Oxidative Stress and Repair

• Oxidative DNA modifications Faiez Al Nimer• Oxidative lipid modifications Huang Liyue• Oxidative protein modifications Sanyal Nikhilesh• Oxidative DNA and protein damage repair Melanie Neely Willis

Oxidative DNA damage

Reactive oxygen species

Strand breaks

Oxidized baseadducts

DNA-DNA andDNA-protein crosslinks

Both nuclear and mitochondrial DNA is attacked by ROS but mtDNA is affected more because it does not contain histonesit is in proximity to ROS generation and has limited antioxidantrepair system

Oxidized base adducts: Hydroxyl radical

Evans et al, Reviews in mutation research, 2004

8-OH-Gua and Thymin glycolGuanine: lowest oxidation potential most oxidized base

Cell viability and disease

For 8-OH-Gua: Results in C→A and G→T transversions, leading to mutations Affects transcription factor binding and gene expression Interferes with methylation of i.e. oncogenes

For thymin glycol: T→C transitions have been noted Blocks replication

DNA oxidation: Neurodegenerative diseases

8-OHDG staining p62 promoter oxidation

Lovell et al, J Neurochem, 1999Du et al, Neurobiol of disease,2009

DNA oxidation: Neurodegenerative diseases

Absence of p62 induces cell death

p62 protects cells from oxidative stress via the antioxidant response element (ARE)

Du et al, Neurobiol of disease,2009

p62 expression

Oxidative Stress and Repair

• Oxidative DNA modifications Faiez Al Nimer• Oxidative lipid modifications Huang Liyue• Oxidative protein modifications Sanyal Nikhilesh• Oxidative DNA and protein damage repair Melanie Neely Willis

Lipid modifications by reactive oxygen species (ROS)

• Overproduction of ROS damages cellular components, including lipids, leading to

decline in physiological function and cell death

Reaction of ROS with lipids lead to

• Lipid oxidation

• Lipid peroxidation

Lipid Oxidation

• Initiation– A radical is formed

• Propagation– Radicals react and transfer their unpaired electron

to other compounds

• Termination– Two radicals combine to stop the reaction

Adibhatla RM; Hatcher JF. Antioxid Redox Signal. 2010

Lipid peroxidation

LHOH or M=O

L

L + O2 LOO

LOO + LH L + LOOH

LOOH + metal

Initiation

Propagation

LOLOO

Hydroperoxide

Peroxy radical

Alkoxy radical

AlkanesMalondialdehyde4 hydroxynananol8-isoprostanes

Commonly measured decomposition products

Adibhatla RM; Hatcher JF. Antioxid Redox Signal. 2010

Oxidative Stress and Repair

• Oxidative DNA modifications Faiez Al Nimer• Oxidative lipid modifications Huang Liyue• Oxidative protein modifications Sanyal Nikhilesh• Oxidative DNA and protein damage repair Melanie Neely Willis

Effects of Reactive oxygen species on Protein backbone

• Oxidation of proteins often targets them for degradation (20S proteasome and other proteases).

• Several age-related disorders are characterised by Protein oxidation. E.g. Parkinson’s disease, Amyotropic Lateral sclerosis.

• Product of the oxidation (Peptide alkoxyl radical) is susceptible to cleavage by diamide or -amidation pathways.

Redox Biochemistry, Wiley Interscience, 2008; p186

Oxidation of amino acid residue side chain

Redox Biochemistry, Wiley Interscience, 2008; p188

Oxidation of amino acid residue side chains : Tyrosine

Larios J M et al. J. Biol. Chem. 2001;276:17437-17441

Qun Gao C et al. Cardiovasc Res 2003;57:426-433

• Tyrosine oxidation can lead to dityrosine formation.

• This leads to protein crosslinking.

• Useful Biomarker for Oxidative damage and aging.

• Increased levels of dityrosine is found in rat hearts where Matrix metalloproteinase-2 (MMP-2) was induced by cytokines.

Tyrosine Dityrosine

Oxidation of amino acid residue side chains : Cysteine

Green J and Paget MS (2004) Nat Rev Microbiol. 2(12):954-66

R’ - Glutathione or another protein

Sulfonic acid

Sulfinic acid

Sulfenic acid

Beneficial effects of ROS on Proteins : e.g. DJ-1

H2O2 oxidises the Cys106 (-SH)to sulfinic acid (-SOOH)

Blackinton et al et al. J. Biol. Chem. 2009;284:6476-6485

Oxidised Cys106Partially oxidised Cys106 (Sulfenic acid)

Cys106 oxidation is controlled by its neighbouring environment.

Beneficial effects of ROS on Proteins : e.g. DJ-1

Cys106 is essential for maintaining nuclear morphology. Shown here are M17neuroblastoma cells

Cys106 helps increase Cell viability under mitochondrial stress

Blackinton et al et al. J. Biol. Chem. 2009;284:6476-6485

Oxidative Stress and Repair

• Oxidative DNA modifications Faiez Al Nimer• Oxidative lipid modifications Huang Liyue• Oxidative protein modifications Sanyal Nikhilesh• Oxidative DNA and protein damage repair Melanie Neely Willis

DNA & Protein Repair Enzymes

Oxidation of pyrimidine and purine bases leads to DNA damage.

Oxidation of methionine and cysteine residues can lead to protein damage and aggregation.

DNA damage is repaired by several enzymes in the Base Excision Repair pathway (BER).

Proteins are repaired by methionine sulfoxide reductases (MsrA, MsrB) and sulfiredoxins.

Base Excision Repair1. Removal of the damaged

base from the sugar by a damage-specific glycosylase

2. Processing of the apyrmidinic-apurinic (AP) site by an AP endonuclease and deoxyribosylphosphate lyase.

3. Replacement of an undamaged nucleotide by DNA polymerase.

4. Repairing nicks in the DNA backbone by DNA ligase.

Crystal Structure Shows DNA Bending

Lukianova OA, David SS. A role for iron-sulfur clusters in DNA repair. Curr Opin Chem Biol. 2005;9:145–151.

Reversibility of Prx HyperoxidationBefore exposure

After 10 minutes exposure to H202

Recovery for 4 hours

10 more minutes of exposure to H202

Recovery for 8 hours

Woo HA, Chae HZ, Hwang SC, Yang KS, Kang SW, Kim K, Rhee SG.Science. 2003 Apr 25;300(5619):592-4.

Mechanism of reduction of Prx-SO2− by Srx.

Jönsson T J et al. J. Biol. Chem. 2009;284:33305-33310