Oxidative Stress Concepts

Oxidative Stress Concepts

Donald BeckerRedox Biology CenterUniversity of Nebraska

Graduate Course 2214/938 (KI/UNL)

June 14, 2010

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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.

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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

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Overview of ROS

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

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ROS sources for bacteriaenvironmental

Imlay, Ann. Rev. Biochem. 2008, 77:755-776.

NADPH oxidase(phagosome)

antibiotics

Competing microbes(pyruvate oxidase)

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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

•NOO2•-

ONOO- (peroxynitrite)

7Hoffman and Brookes, JBC, 284, pp. 16236–16245, 2009.

ROS-site Km (O2) uMComplex I flavin 0.2Complex I QH 0.9Complex III QH 2.0ETF QH 5.0

Sites of mitochondrial ROS formation

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ROS generation by NADPH Oxidases

(contain flavin and cytochrome b)

Neutrophil phagosome Receptor mediated (smooth muscle cells)

Winterbourn, NATURE CHEMICAL BIOLOGY, 4, 278-286, 2008.

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Estimated diffusion distances of ROS

Winterbourn, NATURE CHEMICAL BIOLOGY, 4, 278-286, 2008.

GSH reaction rate constants (M-1 s-1) H2O2 0.9

ONOO- 700HOCl 3x107

•NO2 3x107 •HO 1x1010

2 mM GSH present

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Targets of ROS

Toledano, Nat Rev Mol Cell Biol, 8:813-824, 2007.

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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 fromfurther oxidation of OG. Thymine glycol (Tg) arises from oxidation of thymine. All ofthese base lesions are repaired by the base excision repair pathway. (David, Chap.3, Redox Biochemistry)

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Protein modifications by ROS

Stadtman, Chapter 5, Redox Biochemistry

13Regnier, Journal of Proteome Research 2006, 5, 2159-2168

Frequency of modification by ROS

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Cellular distribution of ROS damaged proteins

Regnier, Journal of Proteome Research 2006, 5, 2159-2168

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Protection against ROS

-Metal sequestration & effluxFerritin

- 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-SO2H)

Methionine sulfoxide reductases

-Detoxify (xenobiotics)Glutathione-S-transferase, Cytochrome P450 enzymes

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

Defenses against ROS

17Hampton, Biochem. J. (2010) 425, 313–325

Is Peroxiredoxin 3 the major H2O2 scavenger in mitochondria?

k (T)=k(target) * [target]

(60 uM) (2 uM)

(20 uM) (2 uM)

(1.4 nM)

(5 mM)

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Catalytic Cycles of Prx

Hampton, Biochem. J. (2010) 425, 313–325

Prx3 Prx5

Prx vs CatKm (H2O2) lower (uM) higher (mM)kcat lower (1-80 s-1) higher (104 s-1)kcat/Km 104-107 M-1s-1 106-107 M-1s-1

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Structural comparison of Prx

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Stress sensing

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Unique roles of H2O2 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.

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Bacterial sensors

PerR

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SoxR system

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OxyR system

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Yap1 Sensor in yeast

With increases in H2O2, Gpx3 catalyzes the oxidation of cysteine residues in Yap1 resulting in disulfide bond. Oxidized Yap1 thenaccumulates in the nucleus where it activates the transcription of antioxidant genes. Finkel, Circ. Res. 2005;97;967-974

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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

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H2O2 signaling

Chapter 4, Redox Biochemistry Book

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Nitric Oxide

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

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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

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Are antioxidants effective in human health and disease?