1. Oxidative Stress Concepts Donald Becker Redox Biology Center University of Nebraska Graduate Course 2214/938 (KI/UNL) June 14, 2010

2. 2 Disease and Aging Rate of living hypothesis- states metabolic rate of species determines its life-time. 1950’s Dr. Harman (University of Nebraska Medical School) speculated the “free radical” theory of ageing results in a pattern of cumulative damage. Free radicals involving oxygen have been renamed as reactive oxygen species (ROS) and encompass a variety of diverse chemical species including superoxide anions, hydroxyl radicals, and hydrogen peroxide.

3. 3 Outline 1.Sources ROS (environmental, metabolic, immune system) 2.Damage that ROS causes 3.Defenses against ROS (enzymes, small molecules, reaction rates) 4. Mechanisms of stress response

4. 4 Overview of ROS Toren Finkel* & Nikki J. Holbrook. (2000) Nature 408, 239-247.

5. 5 ROS sources for bacteria environmental Imlay, Ann. Rev. Biochem. 2008, 77:755-776. NADPH oxidase (phagosome) antibiotics Competing microbes (pyruvate oxidase)

6. 6 ROS sources in mammalian cells Mitochondrial respiration Byproducts of enzyme activity (flavin enzymes, xanthine oxidase) Nitric Oxide Synthase Peroxisomal and endoplasmic reticulum processes NADPH oxidases (NOXs) Environmental-UV radiation, redox cycling (P450) NOS NADPH NADP+ L-Arg NO O 2 - ONOO - (peroxynitrite)

7. 7 Hoffman and Brookes, JBC, 284, pp. 16236–16245, 2009. ROS-site K m (O 2 ) uM Complex I flavin 0.2 Complex I QH 0.9 Complex III QH 2.0 ETF QH 5.0 Sites of mitochondrial ROS formation

8. 8 ROS generation by NADPH Oxidases (contain flavin and cytochrome b) Neutrophil phagosomeReceptor mediated (smooth muscle cells) Winterbourn, NATURE CHEMICAL BIOLOGY, 4, 278-286, 2008.

9. 9 Estimated diffusion distances of ROS Winterbourn, NATURE CHEMICAL BIOLOGY, 4, 278-286, 2008. GSH reaction rate constants (M -1 s -1 ) H 2 O 2 0.9 ONOO- 700 HOCl 3x10 7 NO 2 3x10 7 HO 1x10 10 2 mM GSH present

10. 10 Targets of ROS Toledano, Nat Rev Mol Cell Biol, 8:813-824, 2007.

11. 11 DNA Damage 7,8-Dihydro-8-oxo-2’-deoxyguanosine (OG) arises from oxidation of guanine. Guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) arise either directly from oxidation of guanine or from further oxidation of OG. Thymine glycol (Tg) arises from oxidation of thymine. All of these base lesions are repaired by the base excision repair pathway. (David, Chap.3, Redox Biochemistry)

12. 12 Protein modifications by ROS Stadtman, Chapter 5, Redox Biochemistry

13. 13 Regnier, Journal of Proteome Research 2006, 5, 2159-2168 Frequency of modification by ROS

14. 14 Cellular distribution of ROS damaged proteins Regnier, Journal of Proteome Research 2006, 5, 2159-2168

15. 15 Protection against ROS -Metal sequestration & efflux Ferritin - Small molecules glutathione, ascorbic acid, beta-carotene, tocopherol, flavonoids - Scavenge reactive oxygen species Antioxidant enzymes- catalase (Cat), superoxide dismutase (SOD), peroxiredoxins (Prx), alkyl hydroperoxide reductases (Ahp), thioredoxin (Trx), thioredoxin reductase (TrxR), glutathione reductase (GR), glutathione peroxidase (Gpx), glutaredoxin (Grx) -DNA and protein damage repair enzymes Base excision repair enzymes (glycosylase) Sulfiredoxins (reduces cysteine sulfinic acid, R-SO 2 H) Methionine sulfoxide reductases - Detoxify (xenobiotics) Glutathione-S-transferase, Cytochrome P450 enzymes

16. 16 Beal, Nature. 2006 Oct 19;443(7113):787-95. Defenses against ROS

17. 17 Hampton, Biochem. J. (2010) 425, 313–325 Is Peroxiredoxin 3 the major H 2 O 2 scavenger in mitochondria? k (T)=k(target) * [target] (60 uM) (2 uM) (20 uM) (2 uM) (1.4 nM) (5 mM)

18. 18 Catalytic Cycles of Prx Hampton, Biochem. J. (2010) 425, 313–325 Prx3Prx5 Prxvs Cat K m (H 2 O 2 )lower (uM)higher (mM) k cat lower (1-80 s -1 )higher (10 4 s -1 ) k cat /K m 10 4 -10 7 M -1 s -1 10 6 -10 7 M -1 s -1

19. 19 Structural comparison of Prx

20. 20 Stress sensing

21. 21 Unique roles of H 2 O 2 and Cysteine in ROS signaling Oxidation of Cys residues as the basis for peroxide signaling Toledano, Nat Rev Mol Cell Biol, 8:813-824, 2007.

22. 22 Bacterial sensors PerR

23. 23 SoxR system

24. 24 OxyR system

25. 25 Yap1 Sensor in yeast With increases in H 2 O 2, Gpx3 catalyzes the oxidation of cysteine residues in Yap1 resulting in disulfide bond. Oxidized Yap1 then accumulates in the nucleus where it activates the transcription of antioxidant genes. Finkel, Circ. Res. 2005;97;967-974

26. 26 Sensors in mammalians Keap1 Under normal conditions, Keap1 (cytosolic) interacts with Nrf2 (a transcription factor) to keep it sequestered in the cytosol. This interaction also helps target Nrf2 for proteasomal degradation. With increases in ROS, cysteine residues in Keap1 are oxidized which leads to disulfide bond formation, zinc release, and a conformational change. As a result, Nrf2 is released from Keap1 and enters the nucleus. Thus, the oxidation of Keap1 triggers Nrf2 to accumulate in the nucleus and activate antioxidant functions in the cell (antioxidant responsive elements). Finkel, Circ. Res. 2005;97;967-974 ROS Change intracellular location Keap1/Nrf2

27. 27 H 2 O 2 signaling Chapter 4, Redox Biochemistry Book

28. 28 Nitric Oxide

29. 29 CO is an important regulator of hypoxic sensing by the carotid body

30. 30 Summary ROS sources involve metabolism, environmental factors, and immune response ROS (hydroxyl radicals) induces damage to a variety of biomolecules A robust antioxidant system helps maintain proper ROS levels (peroxiredoxin) Specific sensors for ROS that turn on transcriptional responses have cysteine and/or metal based centers Reactivity of cysteine residues are tuned by the protein Hydrogen peroxide is an important ROS signaling molecule

31. 31 Are antioxidants effective in human health and disease?