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

Hydroxyl Lys Protein carbonyl radical formation: Metal-Catalyzed Oxidation Fenton Reaction H 2 O H 2 O E. Stadtman. Free Rad. Biol. Med . 1990 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

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

Protein carbonyl detection Fenton Anti-DNP DNPH reaction Antibody *OH DNP DNP NH 2 O CH 2 CH CH CH Carbonyl group Western blot BODIPY-hydrazide Tamarit et al. J. Proteomics. 75:3778-3788 derivatization BODIPY-HZ O CH CH H 2 O 2 Bodipy HZ Protein-Flamingo

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

Identification of Control carbonylated proteins 10 Cell models of Friedreich ataxia (FA) 11 9 57% Mitochondrial 43% Cytosolic 6 Protein 7 Oxidation Protein Gene Carbonyl fold detection Heat shock protein mitochondrial SSC1 3.16 10 Heat shock protein mitochondrial HSP78 15.3 11 Heat shock protein cytosolic SSE1 7.1 9 6 F 1 F O ATP synthase a subunit ATP1 8.5 FA 7 F 1 F O ATP synthase b subunit ATP2 4.7 Acetohydroxiacid reductoisomerase ILV5 9.6 Control Pyruvate kinase 1 CDC19 3.6 10 11 3-phosphoglycerate kinase PGK1 2.2 Adenylate kinase ADK1 3.3 9 Actin, a chain ACT1 3.4 6 7 Elongation factor EF-1a TEF2 7.1 Protein Catalase A CTA1 8.1 stain Peroxiredoxin thiol specific AHP1 3.7 10 11 Superoxide dismutase 1 SOD1 2.9 9 6 FA 7

Protein stain Carbonyl detection Identification of carbonylated proteins Striatum from Huntington Disease patients (“post mortem”) Protein Gene Oxidation fold Control HD 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 ALDH7A1 3,5 DH Enolase ENO1 2,5 Sorolla MA, et al. Free Radic Biol Med. 49:612-621. Glyceraldehyde-3-P-DH GAPDH 2,5

Identification of carbonylated proteins. Yeast Aging: Old vs young yeast cells Mass CARBONYLATED PROTEINS (Da) Young 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 Bodipy signal 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 Old VDAC1, Mitochondrial outer membrane porin 30524 Tamarit et al. J. Proteomics. 75:3778-3788

Protein carbonylation in aging (from several models) Glucose Metabolism Miscellaneous Pyruvate DH and TCA Cycle Electron Transport Chain and ATP synthesis Plasma Proteins Plant Metabolism: Photosynthesis and Seed Metabolism Membrane Transport Amino Acid and Protein Metabolism Receptors and Cell Signaling Lipid Metabolism Antioxidant Defense Cytoskeleton 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

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

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

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. 2009. PLoS One.4(10 )

RULES EXPLAINING THE SPECIFICITY OF PROTEIN DAMAGE? Frataxin-deficient cells Oxidation Oxidation Protein Protein Gene Gene fold fold Heat shock protein mitochondrial Heat shock protein mitochondrial SSC1 SSC1 3.16 3.16 Molecular chaperone mitochondrial Molecular chaperone mitochondrial HSP78 HSP78 15.3 15.3 Heat shock protein cytosolic Heat shock protein cytosolic SSE1 SSE1 7.1 7.1 F 1 F O ATP synthase a subunit F 1 F O ATP synthase a subunit ATP1 ATP1 8.5 8.5 Nucleotide-Binding Proteins F 1 F O ATP synthase b subunit F 1 F O ATP synthase b subunit ATP2 ATP2 4.7 4.7 Acetohydroxiacid reductoisomerase Acetohydroxiacid reductoisomerase ILV5 ILV5 9.6 9.6 Pyruvate kinase 1 Pyruvate kinase 1 CDC19 CDC19 3.6 3.6 3-phosphoglycerate kinase 3-phosphoglycerate kinase PGK1 PGK1 2.2 2.2 Adenylate kinase Adenylate kinase ADK1 ADK1 3.3 3.3 Actin, a chain Actin, a chain ACT1 ACT1 3.4 3.4 Elongation factor EF-1a Elongation factor EF-1a TEF2 TEF2 7.1 7.1 Catalase A Catalase A CTA1 CTA1 8.1 8.1 Peroxiredoxin thiol specific Peroxiredoxin thiol specific AHP1 AHP1 3.7 3.7 Superoxide dismutase 1 Superoxide dismutase 1 SOD1 SOD1 2.9 2.9

Hydrogen peroxide stress Pyruvate kinase – yeast- Hydrogen peroxide ILV5, mitochondrial stress Heat shock protein 75 DNA K – bacteria- Chronological Elongation Heat shock protein 60 factor G Phosphoglycerate aging Alcohol DH E kinase – yeast- F 1 F 0 -ATP Pyruvate DH a -Ketoglutarate DH synthase b HSP 72 GAPDH Enolase 2 Hexokinase-1 OMP A GAPDH3 Pyruvate kinase 1 Enolase Aldehyde DH, mitochondrial Frataxin Phosphoglycerate kinase depletion Elongation factor 1-alpha GAPDH 3 HSP 78 mitochondrial Isocitrate DH, mitochondrial HSP 70 cytosolic Alcohol dehydrogenase 1 F 1 F O ATP synthase a subunit Alcohol dehydrogenase 2 F 1 F O ATP synthase b subunit VDAC1, Mitochondrial OMP Pyruvate kinase 3-phosphoglycerate kinase Heat shock cognate 71 Acetohydroxyacid redcutoisomerase Heat shock cognate 71 ATPase ER Adenylate kinase Dihyropyrimidinase-related Actin, a chain HSP 70 protein-2 CK mitochondrial Elongation factor EF-1a alpha-internexin F 1 F O ATP synthase, a GAPDH-3 ATP synthase subunit b Pyruvate kinase 1 SOD 1 alpha-enolase Pyridoxal kinase Catalase A Actin Citrate synthase Peroxiredoxin 1 Aconitase ATP synthase subunit a Huntington Synapsin-1 disease MDMA treatment

Bacteria Yeast Hydrogen Hydrogen peroxide DNA K (HSP70) Pyruvate kinase peroxide Elongation factor G ILV5, mitochondrial GAPDH Pyruvate DH Alcohol DH E HSP 75 a -Ketoglutarate DH OMP A F 1 F 0 -ATP synthase b HSP 60 Enolase Enolase 2 Phosphoglycerate kinase GAPDH3 Frataxin HSP 78 mitochondrial HSP 70 cytosolic ATPase ER Huntington depletion F 1 F O ATP synthase a subunit HSP 70 disease F 1 F O ATP synthase b subunit CK mitochondrial Nucleotide F 1 F O ATP synthase, a Acetohydroxiacid reductoisomerase binding Pyruvate kinase 1 Citrate synthase Pyruvate kinase 1 Pyridoxal kinase proteins GAPDH-3 3-phosphoglycerate kinase SOD 1 Adenylate kinase Catalase A Actin, a chain HSP 72 GAPDH 3 Peroxiredoxin Elongation factor EF-1a Hexokinase-1 Isocitrate DH mt Pyruvate kinase 1 Alcohol DH 1 Phosphoglycerate kinase Alcohol DH 2 Heat shock cognate 71 Elongation factor 1-alpha alpha-enolase Heat shock cognate 71 Isocitrate DH subunit 2, Aging alpha-internexin Dihyropyrimidinase-related protein-2 Synapsin-1 mitochondrial – yeast- ATP synthase subunit b VDAC1, Mitochondrial OMP Actin MDMA ATP synthase subunit a treatment

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