Protein carbonylation: a marker of oxidative stress damage - - PowerPoint PPT Presentation

Protein carbonylation: a marker of oxidative stress damage

ITN-TREATMENT Metabolic Dysfunctions associated with Pharmacological Treatment of Schizophrenia TREATMENT

Protein carbonyl formation: Metal-Catalyzed Oxidation

Physiological consequences can be inferred due to the specificity

• f protein functions

Products (damage) are relatively stable Sensitive assays are available

Why using proteins as markers of oxidative stress?

Carbonylation: Irreversible. Associated with protein dysfunction

H2O H2O

Fenton Reaction

• E. Stadtman.

Free Rad. Biol. Med. 1990

Hydroxyl radical

Lys

Oxidative damage to proteins and disease

• some examples-

 Neurodegenerative diseases

• Alzheimer
• Parkinson
• Sporadic amyotrophic lateral sclerosis
• Friedreich ataxia

 Muscular dystrophy  Iron disorders  Aging  Progeria  Atherosclerosis  Ischemia-reperfusion injury  Acute pancreatitis  Chronic ethanol ingestion

Oxidative damage to proteins: importance beyond the original cause of the disease: worsening cell functions

O CH CH

Anti-DNP Antibody

CH

*OH

CH2 NH2

Fenton reaction

Carbonyl group

Western blot DNPH

DNP DNP

O CH CH

BODIPY-HZ

BODIPY-hydrazide derivatization

Protein carbonyl detection

Bodipy HZ

H2O2

Protein-Flamingo

Tamarit et al. J. Proteomics. 75:3778-3788

MDMA

3,4-Methylenedioxymethamphetamine (MDMA) in mice brain

Identification of carbonylated proteins

Ros-Simó C, et al. J Neurochem. 25(5):736-46

Carbonylated proteins identified in hippocampus of MDMA treated mice

Control

Control MDMA

5 8 pI pI 5 8

Protein stain Carbonyl detection

Cell models of Friedreich ataxia (FA)

Protein Gene Oxidation fold

Heat shock protein mitochondrial SSC1 3.16 Heat shock protein mitochondrial HSP78 15.3 Heat shock protein cytosolic SSE1 7.1 F1FO ATP synthase a subunit ATP1 8.5 F1FO ATP synthase b subunit ATP2 4.7 Acetohydroxiacid reductoisomerase ILV5 9.6 Pyruvate kinase 1 CDC19 3.6 3-phosphoglycerate kinase PGK1 2.2 Adenylate kinase ADK1 3.3 Actin, a chain ACT1 3.4 Elongation factor EF-1a TEF2 7.1 Catalase A CTA1 8.1 Peroxiredoxin thiol specific AHP1 3.7 Superoxide dismutase 1 SOD1 2.9 57% 43% Cytosolic Mitochondrial 6 7 10 11 9 6 7 9 10 11 6 7 10 11 9 6 10 11 9 7

Protein Carbonyl detection

Protein stain

Identification of carbonylated proteins

Control FA Control FA

Protein Gene Oxidation fold

ATPase ER VCP >9 Heat shock protein HSC71 7 Creatine kinase mitochondrial CK-MT1 7,5 F1FO ATP synthase, subunit a ATP5A 2,5 Citrate synthase CS 7,5 Pyruvate kinase 1 PKM2 6 Pyridoxal kinase PDXK 3,5 Cytochrome b-c1, sub. 2 UQCRC2 7,5 Aminoadípic semialdehyde DH ALDH7A1 3,5 Enolase ENO1 2,5 Glyceraldehyde-3-P-DH GAPDH 2,5

Striatum from Huntington Disease patients

(“post mortem”)

Control HD Identification of carbonylated proteins

Sorolla MA, et al. Free Radic Biol Med. 49:612-621.

Protein stain Carbonyl detection

Bodipy signal

CARBONYLATED PROTEINS Mass (Da) SSA2, Heat shock protein 71 69599 HXKA, Hexokinase-1 53738 MLS1, Malate synthase 1, glyoxysomal 62791 PYK1, Pyruvate kinase 1 54544 ALDH4, Potassium-activated aldehyde DH, Mito 56973 PGK, Phosphoglycerate kinase 44738 EF1A, Elongation factor 1-alpha 50032 TAL1, Transaldolase 37036 G3P3, Glyceraldehyde-3-phosphate DH 3 35838 IDH2, Isocitrate DH subunit 2, mitochondrial 39886 ADH1, Alcohol DH 1 36849 ADH2, Alcohol DH 2 37165 VDAC1, Mitochondrial outer membrane porin 30524

Yeast Old Young

Identification of carbonylated proteins.

Tamarit et al. J. Proteomics. 75:3778-3788

Aging: Old vs young yeast cells

Miscellaneous Glucose Metabolism Pyruvate DH and TCA Cycle Electron Transport Chain and ATP synthesis Plant Metabolism: Photosynthesis and Seed Metabolism Amino Acid and Protein Metabolism Lipid Metabolism Antioxidant Defense Systems Plasma Proteins Membrane Transport Receptors and Cell Signaling Cytoskeleton Heat Shock Proteins / Chaperones

Protein carbonylation in aging (from several models)

Cabiscol E; Tamarit J; Ros J. (2013). Protein carbonylation: proteomics, specificity and relevance to aging. Mass Spectrometry Reviews. 33:21-48

Why this oxidative damage specifically targets certain proteins?

Amount? Protein stain Protein carbonyl

• Ex. 1

Frataxin mutants

• Ex. 2

Yeast aging

RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE?

Cytoplasm Secreted

Mitochondria

Endoplasmic Reticulum Cell Membrane Chloroplast Others

Cellular compartment

Subcellular location of carbonylated proteins. Proteins from pathways or functions with fewer than five members were grouped as “Others”. Each group includes proteins with two or more possible locations.

LOCATION

RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE?

Sequences Prone to Carbonylation

Most of the sites—approximately 75% — were grouped in the regions containing sequences rich in the amino acids Arg (R), Lys (K), Pro (P), and Thr (T) (i) the impact in these sites with RKPT-rich sequences was four times greater than in other regions Among the 21 classes of assigned functions, proteins involved in translation and ribosomal structure showed the highest percentage of carbonylatable sites when compared to the mean value of the whole E. coli proteome. This also applies to proteins involved in energy production or in nucleotide transport. Maisonneuve E et al. 2009. PLoS One.4(10) RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE?

Metals: Transition metals such as Iron or Copper

• Fenton chemistry-

Nucleotide-Binding Proteins

Protein Gene Oxidation fold Heat shock protein mitochondrial SSC1 3.16 Molecular chaperone mitochondrial HSP78 15.3 Heat shock protein cytosolic SSE1 7.1 F1FO ATP synthase a subunit ATP1 8.5 F1FO ATP synthase b subunit ATP2 4.7 Acetohydroxiacid reductoisomerase ILV5 9.6 Pyruvate kinase 1 CDC19 3.6 3-phosphoglycerate kinase PGK1 2.2 Adenylate kinase ADK1 3.3 Actin, a chain ACT1 3.4 Elongation factor EF-1a TEF2 7.1 Catalase A CTA1 8.1 Peroxiredoxin thiol specific AHP1 3.7 Superoxide dismutase 1 SOD1 2.9 Protein Gene Oxidation fold Heat shock protein mitochondrial SSC1 3.16 Molecular chaperone mitochondrial HSP78 15.3 Heat shock protein cytosolic SSE1 7.1 F1FO ATP synthase a subunit ATP1 8.5 F1FO ATP synthase b subunit ATP2 4.7 Acetohydroxiacid reductoisomerase ILV5 9.6 Pyruvate kinase 1 CDC19 3.6 3-phosphoglycerate kinase PGK1 2.2 Adenylate kinase ADK1 3.3 Actin, a chain ACT1 3.4 Elongation factor EF-1a TEF2 7.1 Catalase A CTA1 8.1 Peroxiredoxin thiol specific AHP1 3.7 Superoxide dismutase 1 SOD1 2.9

Frataxin-deficient cells

RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE?

HSP 78 mitochondrial HSP 70 cytosolic F1FO ATP synthase a subunit F1FO ATP synthase b subunit Pyruvate kinase 3-phosphoglycerate kinase Acetohydroxyacid redcutoisomerase Adenylate kinase Actin, a chain Elongation factor EF-1a GAPDH-3 SOD 1 Catalase A Peroxiredoxin 1 HSP 72 Hexokinase-1 Pyruvate kinase 1 Aldehyde DH, mitochondrial Phosphoglycerate kinase Elongation factor 1-alpha GAPDH 3 Isocitrate DH, mitochondrial Alcohol dehydrogenase 1 Alcohol dehydrogenase 2 VDAC1, Mitochondrial OMP ATPase ER HSP 70 CK mitochondrial F1FO ATP synthase, a Pyruvate kinase 1 Pyridoxal kinase Citrate synthase Pyruvate kinase ILV5, mitochondrial Heat shock protein 75 Heat shock protein 60 Phosphoglycerate kinase Pyruvate DH a-Ketoglutarate DH Enolase 2 GAPDH3 DNA K Elongation factor G Alcohol DH E F1F0-ATP synthase b GAPDH OMP A Enolase

Frataxin depletion Chronological aging – yeast- Hydrogen peroxide stress –yeast- Huntington disease Hydrogen peroxide stress –bacteria-

Heat shock cognate 71 Heat shock cognate 71 Dihyropyrimidinase-related protein-2 alpha-internexin ATP synthase subunit b alpha-enolase Actin Aconitase ATP synthase subunit a Synapsin-1

MDMA treatment

DNA K (HSP70) Elongation factor G Alcohol DH E F1F0-ATP synthase b Pyruvate kinase ILV5, mitochondrial HSP 75 HSP 60 Phosphoglycerate kinase ATPase ER HSP 70 CK mitochondrial F1FO ATP synthase, a Pyruvate kinase 1 Pyridoxal kinase HSP 72 Hexokinase-1 Pyruvate kinase 1 Phosphoglycerate kinase Elongation factor 1-alpha Isocitrate DH subunit 2, mitochondrial VDAC1, Mitochondrial OMP HSP 78 mitochondrial HSP 70 cytosolic F1FO ATP synthase a subunit F1FO ATP synthase b subunit Acetohydroxiacid reductoisomerase Pyruvate kinase 1 3-phosphoglycerate kinase Adenylate kinase Actin, a chain Elongation factor EF-1a

Frataxin depletion Aging – yeast- Yeast Huntington disease

GAPDH OMP A Enolase Pyruvate DH a-Ketoglutarate DH Enolase 2 GAPDH3 Citrate synthase GAPDH 3 Isocitrate DH mt Alcohol DH 1 Alcohol DH 2 GAPDH-3 SOD 1 Catalase A Peroxiredoxin

Bacteria

Hydrogen peroxide Hydrogen peroxide

Heat shock cognate 71 Heat shock cognate 71 Dihyropyrimidinase-related protein-2 ATP synthase subunit b Actin ATP synthase subunit a

Nucleotide binding proteins

alpha-enolase alpha-internexin Synapsin-1

MDMA treatment

Open question: Is this mechanism designed to stop ATP consumption?

 Of the total entries in the Uniprot database, 21% are classified as NB-proteins  Among the targets identified 60% of carbonylated proteins were NB-proteins

DNA K (HSP 70)

Ascorbate + Iron + O2

• ATP

+ATP 0 15’ 30’ 15’ 30’ Protein Carbonylation

Concluding remark Whether this has evolved to preserve cellular integrity or it is an inevitable consequence of

• xidative stress is still an open question…

The presence of the nucleotide would act as a metal chelator promoting the damage observed as follows

H2O2

Mg

Fe2+

OH

• Fe-proteins

Fe3+ Apoproteins

O-

2

NTPs bound to proteins could explain the specificity/selectivity observed ATP-Mg ATP-Fe2+ ATP-Fe3+

O CH O CH Fenton chemistry

Fabien Delaspre Elena Britti Marta Llovera Rosa Purroy Jordi Tamarit David Alsina Anna Molet

DRG neurons Cardiomyocytes Yeasts