oxidative stress concepts
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Oxidative Stress Concepts. Graduate Course 2214/938 (KI/UNL) June 14, 2010. Donald Becker Redox Biology Center University of Nebraska. Disease and Aging. Rate of living hypothesis- states metabolic rate of species determines its life-time. - PowerPoint PPT PresentationTRANSCRIPT
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?