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Protein Oxidation Products as Biomarkers
C.G. Fraga, P. Oteiza / Free Radical Biology and Medicine 75 (2014) S3–S12
Proteins are major targets for oxidative damage in vivo due to their high abundance and rapid rates of reaction with both one-electron (radical) and two-electron oxidants (e.g. singlet oxygen, hypochlorous acid, peroxynitrous acid, reactive aldehydes). The turnover of both native and modified proteins is critical for maintenance of cell homeostasis, with this occurring via multiple pathways including proteasomes (for cytosolic species), the Lon protease (in mitochondria), and the endo-lysosomal systems (both extra- and intra-cellular species). Evidence has been presented for both enhanced and diminished rates of catabolism of modified proteins, as well as altered turnover of native (unmodified) proteins as a result of damage to these systems, potentially as a result of the accumulation of damaged proteins. In recent studies we have shown that long-lived reactive species forms on proteins (hydroperoxides, chloramines and aldehydes) can modify the activity of proteasomal and lysosomal enzymes. Some of the above species are efficient inhibitors of the tryptic and chymotryptic activities of the 26 S proteasome, as well as lysosomal cathepsin and acid lipase activities. These are key species in the turnover of both proteins and lipoproteins. The loss of enzyme activity is accompanied in many cases, by oxidation of critical thiol residues via molecular reactions. For reactive aldehydes (either free or proteinbound) direct enzyme inhibition can occur as well as modulation of protein levels and, in the case of lysosomes, changes in lysosomal numbers. Overall, these data indicate that the formation of reactive species on proteins can modulate cell function by multiple pathways including interference with the turnover of native proteins (including critical cell signalling molecules) and alterations in the rate of clearance of modified proteins. Both pathways may contribute to the development of a number of human pathologies associated with oxidative damage.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.841
S4-3
Proteasome activation as a novel anti-aging strategy Efstathios Gonos
S7
S4-4
Protein Oxidation Products as Biomarkers Tilman Grune German Institute of Human Nutrition, Department of Molecular Toxicology, Nuthetal, Germany Oxidative stress is associated with aging and most degenerative diseases: it contributes to clinical complications, organ failure and mortality. Furthermore, oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Interestingly, it is unknown whether and to what extent oxidative stress contributes to the clinical worsening and most importantly, no common strategy exists about its measurement. This is partially due to the fact that many methods of measuring oxidative stress have proven unreliable and no single method exists enabling objective determination and characterization of oxidative stress in clinical settings whether in aging or in chronic disease. Some methods basing on the measurement of lipid peroxidation, as the determination of F2-isoprostanes or malondialdehyde, or on the measurement of DNA/RNA damage, as 8-hydroxydesoxyguanosine or 8-hydroxydesoxyguanine, are widely used to determine oxidative stress and gain more and more acceptance or are reliable supportive parameters. A set of methods exists for clinical set-ups based on the measurement of protein oxidation or oxidative protein modification. Some of these methods are gaining more and more acceptance due to the development of easy to use and stable methods. This includes the long known method of protein carbonyl determination in various methodological variations, but also the use of the determination of protein-based nitrotyrosine or HNEmodified proteins. All these methods can be used in clinical set-ups, but special care has to be taken on chemical and biological sample stability. It should be mentioned, that no single method, however, is yet alone able to characterize oxidative stress under clinical conditions and, therefore, various combinations of damage parameters are the most promising tools.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.843
National Hellenic Research Foundation (IBMCB), Molecular & Cellular Ageing, Greece Aging and longevity are two multifactorial biological phenomena whose knowledge at molecular level is still limited. We have studied proteasome function in replicative senescence and cell survival (Mol Aspects Med 35, 1-71, 2014). We have observed reduced levels of proteasome content and activities in senescent cells due to the down-regulation of the catalytic subunits of the 20 S complex (J Biol Chem 278, 28026-28037, 2003). In support, partial inhibition of proteasomes in young cells by specific inhibitors induces premature senescence which is p53 dependent (Aging Cell 7, 717-732, 2008). Stable over-expression of catalytic subunits or POMP resulted in enhanced proteasome assembly and activities and increased cell survival following treatments with various oxidants. Importantly, the developed “proteasome activated” human fibroblasts cell lines exhibit a delay of senescence by approximately 15% (J Biol Chem 280, 11840-11850, 2005; J Biol Chem 284, 30076-30086, 2009). Our current work proposes that proteasome activation is an evolutionary conserved mechanism, as it can delay aging in various in vivo systems. Moreover, additional findings indicate that the recorded proteasome activation by many inducers is Nrf2dependent (J Biol Chem 285, 8171-8184, 2010). Finally, we have studied the proteolysis processes of various age-related proteins and we have identified that CHIP is a major p53 E3 ligase in senescent fibroblasts (Free Rad Biol Med 50, 157-165, 2011).
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.842
Symposium 5: Protein lipoxidation: biological role and analysis (COST sponsored) S5-1
Chemical strategies and tandem mass spectrometry for the detection and identification of protein modifications by electrophilic lipoxidation products Claudia Maier Oregon State University, Corvallis, Oregon, USA
The post-translational modification of proteins by electrophilic oxylipids is emerging as an important mechanism that contributes to the complexity of proteomes. Originally considered solely as markers of oxidative insult, more recently the modifications of proteins by lipid peroxidation products are being recognized as having roles in cell signaling with relevance to redox homeostasis, adaptive response and inflammatory resolution. We report on mass spectrometry-based methods in conjunction with chemoselective strategies that are capable of
Proteins are major targets for oxidative damage in vivo due to their high abundance and rapid rates of reaction with both one-electron (radical) and two-electron oxidants (e.g. singlet oxygen, hypochlorous acid, peroxynitrous acid, reactive aldehydes). The turnover of both native and modified proteins is critical for maintenance of cell homeostasis, with this occurring via multiple pathways including proteasomes (for cytosolic species), the Lon protease (in mitochondria), and the endo-lysosomal systems (both extra- and intra-cellular species). Evidence has been presented for both enhanced and diminished rates of catabolism of modified proteins, as well as altered turnover of native (unmodified) proteins as a result of damage to these systems, potentially as a result of the accumulation of damaged proteins. In recent studies we have shown that long-lived reactive species forms on proteins (hydroperoxides, chloramines and aldehydes) can modify the activity of proteasomal and lysosomal enzymes. Some of the above species are efficient inhibitors of the tryptic and chymotryptic activities of the 26 S proteasome, as well as lysosomal cathepsin and acid lipase activities. These are key species in the turnover of both proteins and lipoproteins. The loss of enzyme activity is accompanied in many cases, by oxidation of critical thiol residues via molecular reactions. For reactive aldehydes (either free or proteinbound) direct enzyme inhibition can occur as well as modulation of protein levels and, in the case of lysosomes, changes in lysosomal numbers. Overall, these data indicate that the formation of reactive species on proteins can modulate cell function by multiple pathways including interference with the turnover of native proteins (including critical cell signalling molecules) and alterations in the rate of clearance of modified proteins. Both pathways may contribute to the development of a number of human pathologies associated with oxidative damage.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.841
S4-3
Proteasome activation as a novel anti-aging strategy Efstathios Gonos
S7
S4-4
Protein Oxidation Products as Biomarkers Tilman Grune German Institute of Human Nutrition, Department of Molecular Toxicology, Nuthetal, Germany Oxidative stress is associated with aging and most degenerative diseases: it contributes to clinical complications, organ failure and mortality. Furthermore, oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Interestingly, it is unknown whether and to what extent oxidative stress contributes to the clinical worsening and most importantly, no common strategy exists about its measurement. This is partially due to the fact that many methods of measuring oxidative stress have proven unreliable and no single method exists enabling objective determination and characterization of oxidative stress in clinical settings whether in aging or in chronic disease. Some methods basing on the measurement of lipid peroxidation, as the determination of F2-isoprostanes or malondialdehyde, or on the measurement of DNA/RNA damage, as 8-hydroxydesoxyguanosine or 8-hydroxydesoxyguanine, are widely used to determine oxidative stress and gain more and more acceptance or are reliable supportive parameters. A set of methods exists for clinical set-ups based on the measurement of protein oxidation or oxidative protein modification. Some of these methods are gaining more and more acceptance due to the development of easy to use and stable methods. This includes the long known method of protein carbonyl determination in various methodological variations, but also the use of the determination of protein-based nitrotyrosine or HNEmodified proteins. All these methods can be used in clinical set-ups, but special care has to be taken on chemical and biological sample stability. It should be mentioned, that no single method, however, is yet alone able to characterize oxidative stress under clinical conditions and, therefore, various combinations of damage parameters are the most promising tools.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.843
National Hellenic Research Foundation (IBMCB), Molecular & Cellular Ageing, Greece Aging and longevity are two multifactorial biological phenomena whose knowledge at molecular level is still limited. We have studied proteasome function in replicative senescence and cell survival (Mol Aspects Med 35, 1-71, 2014). We have observed reduced levels of proteasome content and activities in senescent cells due to the down-regulation of the catalytic subunits of the 20 S complex (J Biol Chem 278, 28026-28037, 2003). In support, partial inhibition of proteasomes in young cells by specific inhibitors induces premature senescence which is p53 dependent (Aging Cell 7, 717-732, 2008). Stable over-expression of catalytic subunits or POMP resulted in enhanced proteasome assembly and activities and increased cell survival following treatments with various oxidants. Importantly, the developed “proteasome activated” human fibroblasts cell lines exhibit a delay of senescence by approximately 15% (J Biol Chem 280, 11840-11850, 2005; J Biol Chem 284, 30076-30086, 2009). Our current work proposes that proteasome activation is an evolutionary conserved mechanism, as it can delay aging in various in vivo systems. Moreover, additional findings indicate that the recorded proteasome activation by many inducers is Nrf2dependent (J Biol Chem 285, 8171-8184, 2010). Finally, we have studied the proteolysis processes of various age-related proteins and we have identified that CHIP is a major p53 E3 ligase in senescent fibroblasts (Free Rad Biol Med 50, 157-165, 2011).
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.842
Symposium 5: Protein lipoxidation: biological role and analysis (COST sponsored) S5-1
Chemical strategies and tandem mass spectrometry for the detection and identification of protein modifications by electrophilic lipoxidation products Claudia Maier Oregon State University, Corvallis, Oregon, USA
The post-translational modification of proteins by electrophilic oxylipids is emerging as an important mechanism that contributes to the complexity of proteomes. Originally considered solely as markers of oxidative insult, more recently the modifications of proteins by lipid peroxidation products are being recognized as having roles in cell signaling with relevance to redox homeostasis, adaptive response and inflammatory resolution. We report on mass spectrometry-based methods in conjunction with chemoselective strategies that are capable of