Download Oxidative Stress Concepts

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.
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
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Toren Finkel* & Nikki J. Holbrook. (2000) Nature 408, 239-247.
ROS sources for bacteria
environmental
Competing microbes
(pyruvate oxidase)
NADPH oxidase
(phagosome)
antibiotics
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Imlay, Ann. Rev. Biochem. 2008, 77:755-776.
ROS sources in mammalian cells
• Mitochondrial respiration
• Byproducts of enzyme activity (flavin enzymes, xanthine
oxidase)
NADPH
• Nitric Oxide Synthase
L-Arg
NOS
O2•-
•NO
NADP+
ONOO- (peroxynitrite)
• Peroxisomal and endoplasmic reticulum processes
• NADPH oxidases (NOXs)
• Environmental-UV radiation, redox cycling (P450)
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Sites of mitochondrial ROS formation
ROS-site
Complex I flavin
Complex I QH
Complex III QH
ETF QH
Km (O2) uM
0.2
0.9
2.0
5.0
Hoffman and Brookes, JBC, 284, pp. 16236–16245, 2009.
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ROS generation by NADPH Oxidases
Neutrophil phagosome
(contain flavin and cytochrome b)
Receptor mediated (smooth muscle cells)
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Winterbourn, NATURE CHEMICAL BIOLOGY, 4, 278-286, 2008.
Estimated diffusion distances of ROS
2 mM GSH present
GSH reaction rate constants (M-1 s-1)
H2O2
0.9
ONOO- 700
HOCl
3x107
•NO2
3x107
•HO
1x1010
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Winterbourn, NATURE CHEMICAL BIOLOGY, 4, 278-286, 2008.
Targets of ROS
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Toledano, Nat Rev Mol Cell Biol, 8:813-824, 2007.
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)
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Protein modifications by ROS
Stadtman, Chapter 5, Redox Biochemistry
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Frequency of modification by ROS
Regnier, Journal of Proteome Research 2006, 5, 2159-2168
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Cellular distribution of ROS damaged
proteins
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Regnier, Journal of Proteome Research 2006, 5, 2159-2168
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-SO2H)
Methionine sulfoxide reductases
-Detoxify
(xenobiotics)
Glutathione-S-transferase, Cytochrome P450 enzymes
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Defenses against ROS
Beal, Nature. 2006 Oct 19;443(7113):787-95.
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Is Peroxiredoxin 3 the major H2O2
scavenger in mitochondria?
(60 uM)
(2 uM)
(20 uM)
(2 uM)
(1.4 nM)
(5 mM)
k (T)=k(target) * [target]
Hampton, Biochem. J. (2010) 425, 313–325
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Catalytic Cycles of Prx
Prx3
Km (H2O2)
kcat
kcat/Km
Prx5
Prx
lower (uM)
lower (1-80 s-1)
104-107 M-1s-1
vs
Cat
higher (mM)
higher (104 s-1)
106-107 M-1s-1
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Hampton, Biochem. J. (2010) 425, 313–325
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
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Toledano, Nat Rev Mol Cell Biol, 8:813-824, 2007.
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 then
accumulates 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
ROS
Change
intracellular
location
Keap1/Nrf2
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).
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Finkel, Circ. Res. 2005;97;967-974
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?
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