Protein carbonylation: a marker of oxidative stress damage

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1 Protein carbonylation: a marker of oxidative stress damage ITN-TREATMENT Metabolic Dysfunctions associated with Pharmacological Treatment of Schizophrenia TREATMENT

Associated with protein dysfunction Why using proteins as markers of oxidative stress?

2 Protein carbonyl formation: Metal-Catalyzed Oxidation Lys Fenton Reaction Hydroxyl radical H 2 O H 2 O E. Stadtman. Free Rad. Biol. Med Carbonylation: Irreversible. Associated with protein dysfunction Why using proteins as markers of oxidative stress? Physiological consequences can be inferred due to the specificity of protein functions Products (damage) are relatively stable Sensitive assays are available

3 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

4 Protein carbonyl detection Fenton reaction DNPH Anti-DNP Antibody *OH NH 2 O DNP DNP CH 2 CH Carbonyl group CH CH Western blot BODIPY-hydrazide derivatization Tamarit et al. J. Proteomics. 75: O BODIPY-HZ CH CH H 2 O 2 Protein-Flamingo Bodipy HZ

proteins identified in hippocampus of MDMA

5 Identification of carbonylated proteins 3,4-Methylenedioxymethamphetamine (MDMA) in mice brain Protein stain Carbonyl detection Control MDMA Control 5 pi 8 Carbonylated proteins identified in hippocampus of MDMA treated mice MDMA 5 pi 8 Ros-Simó C, et al. J Neurochem. 25(5):736-46

6 Identification of carbonylated proteins Cell models of Friedreich ataxia (FA) 57% Mitochondrial 43% Cytosolic 6 Protein Gene Oxidation fold Heat shock protein mitochondrial SSC Control Protein Carbonyl detection Heat shock protein mitochondrial HSP Heat shock protein cytosolic SSE1 7.1 F 1 F O ATP synthase a subunit ATP1 8.5 F 1 F O ATP synthase b subunit ATP FA Acetohydroxiacid reductoisomerase ILV5 9.6 Pyruvate kinase 1 CDC phosphoglycerate kinase PGK Control Adenylate kinase ADK1 3.3 Actin, a chain ACT1 3.4 Elongation factor EF-1a TEF2 7.1 Catalase A CTA Protein stain Peroxiredoxin thiol specific AHP1 3.7 Superoxide dismutase 1 SOD FA

subunit a ATP5A 2,5 Citrate synthase CS 7,5 Pyruvate kinase 1 PKM2 6 Pyridoxal kinase PDXK 3,5 Cytochrome b-c1, sub.

7 Identification of carbonylated proteins Protein stain Carbonyl detection Striatum from Huntington Disease patients ( post mortem ) Protein Gene Oxidation fold ATPase ER VCP >9 Heat shock protein HSC71 7 Creatine kinase mitochondrial CK-MT1 7,5 F 1 F O 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 Control HD Sorolla MA, et al. Free Radic Biol Med. 49:

Yeast Identification of carbonylated proteins.

protein 71 69599 HXKA, Hexokinase-1 53738 MLS1, Malate synthase 1, glyoxysomal 62791 PYK1, Pyruvate kinase 1

factor 1-alpha 50032 TAL1, Transaldolase 37036 G3P3, Glyceraldehyde-3-phosphate DH 3 35838 IDH2, Isocitrate

8 Yeast Identification of carbonylated proteins. Aging: Old vs young yeast cells CARBONYLATED PROTEINS Mass (Da) Young Old Bodipy signal SSA2, Heat shock protein HXKA, Hexokinase MLS1, Malate synthase 1, glyoxysomal PYK1, Pyruvate kinase ALDH4, Potassium-activated aldehyde DH, Mito PGK, Phosphoglycerate kinase EF1A, Elongation factor 1-alpha TAL1, Transaldolase G3P3, Glyceraldehyde-3-phosphate DH IDH2, Isocitrate DH subunit 2, mitochondrial ADH1, Alcohol DH ADH2, Alcohol DH VDAC1, Mitochondrial outer membrane porin Tamarit et al. J. Proteomics. 75:

Protein carbonylation in aging (from several models) Plasma Proteins Membrane Transport Receptors and Cell Signaling Miscellaneous Glucose Metabolism Pyruvate DH and TCA Cycle Electron Transport

9 Protein carbonylation in aging (from several models) Plasma Proteins Membrane Transport Receptors and Cell Signaling 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 Cytoskeleton Antioxidant Defense Systems Heat Shock Proteins / Chaperones Cabiscol E; Tamarit J; Ros J. (2013). Protein carbonylation: proteomics, specificity and relevance to aging. Mass Spectrometry Reviews. 33:21-48

10 Why this oxidative damage specifically targets certain proteins? RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE? Amount? Protein stain Protein carbonyl Ex. 1 Frataxin mutants Ex. 2 Yeast aging

Endoplasmic Reticulum Mitochondria Subcellular location of carbonylated proteins.

11 RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE? LOCATION Cellular compartment Secreted Cell Membrane Others Chloroplast Cytoplasm Endoplasmic Reticulum Mitochondria 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.

12 RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE? Metals: Transition metals such as Iron or Copper -Fenton chemistry- 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 PLoS One.4(10)

13 RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE? Frataxin-deficient cells Protein Gene Oxidation fold Heat shock protein mitochondrial SSC Molecular chaperone mitochondrial HSP Heat shock protein cytosolic SSE1 7.1 Nucleotide-Binding Proteins F 1 F O ATP synthase a subunit ATP1 8.5 F 1 F O ATP synthase b subunit ATP2 4.7 Acetohydroxiacid reductoisomerase ILV5 9.6 Pyruvate kinase 1 CDC 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

Hydrogen peroxide stress bacteria- Frataxin depletion DNA K Elongation factor G Alcohol DH E F 1 F 0 -ATP synthase b GAPDH OMP A Enolase HSP 78 mitochondrial HSP 70 cytosolic F 1 F O ATP synthase a

14 Hydrogen peroxide stress bacteria- Frataxin depletion DNA K Elongation factor G Alcohol DH E F 1 F 0 -ATP synthase b GAPDH OMP A Enolase HSP 78 mitochondrial HSP 70 cytosolic F 1 F O ATP synthase a subunit F 1 F O 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 Pyruvate kinase ILV5, mitochondrial Heat shock protein 75 Heat shock protein 60 Phosphoglycerate kinase Pyruvate DH a-ketoglutarate DH Enolase 2 GAPDH3 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 Hydrogen peroxide stress yeast- Chronological aging yeast- 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 MDMA treatment ATPase ER HSP 70 CK mitochondrial F 1 F O ATP synthase, a Pyruvate kinase 1 Pyridoxal kinase Citrate synthase Huntington disease

Hydrogen peroxide GAPDH OMP A Enolase Bacteria DNA K (HSP70) Elongation factor G Alcohol DH E F 1 F 0 -ATP synthase b Yeast Pyruvate kinase ILV5, mitochondrial HSP 75 HSP 60 Phosphoglycerate kinase

15 Hydrogen peroxide GAPDH OMP A Enolase Bacteria DNA K (HSP70) Elongation factor G Alcohol DH E F 1 F 0 -ATP synthase b Yeast Pyruvate kinase ILV5, mitochondrial HSP 75 HSP 60 Phosphoglycerate kinase Hydrogen peroxide Pyruvate DH a-ketoglutarate DH Enolase 2 GAPDH3 Frataxin depletion GAPDH-3 SOD 1 Catalase A Peroxiredoxin alpha-enolase alpha-internexin Synapsin-1 MDMA treatment HSP 78 mitochondrial HSP 70 cytosolic F 1 F O ATP synthase a subunit F 1 F O ATP synthase b subunit Acetohydroxiacid reductoisomerase Pyruvate kinase 1 3-phosphoglycerate kinase Adenylate kinase Actin, a chain Elongation factor EF-1a Nucleotide binding proteins Heat shock cognate 71 Heat shock cognate 71 Dihyropyrimidinase-related protein-2 ATP synthase subunit b Actin ATP synthase subunit a ATPase ER HSP 70 CK mitochondrial F 1 F O 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 Huntington disease Citrate synthase GAPDH 3 Isocitrate DH mt Alcohol DH 1 Alcohol DH 2 Aging yeast-

Protein Carbonylation -ATP +ATP DNA K (HSP 70) 0 15 30 15 30 Ascorbate +

as NB-proteins Among the targets identified 60% of carbonylated proteins

16 Protein Carbonylation -ATP +ATP DNA K (HSP 70) Ascorbate + Iron + O 2 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 Open question: Is this mechanism designed to stop ATP consumption?

The presence of the nucleotide would act as a metal chelator promoting the damage observed as follows Fe 3+ Apoproteins Fe-proteins O - 2 Fe 2+ Fenton chemistry H 2 O 2 Mg OH ATP-Mg ATP-Fe 2+

17 The presence of the nucleotide would act as a metal chelator promoting the damage observed as follows Fe 3+ Apoproteins Fe-proteins O - 2 Fe 2+ Fenton chemistry H 2 O 2 Mg OH ATP-Mg ATP-Fe 2+ ATP-Fe3+ CH O CH O NTPs bound to proteins could explain the specificity/selectivity observed Concluding remark Whether this has evolved to preserve cellular integrity or it is an inevitable consequence of oxidative stress is still an open question

18 Fabien Delaspre Elena Britti Marta Llovera Rosa Purroy Jordi Tamarit DRG neurons Cardiomyocytes Yeasts David Alsina Anna Molet

Cellular Respiration and Fermentation

CAMPBELL BIOLOGY IN FOCUS URRY CAIN WASSERMAN MINORSKY REECE 7 Cellular Respiration and Fermentation Lecture Presentations by Kathleen Fitzpatrick and Nicole Tunbridge, Simon Fraser University SECOND EDITION