Nitric oxide (NO): Biochemistry and Signaling

Transcription

1 Graduate course No. 2214, Karolinska Institutet, June 2014 Nitric oxide (NO): Biochemistry and Signaling Moran Benhar Department of Biochemistry Rappaport Faculty of Medicine Technion Haifa, Israel

2 Overview Nitric oxide - properties and key reactions Role of nitric oxide in physiology/pathology Synthesis and breakdown of nitric oxide Protein S-nitrosylation and denitrosylation: cellular functions and methods of analysis

3 Identification of nitric oxide as a biological messenger molecule 1977: Freid Murad discovered that nitroglycerin acts on smooth muscle cells by releasing nitric oxide. 1980: Robert Furchgott recognized that endothelia cells release a substance which promotes acetylcholine-induced vasorelaxation. This unknown substance was called endothelium-derived relaxing factor or EDRF. 1987: Lois Ignarro and Salvador Moncada identified EDRF as identical to nitric oxide. Murad Furchgott Ignarro Moncada

4 Nitric oxide (nitrogen monoxide) basic biochemistry Colorless gas Soluble in water but more soluble in organic solvents (easy passage between cell membranes) Intracellular concentration < micromolar Short lived ( < sec) Related species: NO + NO - Oxidation of NO 2NO + O 2 2NO 2 (nitrogen dioxide) 3 bonds 2.5 bonds 2 bonds. N N.. N O.. O O 4NO + O 2 + 2H 2 O 4H + + 4NO 2 - (nitrous acid). NO radical: NO or NO

5 Nitric oxide as a free radical scavenger NO reacts very slowly with most molecules (e.g., RSH) but very rapidly with other free radicals (k > 10 9 ) NO + OH HNO 2 NO + RO 2 ROONO NO + RS RSNO (nitrosothiol) NO + O - 2 ONOO - (peroxynitrite) protective damaging

6 Nitric oxide - interaction with metals (heme) NO activates soluble guanylate cyclase via heme ligation H 2 N NO NH 2 Heme Fe Heme binding region His 105 Dimer interface HOOC COOH GTP cgmp Catalytic site Other: aconitase, cytochrome c oxidase

7 Nitric oxide signaling through cgmp O N NH HO O P O O P O O P O N N NH 2 O - O - O - O OH OH Guanosine triphosphate (GTP) Guanylate cyclase N O N NH O N NH 2 OH O P O O O - cyclic Guanosine monophosphate (cgmp) Francis SH, et al., Pharmacol Rev. (2010)

8 Francis SH, et al., Pharmacol Rev. (2010) Cellular targets of cgmp

9 Nitric oxide physiological/pathological roles Physiological role Excess implicated in tissue injury in Effect of knockout of the relevant gene in mice Nervous system Response to excitatry amino acids (especially glutamtae); neurotransmissiton/neuromodulation; Synaptic plasticity (long term memory) Epilepsy, stroke, excitotoxicity (implicated in multiple neurodegenerative disorder) nnos: obstruction of the pylorus, less sensitive to brain damage by ischemia-reperfusion or trauma; inapporpiate and excessive sexual and aggressive behavior Vascular & immune systems Control of blood pressure; inhibition of platelet aggregation; killing of microorganisms; generally antiatherosclerotic Septic shock (vasodilation, low blood pressure); chronic inflammation (rheumatoid arthritis, ulcerative colitis); increased cancer risk; transplant rejection inos: icreased suseptibility to tuberculosis, Listeria and Leishmania infection and lymphoma cell proliferation; resistance to endotoxin-induced hypotension enos: deficient vasodilation in response to acetylcholine; heypertension Other systems Penile erection, bladder control, lung vasodilation, gastrointestinal function, wound healing; Asthma

10 NO + O 2 - ONOO - k ~ 10 9 Peroxynitrite Mechanisms of damage: protein modification: Protein oxidation and nitration (Cys, Tyr), inactivation of enzymes, lipid peroxidation Pacher et al., Pysiol. Rev. (2007)

11 Pacher et al., Pysiol. Rev. (2007) Roles of NO and peroxynitrite in the pathophysiology of stroke

12 Nitric oxide redox scheme - O O N + NO 3 - O - +2e - -2e - O - O N: - NO 2 H H H +1e -. +1e :N O - +2e - +2e N O N O - -1e - -1e - -2e - H -2e - NO HNO H 2 NOH H H N H NH 3 nitrate nitrite nitric oxide nitroxyl hydroxylamine ammonia -e - +e - - O. NH 3 From amino acid catabolism, enters the urea cycle. N + O NH 2 OH A potent mutagen that reacts with cytosine. The cytosine adduct then pairs with adenine rather than guanine. NO 2 nitrogen dioxide HNO Biology and chemistry relatively unknown. NO Generated endogenously via NOS. A potent vasorelaxant and involved in neurotransmission and immune response. NO 2 A potent oxidant. A major toxic constituent of air pollution. NO 2 - Used as a preservative. Toxic. Converted to NH 3 via nitrite reductases. NO 3 - Substrate for nitrate reductases

13 Synthesis of Nitric Oxide Nitric oxide is synthesized from L-arginine This reaction is catalyzed by nitric oxide synthase (NOS) COO - COO - COO - + H 3 N C H (CH 2 ) 3 NH 1 NADPH + O 2 + H 3 N C H (CH 2 ) 3 NH 0.5 NADPH + H 3 N C H (CH 2 ) 3 NH +NO C H 2 N NH 2 + C H 2 N N + H OH O C NH2 Arginine N ω -Hydroxyarginine Citrulline

14 Nitric oxide synthase (NOS) L-Arg +O 2 L-Cit +NO Heme e - calmodulin FMN FAD NADPH BH4 Oxygenase domain Reductase domain

15 Sources of Nitric Oxide Nitric Oxide Synthases (NOS) in mammalian tissues arginine + O 2 + NADPH citrulline +. NO + NADP + NOS NOS1 (nnos) Neuronal Constitutive Ca 2+ NOS2 (inos) Macrophage inducible NOS3 (enos) Endothelial Constitutive Ca 2+ Reduction of nitrogen oxides (nitrate and nitrite) microbial denitrification pathways (soil) nitrite reduction by oxidoreductases (gut)

16 Removal of NO The reaction of NO with deoxyhemoglobin Hb-Fe 2+ + NO Hb-Fe 2+ NO The reaction of NO with oxyhemoglobin Hb-Fe 2+ -O 2 + NO Hb-Fe 3+ + NO 3 - In microorganisms removal by NO reductases and NO dioxygenases

17 Two major mechanisms of NO-based signaling Lima et al., Circ. Res. (2007)

18 S-nitrosylation (S-nitrosation) Thiol (SH) S-nitrosothiol (SNO) Cysteine COO - S-Nitrosocysteine COO HN C CH 2 H +. NO e - (metal, O 2 ) + 3 HN C CH 2 H SH SNO RSH +. NO RSNO RS - + NO + RSNO RS. +. NO RSNO Slow Fast Fast

19 Potential mechanisms of GSNO and protein SNO formation (and degradation) Dinitrosyl iron complexes Smith BC & Marletta MA. Curr Opin Chem Biol. (2012)

20 Examples of S-nitrosylated proteins Protein Targeted Cysteine Effect NMDA receptor (NR1 and NR2A subunits) Ryanodine receptor (RyR1) C744, C798 of NR1, and C87, C320, C399 of NR2A C3635 Inhibition of excessive NMDA current (anti-apoptotic) Enhances Ca 2+ flux and muscle contractility Caspase-3 C163 Inhibition of caspase-3 activity (anti-apoptotic) Metalloproteinase-9 (MMP-9) C99 Activation of MMP (proapoptotic) Parkin (E3 ligase) C241, C260 Inhibition of E3 ligase activity (pro-apoptotic) GAPDH C150 Binding to Siah1, nuclear translocation (pro-apoptotic) I B kinase (IKK C179 Inhibition of TNF -induced IKK activation SHP-2 (tyrosine phosphatase) C453 Inhibition of enzyme activity OxyR C199 Activates transcription NSF C91 Inhibits exocytosis Dynamin C607 Promotes endocytosis

21 Abnormal S-nitrosylation in disease Cancer: S-nitrosylation of Ras proteins is involved in tumor maintenance Lim KH et al. Nature (2008) Neurodegeneration: S-nitrosylation of protein disulfide isomerase links protein misfolding to neurodegeneration Uehara T et al. Nature (2006) Muscle disorder: S-nitrosylation of RyR1 underlies environmental heat stroke and sudden death Durham WJ et al. Cell (2008) sickle cell disease, pulmonary hypertension, cystic fibrosis, asthma, heart failure

22 Protein S-nitrosylation: General principles and salient features 1. NO targets one or few Cys S-nitrosylation motifs: Acid-base motif : (E,D/K,R,H) -C- (E,D/K,R,H) Hydrophobic motif : local hydrophobicity might promote specific S-nitrosylation 2. Effect on function Protein activity, subcellular location, protein-protein interaction, protein stability 3. Reversible and regulated Nitrosylation is a reversible covalent modification; Nitrosylation & denitrosylation are stimulus (receptor)-coupled, operating on physiological time scales

23 Compartmentalized S-nitrosylation Martínez-Ruiz A. et al., Antioxid Redox Signal. (2013)

24 Transnitrosylation between proteins Stamler JS and Hess DT. Nat. Cell Biol. (2010)

25 Mechanisms of NO-based signaling

26 Denitrosylation mechanisms SNO GSH Protein Trx (SH) 2 NADP + NAD + NADH TrxR GSNOR GSNHOH GSNO SH Protein NO / NO - Trx S 2 NADPH Other denitrosylases have been characterized in vitro and/or in cells including: TRP14, protein disulfide isomerase, and carbonyl reductase.

27 Regulation of SNO homeostasis by S-nitrosoglutathione reductase (GSNOR) Foster MW et al., Trends Mol. Med. (2009)

28 Denitrosylation mechanisms SNO GSH Protein Trx (SH) 2 NADP + NAD + NADH TrxR GSNOR GSNHOH GSNO SH Protein NO / NO - Trx S 2 NADPH Nikitovic, D. & Holmgren, A. J. Biol. Chem. 271 (1996) Stoyanovsky, DA. et al., JACS (2005) Benhar, M. et al., Science (2008). Pader, I. et al., PNAS (2014)

29 S-nitrosylation of caspases Caspases: a family of cysteine proteases and central components of the cell death program / apoptosis Nitric oxide inhibits caspase activity through S-nitrosylation of the active site cysteine Caspase-3 is S-nitrosylated in vivo by endogenously produced NO (lymphocytes, endothelial cells, ) Caspase-3 Caspase-6 Caspase-7 Caspase-8 Caspase-9 Caspase-10 Caspase-2 --KLFIIQACRGTELD---KIFIIQACRGNQHD---KLFFIQACRGTELD---KVFFIQACQGDNYQ---KLFFIQACGGEQKD---KLFFIQACQGEEIQ---KMFFIQACRGDETD--

30 Regulation of apoptosis by reversible S-nitrosylation Benhar M. et al., Nat. Rev. Mol. Cell Biol (2009)

31 Mechanisms and consequences of reversible S-nitrosylation NO/SNO can affect the Trx/TrxR system Disease Benhar M. et al., (2009)

32 Trx Reduction of disulfides (RS-SR) Reduction RSNO (denitrosylation) dntp s (ribonucleotide Reductase) Peroxiredoxins MsrA, MsrB NFkB, Fos, Jun Ref-1, p53 ASK-1 phosphatases? DNA synthesis or repair Antioxidant defense Gene regulation Cell signaling? Cell growth, survival, death Major research goal: Identification of S-nitrosylated targets of thioredoxin

33 Benhar M. et al., (2009) Studying S-nitrosylation in the macrophage: Relevance to host microbe interaction

34 SNO-protein detection using the biotin-switch method Jaffrey SR et al. Nature Cell Biol. 2001

35 Identification of Trx substrates using the biotin switch assay coupled to quantitative proteomics Benhar M. et al., Biochemistry (2010)

36 Global/proteomic profiling of S-nitrosylation Proteomic profiling of S-nitrosylation remain challenging using current methods (i.e., biotin switch assay), particularly under physiologicallyrelevant conditions (due to its chemical lability, low stoichiometry) Substrate trapping by Trx as a novel approach to identify proteins regulated by reversible nitrosylation

37 Mechanism of Trx-mediated protein denitrosylation Substrate Substrate Substrate SNO NO S SH SH SH S SH S S cys32 Trx cys35 cys32 Trx cys35 cys32 Trx cys35 Thioredoxin reductase NADPH

38 Trapping nitrosylated targets of thioredoxin Cell treatment (NO donor or LPS/IFN- ) Cell lysis with digitonin SH SNO SH x Trx S S x Trx + NO / HNO Pull-down of Trx-target adducts Release of targets by DTT SDS-PAGE LC-MS/MS

39 Trx(C35S)-dependent SNO protein trapping from THP-1 cells

40 Trx(C35S)-dependent SNO protein trapping from RAW264.7 macrophages LPS TLR4 NF- B MAPK inos SNO S x Trx LC-MS/MS

41 Exemplary proteins identified by mass spectrometry THP-1 cells (CysNO) RAW264.7 cells (LPS/IFN-g) ~ 600 proteins ~ 400 proteins Caspase-3 HDAC1 and 2 STAT6 PARP-1 Gpx-1 CDK-5 STAT3 PRMT1 PA28b MEK1 GAPDH Elongation factor 2 Annexin A1,2,4,7, proteins SHIP1 and 2 DUS3 Cofilin HSP105 Caspase-1 STAT1 Hexokinase 1-3 PCNA NFAT NF-kB Enrichment of low abundance proteins, signaling proteins

42 Regulation of MEK1 S-nitrostylation HEK cells SNO Inactive MEK In vitro kinase assay

43 Nitrosothiol trapping by thioredoxin as a novel approach to explore the nitrosoproteome MEK1, STAT3 Transcription Stress response,

44 Summary NO is involved in a broad spectrum of physiological and pathological processes The cellular effects of NO are mediated by both cgmp and S-nitrosylation NOS and denitrosylases (GSNOR, Trx) govern S-nitrosylation and thereby regulate protein and cell function Abnormal levels of protein S-nitrosylation, which affect various SNO proteins, have been associated with diverse disorders Further research is needed to: (i) Characterize the SNO proteome of relevant healthy and diseased cells and tissues (ii) Establish a casual relationship between aberrant S-nitrosylation and specific pathological conditions (iii) Determine the relative contribution of aberrant nitrosylation vs. denitrosylation in cellular dysfunction and disease

45 Acknowledgements Technion Shani Ben-Lulu Rotem Engelman Pnina Shomer, Ph.D. Arie Admon, Ph.D. Tamar Ziv, Ph.D. Karolinska Institutet Elias S.J. Arnér, M.D., Ph.D. Jianqiang Xu Irina Pader Technion Medical School / Haifa Funding Israel Science Foundation Israel Cancer Association Israel Cancer research Fund European Commission