PROTEIN S-NITROSYLATION AND ITS RELEVANCE TO REDOX CONTROL OF CELL SIGNALING KYAW HTET HLAING (M.B.B.S, UM 2) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. Kyaw Htet Hlaing 24 Dec 2012 Acknowledgements I wish to express my deepest gratitude to my supervisor, Associate Professor Marie-Véronique Clément, Department of Biochemistry, for introducing me into the field of “Redox Control of Cell Signaling”, and guiding me along the arduous journey of my Ph.D. study. I am truly grateful for her warm encouragement and constant optimism in the face of “reality of day-to-day life of a graduate student” over the years. This thesis has not been complete without her unending support and kind understanding. I also like to thank my TAC members, Dr Andrew Jenner and Professor Kini R Manjunatha, for their comments, useful advice and feedbacks throughout my study. My heart-felt thanks to my lab members for listening to both of my happy and frustrating stories. Spending time together with them has made my life in the lab most enjoyable. I want to thank Luo Le in particular for taking time to read the draft of my thesis and giving me useful feedback. Also my special thank to Ms Lee Mui Khin for keeping things in order and making sure that I always get what I need in time. Lastly, my deepest gratitude to my family for their encouragement and support all along. I wish to express my special thank to my older sister, Ms Wint Wint Htet Hlaing, for helping me out financially when in need and motivating me when confronted with various setbacks during my study.   i   Contents Acknowledgements i Contents ii Summary vii List of Figures ix List of Tables xiii Abbreviations xiv CHAPTER 1: INTRODUCTION 1.1 BIOCHEMISTRY OF FREE RADICALS 1.2 SOURCES AND FORMATION OF REACTIVE OXYGEN AND NITROGEN SPECIES 1.3 1.2.1 Superoxide 1.2.2 Hydrogen Peroxide and Hydroxyl Radical 1.2.3 Nitric Oxide and its derivatives EFFECTS OF REACTIVE OXYGEN AND NITROGEN SPECIES ON CELLULAR STRACTURE AND SIGNALING 1.4 1.3.1 Cellular Toxicity 1.3.2 Physiological Function: Redox Signaling 10 MECHANISMS OF REDOX-BASED REGULATION OF CELL SIGNALING: FUNCTIONAL CONSEQUENCES OF OXIDATION OF “REACTIVE CYSTEINE”   14 1.4.1 Inhibition of Activity 15 1.4.2 Activation of Protein Functions 16 ii   1.4.3 Multimerization of Subunits 17 1.4.4 Release of Regulatory Proteins 17 1.4.5 Oxidation of Transcription Factors 18 1.5 TYPES OF REVERSIBLE CYSTEINE OXIDATION 1.6 DIFFERENTIAL REDOX-MODIFICATION 19 AND FUNCTIONAL CONSEQUENCES 1.7 21 REDOX-MODIFICATION: PHYSIOLOGICAL SIGNALING VERSUS CELLULAR TOXICITY 22 1.8 PROTEIN S-NITROSYLATION 24 1.8.1 25 1.9 Factors influencing protein S-nitrosylation ABERRATION OF REDOX SIGNALING AND CARCINOGENESIS 31 1.10 RATIONALE OF THESIS 36 CHAPTER 2: MATERIALS AND METHODS 39 2.1 MATERIALS 39 2.1.1 Chemicals 39 2.1.2 Antibodies 41 2.1.3 Cell Lines and Cell Culture 42 2.2       METHODS                   2.2.1 Whole Cell Lysate Preparation 2.2.2 Sodium Dodecyl sulphate polyacrylamide gel electrophoresis 43   43 (SDS-PAGE) and Western Immunoblotting 43 2.2.3 Transient Transfection 45 2.2.4 siRNA Transfection 45 iii   2.2.5 Detection of S-nitrsoylated and Oxidized PTEN Oxidation/Reduction Assay 46 2.2.6 Biotin Switch Technique (BST) 47 2.2.6.1 Detection of Total Protein and PTEN S-nitrosylation 47 2.2.6.2 Detection of Total Protein and PTEN Oxidation 48 2.2.7 Lucigenin Chemiluminiscence Assay for Detection of Intracellular Superoxide 2.2.8 49 Fluorescence Flow Cytometry Assay for Detection of Intracellular Hydrogen Peroxide, Nitric Oxide and Calcium 50 2.2.9 51 Statistical Analysis CHAPTER 3: RESULTS 3.1 52 INCREASE IN INTRACELLULAR O2˙- INDUCES GENERALIZED PROTEIN S-NITROSYLATION 3.1.1 52 Serum withdrawal causes a reduction in basal production of intracellular O2˙3.1.2 53 Pharmacological inhibition of Cu-Zn SOD leads to an increase in intracellular O2˙- without concurrent rise in H2O2 level 54 3.1.3 Detection of protein S-nitrosylation 57 3.1.3.1 Oxidation/reduction assay 57 3.1.3.2 Biotin Switch Technique 58 3.1.4 Both pharmacological inhibition and siRNA gene silencing of Cu-Zn SOD induce protein S-nitrosylation 3.2 64 PHYSIOLOGICALLY RELEVENT CONCENTRATIONS OF H2O2 INDUCES   by PROTEIN S-NITROSYLATION WHEREAS HIGH iv   CONCENTRATION OF H2O2 CAUSES NON-SNO OXIDATIVE MODIFICATIONS 3.3 67 PROTEIN S-NITROSYLATION INDUCED BY GROWTH FACTORS 76 3.4 OXIDATIVE MODIFICATION OF TUMOR SUPPRESSOR PTEN BY ROS AND GROWTH FACTORS 83 3.5 88 PROCESS OF PROTEIN S-NITROSYLATION 3.5.1 Intracellular NO˙ is decreased with an increase in O2˙- generation whereas it is actively synthesized by H2O2 and growth factors 3.5.2 Identification of S-nitrosylation species for oxidants- and growth factors-induced S-nitrosylation 92 3.5.3 Peroxynitrite: oxidation vs nitration 99 3.5.4 Role of calcium in protein S-nitrosylation caused by ROS and PDGF 3.6 103 3.5.5 GSNOR inhibition enhances protein S-nitrosylation 3.5.6 Inhibition of O2˙- production enhances protein S-nitrosylation through 111 PROTEIN S-NITROSYLATION IN SIGNAL TRANSDUCTION 113 Scavenging PNOO˙ prevents PDGF activation of Akt kinase whereas GSNOR inhibition enhances it 113 3.6.2 O2˙-/ NO˙ Balance in Signal Transduction 3.6.3 ONOO- mediates Akt activation by O2˙- and low concentration of H2 O2 119 S-NITROSYLATION AND TUMOR MAINTENANCE 121 3.7.1   107 an increase in intracellular NO˙ 3.6.1 3.7 88 115 Maintenance of protein S-nitrosylation in the absence of serum is v   associated with sustained signal transduction in precancerous and cancer cells. 3.7.2 121 Protein de-nitrosylation in cancer CHAPTER  4:  DISCUSSION   4.1   126       129 S-NITROSYLATION IS THE COMMON MECHANISM OF PROTEIN OXIDATION USED BY O2˙- AND PHYSIOLOGICALLY RELEVANT CONCENTRATION OF H2O2 4.2 129 4.1.1 O2˙- and SNO Modification 129 4.1.2 H2O2 and SNO Modification 130 4.1.3 Redox Signaling: O2˙- vs H2O2 131 REDOX SIGNALING BY GROWTH FACTORS IS THROUGH S-NITROSYLATION 4.2.1 132 PTEN: an example of oxidative modification of protein upon growth factor induction of cell proliferation 4.3 PEROXYNITRITE: A POTENTIAL 133 PHYSIOLOGICALLY RELEVANT S-NITROSYLATING INTERMEDIATE 4.4 O2- AND NO˙: STRIKING THE RIGHT BALANCE FOR SIGNAL TRANSDUCTION 4.5 134 145 PROTEIN S-NITROSYLATION AND ROS-DRIVEN CARCINOGENESIS 149 4.6. 152 CONCLUSION References 155 Publication and Presentation 195   vi   Summary Discovery of the function of oxidants as signaling molecules marks the beginning of the field of redox control of cell signaling. Understanding the mechanism of how free radicals regulate signaling is critical to distinguish between normal physiology and cellular toxicity both caused by reactive species. It is now known that free radicals influence various cellular processes by altering the function of critical proteins as a result of reversible oxidation of “reactive cysteine” within the proteins. Different types of oxidative modification such as S-nitrosylation, Sglutathionylation, di-sulphide bond formation, sulphenic acid formation, have been proposed to mediate redox control of cell signaling. However, physiological relevance of these modifications is somehow missing. Furthermore, there has been a debate about relative importance of O2˙- versus H2O2 in mediating enhanced cell proliferation. Following up on our previous study that demonstrates that O2˙- activates survival kinase Akt through S-nitrosylation of the tumor suppressor PTEN, our current study deciphers the mechanistic aspect of how oxidative signal by O2˙- is transformed into nitrosative signal. We also provide evidence that physiologically relevant concentration of H2O2 predominately induces protein S-nitrosylation over non-SNO modifications. We demonstrate that protein S-nitrosylation induced by O2˙and H2O2 is both mediated by common S-nitrosylating species, ONOO- although the pathways to formation of ONOO- are different in each case. Moreover, we show that oxidation of proteins that occurs following incubation with PDGF, EGF and 10% FBS is by protein S-nitrosylation. Particularly in the case of PDGF, the growth factor does not generate a high level intracellular H2O2 regardless of concentration of PDGF used and it consistently induces protein S   vii   nitrosylation. Again, we find that the relevant S-nitrosylating species that mediates growth factors-induced protein S-nitrosylation is ONOO-. Removal of ONOOprevents protein S-nitrosylation as well as activation of Akt induced by O2˙-, H2O2 and PDGF demonstrating protein S-nitrosylation is of relevance to redox control of cell signaling. We also highlight the consequences of disturbing O2˙-/NO˙ balance in cell signaling. On one hand, removal of NO˙ is effective in preventing S-nitrosylation but it increases the levels of intracellular O2˙- and H2O2 potentially causing oxidative stress with damaging consequences. On the other hand, we demonstrate the ineffectiveness of removing O2˙- alone to stop pro-survival signaling as the latter could continue by ONOO--independent but NO˙-dependent S-nitrosylation. Lastly, we show that increased ROS and RNS production in breast cancer cell line (MCF7) correlate with sustained protein S-nitrosylation and Akt activation in the absence of serum. 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Phosphatidylinositol-3,4,5-trisphosphate PI3-K Phosphatidylinositol 3 - kinase PP2A Protein phosphatase 2A PTEN Phosphatase and Tensin Homolog Deleted on Chromosome 10   xv   PTP Protein tyrosine phosphatase RBS Reactive bromine species RCS Reactive chlorine species RNS Reactive nitrogen species ROS Reactive oxygen species RSS Reactive sulphur species Ser Serine SNO S- nitrosylation S- S Di-sulphide bond... of these events is not fully understood 1.4 MECHANISMS OF REDOX- BASED REGULATION OF CELL SIGNALING: FUNCTIONAL CONSEQUENCES OF OXIDATION OF “REACTIVE CYSTEINE” The notion of ROS/RNS acting as signaling molecules comes from evidence that reaction of these oxidants with signaling proteins results in alteration of protein functions (Janssen-Heininger YM et al, 2008) Majority of the targets proteins by... transduction is often referred to as Chapter  1:  Introduction   10   Redox Signaling It is established that cells are capable of generating low concentrations of ROS when stimulated by various ligands such as cytokines, growth factors and hormones (Petry A et al, 2010) Intentional generation of ROS was first observed in immune cells such as neutrophils and macrophages but certain cytokines such as TNF-α,... Synthesis of endogenous NO˙ is highly regulated by the activity of isoforms of nitric oxide synthase (NOS) There are three types of NOS Neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3) are constitutively expressed in nervous system tissues and endothelia cells respectively (Bredt DS et al, 1990; Knowles RG et al, 1989; Palmer LA et al, 1988) Inducible NOS (iNOS or NOS2) was first identified... Cellular Toxicity Both ROS and RNS are known to cause damage to cell structures, nucleic acids, lipids and proteins Their harmful effects are termed “oxidative stress” and “nitrosative stress” respectively The primary ROS, O2˙- is not reactive itself Only under stress conditions, an excess of O2˙- releases “free iron” from iron containing molecules, for example, (4Fe- 4S) cluster-containing enzymes of dehydratase-lyase... includes phosphatases, kinases, ion channels, chaperone proteins and transcription factors Function of these proteins is modified as a result of oxidation of reactive cysteine (s) within the proteins The summary of possible functional consequences by cysteine oxidation is depicted in figure 2 and details are given in the respective sections that follow Chapter  1:  Introduction   14   (Taken from Janssen-Heininger... targets and vicinity of targets to the site of ROS/RNS production (D'Autréaux B and Toledano MB, 2007) Proteins vary widely in their oxidant sensitivity and only a small number of highly sensitive proteins are suitable for redox signaling The redox sensivity of a particular protein is determined by the presence of oxidizable amino acids The most important amino acid is cysteine that contains sulfhydryl... is by stimulusinduced activation of membrane-bound enzyme systems such as the NADPH oxidase complex (NOX) Superoxide generation by the NOX complex is deliberate and it was best characterized in phagocytic cells such as neutrophils that undergo a series of reactions called the respiratory burst in response to microorganisms or inflammatory mediators (Babio BM et al, 2002) The enzyme complex consists... below) of caspases under basal condition is associated with inhibition of their activities and prevention of apoptosis (Li J et al, 1997) but on the other hand, denitrosylation (reducing) of caspases following a variety of apoptotic stimuli triggers apoptosis (Kim TE and Tannenbaum SR, 2004; Mannick JB, 1999) Recently, it was suggested that antiapoptotic effect of the antioxidant protein, thioredoxin... maintenance (Heo J and Campbell SL, 2004; Raines KW et al, 2007) N-Ras is S- nitrosylated at cysteine 118 in eNOS dependent manner in T-cells Mutation of this redox sensitive cysteine residue leads to abrogation of Angiotensin II signaling and T-cells response mediated by N-Ras activation (Ibiza S et al, 2008) In skeletal muscles, high-conductance Ca2+ release channels or ryanodine receptors (RyR) are activated . PROTEIN S-NITROSYLATION AND ITS RELEVANCE TO REDOX CONTROL OF CELL SIGNALING KYAW HTET HLAING (M.B.B.S, UM 2) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY. protein S-nitrosylation is of relevance to redox control of cell signaling. We also highlight the consequences of disturbing O 2 ˙ - /NO˙ balance in cell signaling. On one hand, removal of NO˙. Discovery of the function of oxidants as signaling molecules marks the beginning of the field of redox control of cell signaling. Understanding the mechanism of how free radicals regulate signaling