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Oxi-DIGE: A novel proteomic approach for detecting and quantifying carbonylated proteins
P. White et al. / Free Radical Biology and Medicine 75 (2014) S21–S53
(or even a shortened lifespan). Altogether our data suggest AF-1 expression during aging to be a mechanism that affects healthy aging and agerelated stress resistance depending on the gender of the fly.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.737
S23
Baraibar Martin, Ladouce Romain, Friguet Bertrand Sorbonne Universités, UPMC Univ Paris 06 (CNRS UMR 8256, INSERM U1164), Biological adaptation and aging - IBPS, France
Abstract P6
Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes Baraibar Martina, Hyzewicz Janeka, Rogowska-Wrzesinska Adelinab, Bulteau Anne-Laurec, Prip-Buus Carinac, Butler-Browne Gilliand, Friguet Bertranda a
Sorbonne Universités, UPMC Univ Paris 06 (CNRS UMR 8256, INSERM U1164), Biological adaptation and aging - IBPS, France b University of Southern Denmark (University of Southern Denmark), Department of Biochemistry and Molecular Biology, Denmark c Institut Cochin (INSERM), INSERM, U1016, France d Institut de Myologie (CHU Pitié-Salpétrière, Sorbonne Universités, UPMC Univ Paris 06), UMRS INSERM U974, CNRS UMR 7215, France
Abstract Accumulation of damaged macromolecules, including irreversibly oxidized proteins, is a hallmark of cellular and organismal ageing. Failure of protein homesotasis is a major contributor to the age-related accumulation of damaged proteins. In skeletal muscle, tissue maintenance and regeneration is assured by resident adult stem cells known as satellite cells. During senescence their replication and differentiation is compromised contributing to sarcopenia. In this study we have addressed the impact of oxidatively modified proteins in the impaired metabolism of senescent human satellite cells. By using a targeted proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during replicative senescence of satellite cells. Inactivation of the proteasome in aged cells appeared as a key contributor to the accumulation of such damaged proteins. Untargeted metabolomic profiling and functional analyses indicated glucose metabolism impairment in senescent cells, although mitochondrial respiration remained unaffected. A metabolic shift leading to increased mobilization of non-carbohydrate substrates as branched chain amino acids or long chain fatty acids was observed in senescent cells. In addition, phospho-and glycerolipids metabolism was altered. Increased levels of acyl-carnitines indicated augmented turnover of storage and membrane lipids for energy production. Such changes reflect alterations in membrane composition and dysregulation of sphingolipids signaling during senescence. This study establishes a new concept connecting oxidative protein modifications with the altered cellular metabolism associated with the senescent phenotype. In addition, these findings highlight the molecular mechanisms implicated in satellite cells dysfunction during ageing, paving the road for future therapeutic interventions aimed at preventing oxidative modifications of proteins and/or stimulating their elimination.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.738
P7
Oxi-DIGE: A novel proteomic approach for detecting and quantifying carbonylated proteins
Proteins are involved in key cellular functions and our health and wellness depends on their quality. Accumulation of oxidatively damaged proteins is a hallmark of deleterious processes such increased oxidative stress, chronic inflammation, ageing and age-related diseases. Thus, quantifying and identifying oxidized proteins is a biomarker of choice for monitoring biological ageing and/or the efficiency of anti-oxidant, antinflammatory and anti-ageing therapies. However, the absence of reliable tools for analyses has inhibited its establishment as the gold standard for measuring the efficacy of anti-ageing and age related diseases interventions. Herein, we present a novel proteomics technology, named OxiDIGEn, which provides a significant improvement in terms of specificity, reproducibility and statistical support for proteomic analysis of carbonylated proteins. In Oxi-DIGE, protein carbonyls are labelled with fluorescent hydrazide probes that bind specifically to carbonyl groups in proteins. Experimental groups (e.g. control and experimental samples) are labelled with different flurophore-binded hydrazides that fluoresce light at different wavelengths, producing different colour fluorescence. Thus samples from different experimental groups are co-resolved on a single 2D gel. Increased accuracy is provided due to: (i) reduced false positives by using an exogenous synthetic fluorescent tag; (ii) multiplexing, that is the possibility to run multiple samples on the same gel, (iii) the use of an internal standard on each gel which eliminates inter-gel variations and provides an increased statistical confidence. In addition, the resolution of the carbonyl groups is improved, forming distinct spots that can be identified by mass spectrometry. n Patent Application (M. Baraibar, R. Ladouce., B. Friguet, A method for detecting and/or quantifying carbonylated proteins (WO/2012/175519) filed by UPMC and referring to the technology described in this abstract.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.739
P8
Is Peroxiredoxin II’s peroxidase activity strongly inhibited in human erythrocytes? Benfeitas Ruia, Selvaggio Gianlucaa, Antunes Fernandob, Coelho Pedroa, Salvador Armindoa a
CNC (Center for Neuroscience and Cell Biology), University of Coimbra, Portugal b DQB (Centro de Química e Bioquímica), Departamento de Química e Bioquímica, Portugal
Abstract H2O2 elimination in human erythrocytes is mainly carried out by catalase (Cat), glutathione peroxidase (GPx1) and the more recently discovered peroxiredoxin 2 (Prx2). However, the contribution of Prx2 to H2O2 consumption is still unclear. Prx2’s high reactivity with H2O2 (kPrx2 ¼10 107 M-1s-1, kCat ¼7 107 M-1s-1, kGPx1 ¼ 4 107 M-1s-1) and high abundance ([Prx2]¼ 570 μM, [Cat] ¼ 32 μM, [GPx1] ¼ 1 μM) suggest that under low H2O2 supply rates it should consume 499% of the H2O2. However, extensive evidence indicates that in intact erythrocytes Prx2 contributes no more than Cat to H2O2 consumption. In order for this to be attained, Prx2’s effective rate constant with H2O2would have to be just 10e M-1s-1, much lower than that determined in multiple experiments with the purified proteins. Nevertheless, nearly all Prx2 is oxidized within
(or even a shortened lifespan). Altogether our data suggest AF-1 expression during aging to be a mechanism that affects healthy aging and agerelated stress resistance depending on the gender of the fly.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.737
S23
Baraibar Martin, Ladouce Romain, Friguet Bertrand Sorbonne Universités, UPMC Univ Paris 06 (CNRS UMR 8256, INSERM U1164), Biological adaptation and aging - IBPS, France
Abstract P6
Impaired metabolism of senescent muscle satellite cells is associated with oxidative modifications of glycolytic enzymes Baraibar Martina, Hyzewicz Janeka, Rogowska-Wrzesinska Adelinab, Bulteau Anne-Laurec, Prip-Buus Carinac, Butler-Browne Gilliand, Friguet Bertranda a
Sorbonne Universités, UPMC Univ Paris 06 (CNRS UMR 8256, INSERM U1164), Biological adaptation and aging - IBPS, France b University of Southern Denmark (University of Southern Denmark), Department of Biochemistry and Molecular Biology, Denmark c Institut Cochin (INSERM), INSERM, U1016, France d Institut de Myologie (CHU Pitié-Salpétrière, Sorbonne Universités, UPMC Univ Paris 06), UMRS INSERM U974, CNRS UMR 7215, France
Abstract Accumulation of damaged macromolecules, including irreversibly oxidized proteins, is a hallmark of cellular and organismal ageing. Failure of protein homesotasis is a major contributor to the age-related accumulation of damaged proteins. In skeletal muscle, tissue maintenance and regeneration is assured by resident adult stem cells known as satellite cells. During senescence their replication and differentiation is compromised contributing to sarcopenia. In this study we have addressed the impact of oxidatively modified proteins in the impaired metabolism of senescent human satellite cells. By using a targeted proteomics analysis we have found that proteins involved in protein quality control and glycolytic enzymes are the main targets of oxidation (carbonylation) and modification with advanced glycation/lipid peroxidation end products during replicative senescence of satellite cells. Inactivation of the proteasome in aged cells appeared as a key contributor to the accumulation of such damaged proteins. Untargeted metabolomic profiling and functional analyses indicated glucose metabolism impairment in senescent cells, although mitochondrial respiration remained unaffected. A metabolic shift leading to increased mobilization of non-carbohydrate substrates as branched chain amino acids or long chain fatty acids was observed in senescent cells. In addition, phospho-and glycerolipids metabolism was altered. Increased levels of acyl-carnitines indicated augmented turnover of storage and membrane lipids for energy production. Such changes reflect alterations in membrane composition and dysregulation of sphingolipids signaling during senescence. This study establishes a new concept connecting oxidative protein modifications with the altered cellular metabolism associated with the senescent phenotype. In addition, these findings highlight the molecular mechanisms implicated in satellite cells dysfunction during ageing, paving the road for future therapeutic interventions aimed at preventing oxidative modifications of proteins and/or stimulating their elimination.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.738
P7
Oxi-DIGE: A novel proteomic approach for detecting and quantifying carbonylated proteins
Proteins are involved in key cellular functions and our health and wellness depends on their quality. Accumulation of oxidatively damaged proteins is a hallmark of deleterious processes such increased oxidative stress, chronic inflammation, ageing and age-related diseases. Thus, quantifying and identifying oxidized proteins is a biomarker of choice for monitoring biological ageing and/or the efficiency of anti-oxidant, antinflammatory and anti-ageing therapies. However, the absence of reliable tools for analyses has inhibited its establishment as the gold standard for measuring the efficacy of anti-ageing and age related diseases interventions. Herein, we present a novel proteomics technology, named OxiDIGEn, which provides a significant improvement in terms of specificity, reproducibility and statistical support for proteomic analysis of carbonylated proteins. In Oxi-DIGE, protein carbonyls are labelled with fluorescent hydrazide probes that bind specifically to carbonyl groups in proteins. Experimental groups (e.g. control and experimental samples) are labelled with different flurophore-binded hydrazides that fluoresce light at different wavelengths, producing different colour fluorescence. Thus samples from different experimental groups are co-resolved on a single 2D gel. Increased accuracy is provided due to: (i) reduced false positives by using an exogenous synthetic fluorescent tag; (ii) multiplexing, that is the possibility to run multiple samples on the same gel, (iii) the use of an internal standard on each gel which eliminates inter-gel variations and provides an increased statistical confidence. In addition, the resolution of the carbonyl groups is improved, forming distinct spots that can be identified by mass spectrometry. n Patent Application (M. Baraibar, R. Ladouce., B. Friguet, A method for detecting and/or quantifying carbonylated proteins (WO/2012/175519) filed by UPMC and referring to the technology described in this abstract.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.739
P8
Is Peroxiredoxin II’s peroxidase activity strongly inhibited in human erythrocytes? Benfeitas Ruia, Selvaggio Gianlucaa, Antunes Fernandob, Coelho Pedroa, Salvador Armindoa a
CNC (Center for Neuroscience and Cell Biology), University of Coimbra, Portugal b DQB (Centro de Química e Bioquímica), Departamento de Química e Bioquímica, Portugal
Abstract H2O2 elimination in human erythrocytes is mainly carried out by catalase (Cat), glutathione peroxidase (GPx1) and the more recently discovered peroxiredoxin 2 (Prx2). However, the contribution of Prx2 to H2O2 consumption is still unclear. Prx2’s high reactivity with H2O2 (kPrx2 ¼10 107 M-1s-1, kCat ¼7 107 M-1s-1, kGPx1 ¼ 4 107 M-1s-1) and high abundance ([Prx2]¼ 570 μM, [Cat] ¼ 32 μM, [GPx1] ¼ 1 μM) suggest that under low H2O2 supply rates it should consume 499% of the H2O2. However, extensive evidence indicates that in intact erythrocytes Prx2 contributes no more than Cat to H2O2 consumption. In order for this to be attained, Prx2’s effective rate constant with H2O2would have to be just 10e M-1s-1, much lower than that determined in multiple experiments with the purified proteins. Nevertheless, nearly all Prx2 is oxidized within