK can directly inhibit the enzymatic activity of NADK, recombinant GST-NADK could be treated with recombinant 14-3- 3 beta and zeta, followed by measurement of enzyme activity.

6.5 Simultaneous overexpression of 14-3-3 zeta (or beta) and NADK with two different tags in HEK293 cells would also provide a tool to study the interaction of NADK with 14-3-3 proteins. One of the proteins could be purified to determine whether the other one was copurified with it. In this way, it would be possible to study the effect of phosphorylation of NADK on its interaction with 14-3-3 proteins by performing site-directed mutagenesis of S46, S48 and S64 in NADK.

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Claims to original research

1. PMA activates NADK in intact human neutrophils by a process that is dependent on PKC and independent of NOX-2 and the respiratory burst. This phenomenon appears to be restricted to primary phagocytic cells, including neutrophils and monocytes, and is not observed in a variety of cell lines related to these cells.

2. PMA-activated NADK, but not non-activated NADK, is selectively recognized by a commercial monoclonal antibody that was raised against GST-tagged NADK. PMA-induced recognition by this antibody is blocked by PKC inhibitors. Recombinant human NADK-GST is recognized by this antibody only after treatment with PKC-, suggesting that the epitope recognized by the antibody must be phosphorylated.

3. PMA-enhanced NADK activity is also observed in neutrophil lysates and the increased activity is preserved though a variety of chromatographic purifications.

4. PKC- phosphorylates recombinant NADK on serines 46, 55, and 64, as determined by mass spectrometric analysis.

5. There are two major isoforms of NADK in neutrophils with molecular masses of approximately 43 and 49 kDa. The 49 kDa form corresponds to that which has previously been reported for human NADK. The 43 kDa form retains the N- and C – terminal regions of NADK, but is missing an as yet unidentified central part of the molecule.

6. PMA induces the PKC-dependent phosphorylation of NADK in neutrophils on serine- 46 and to a lesser extent on serine-64.

7. Phosphorylation of NADK by PKC does not directly increase its enzymatic activity, but may affect its interaction with regulatory proteins.

8. NADK in unstimulated neutrophils is complexed by the beta and zeta isoforms of the regulatory protein 14-3-3. This interaction is disrupted by treatment of neutrophils with PMA, suggesting that PMA-induced phosphorylation of NADK results in dissociation of the complex, releasing NADK from the inhibitory effect of 14-3-3 and thereby increasing enzyme activity.

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Appendix

Figure S.1. MS/MS spectra of 43k NADK form vehicle-treated neutrophils. MS/MS spectra obtained from peptides carrying A. phospho-S48 and B. phospho-S64 in 43k Da NADK immunoprecipitated from vehicle-treated neutrophils using the poly (B) aNADK Ab.

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Figure S.2. MS/MS spectra of 49k NADK form vehicle-treated neutrophils. MS/MS spectra correspond to peptides carrying A. phospho-S46, B. phospho-S48 and C. phospho-S64 in 49k Da NADK immunoprecipitated from vehicle-treated neutrophils using the poly (B) aNADK Ab.

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Figure S.3. MS/MS spectra of 43k NADK form PMA-treated neutrophils. MS/MS spectra correspond to peptides containing A. phospho-S46, B. phospho-S48 and C. phospho-S64 residues in 43k Da NADK purified from PMA-treated neutrophils performing IP using the poly (B) aNADK Ab.

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Figure S.4. MS/MS spectra of 49k NADK form PMA-treated neutrophils. MS/MS spectra produced by peptides containing A. phospho-S46, B. phospho-S48 and C. phospho-S64 residues in 49k Da NADK purified from PMA-treated neutrophils performing IP using the poly (B) aNADK Ab.

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Figure S.5. MS/MS spectra of recombinant GST-NADK. MS/MS spectra produced obtained from A. phospho-S46, B. phospho-S64 in recombinant GST-NADK treated with PKC-δ

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