- Email: [email protected]
Long-lived reactive species formed on proteins induce changes in protein and lipid turnover
S6
C.G. Fraga, P. Oteiza / Free Radical Biology and Medicine 75 (2014) S3–S12
S3-3
Oxysterols in the orchestra of liver cell metabolism Gaetano Serviddio, Francesco Bellanti, Gianluigi Vendemiale Centro Universitario per la Ricerca e la Cura delle Malattie Epatiche (C.U.R.E.), Institute of Internal Medicine, University of Foggia, Italy Non-alcoholic fatty liver disease (NAFLD) is a chronic hepatic disorder affecting up to 25% of the general population. Several intracellular events leading to NAFLD and progression to non-alcoholic steatohepatitis (NASH) have been identified, including lipid accumulation, mitochondrial dysfunction and oxidative stress. Emerging evidence has suggested the cooperation of both hepatic free fatty acids (FFAs) and cholesterol (FC) accumulation in NAFLD development. Oxysterols, oxidized derivatives of cholesterol were reported as activating ligands of Liver X Receptors. Interestingly, serum levels of agonist oxysterols such as 25-HC and 27-HC are significantly increased in NAFLD patients. By contrast, 22-s- HC is considered an antagonist ligand of LXRα. 22-s-Hc down-regulated expression of the FAS gene through an LXRE located in the promoter and abolished the effect of the synthetic LXRα agonist. In addition it has been reported that 22-s-HC attenuated hepatic steatogenesis in a mouse model of high-fat-induced fatty liver. Very recently, it has been demonstrated that the inhibition of LXRα by 22-s-HC dramatically represses steatosis and HIF-1 mediated activation of MCP-1 in ethanol-induced fatty liver injury in hepatocytes as well as in Kupferr cells. Our data demonstrated that High fat diet (HF) caused liver steatosis while an atherogenic diet (ATH) diet induced hepatocellular ballooning, but only the Athþ HF diet resulted in steatohepatitis with associated mitochondrial dysfunction and impaired mitochondriogenesis. We identified in the the oxysterol cholestane-3beta,5alpha,6beta-triol (Triol) the molecule able to impair mitochondrial respiration and mitochondriogenesis by down-regulation of PGC1-alpha, mTFA and NRF1 signal. It seems that the role of oxysterols in the pathogenesis of human diseases, particularly in fat induced injury, should take into account the possible disruption of the balance between activation and inhibition of LXR signalling. Development of LXRα that specifically control inflammation and hepatic lipogenesis may provide novel therapeutics that block the development and progression of NAFLD.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.838
S3-4
NASH (Nonalcoholic steatohepatitis): A case of multiorganelle failure Stephen Caldwell University of Virginia (Medical Center), Hepatology, Charlottesville, USA The clinical term ‘multiorgan failure’ lends itself, modified to ‘multiorganelle failure’, to the cascading events in cellular systems leading to hepatocyte injury, cell death, inflammation and fibrosis and ultimately to cirrhosis in NASH (non-alcoholic steatohepatitis). NASH is one of the most common forms of liver disease and constitutes the severe form of NAFLD (non-alcoholic fatty liver disease). The key features that distinguish potentially progressive NASH from relatively stable non-NASH fatty liver (NNFL, often referred to as simple steatosis) are cellular ballooning, inflammation and fibrosis. These findings, together with steatosis or accumulation of greater than normal hepatic lipid, usually constitute histological NASH seen on liver biopsy or in laboratory samples. Cellular ballooning is not specific to NASH but it is perhaps the most emblematic finding on histological samples. The ballooned hepatocyte has evidence of cytoskeletal injury (depletion and
condensation as Mallory-Denk bodies), accumulation of partially oxidized small fat droplets, mitochondrial morphological changes presumably related to organelle dysfunction, dilated endoplasmic reticulum and autophagosomes - an attempt at cellular repair. Ballooning itself likely results from a combination of cytoskeletal injury resulting in loss of normal cell shape and from accumulation of injured and somewhat derelict small fat droplets and dilated endoplasmic reticulum. Cellular injury in NASH and especially cellular ballooning can be viewed as a process of ‘multi-organelle failure’ beginning with generation of super oxide and failure to contain the subsequent oxidative injury and by-products in an environment rich in lipid fuel. These events lead to activation of imunologic pathways. Dysfunction of the small fat droplet appears to be a central mechanism and the oxidative injury can be viewed as the process of rancidification – the chemical decomposition of oils, lipids and fats.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.839
Symposium 4: Proteolysis, mitochondria and ageing
S4-1
What Goes Wrong with Lon in Ageing? Kelvin J.A. Davies University of Southern California, Andrus Gerontology Center of the Davis School of Gerontology, and Division of Molecular & Computational Biology, College of Letters, Arts & Sciences, USA
We have shown previously that the product of the human lon gene, the Lon protease, selectively degrades oxidized mitochondrial proteins, thus preventing their aggregation and cross-linking. We have also shown that lon is a stress-responsive gene, whose protease product is a stress-responsive protein that is induced by multiple stressors, including heat shock, serum starvation, and oxidative stress. Lon induction, by pre-treatment with lowlevel stress, protects against oxidative protein damage, diminished mitochondrial function, and loss of cell proliferation, induced by toxic levels of hydrogen peroxide. Blocking Lon induction, with lon siRNA, also blocks this induced protection. All of these results were obtained in young, healthy cells. In senescent cells, and in older primary cells, however, Lon activity declines, and adaptational responses become sluggish or even ineffectual. Studies in Drosophila melanogaster flies and in mice now suggest that declining Lon activity and declining responsiveness to stress, may contribute to the ageing process, and to various age-associated diseases. We propose that Lon is a generalized stress-protective enzyme whose decline may contribute to the increased levels of protein damage and mitochondrial dysfunction observed in ageing and various age-related diseases. We further propose that impaired ability to induce lon, and other stress-responsive genes, now needs to be included in any Free Radical Theory of Ageing.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.840
S4-2
Long-lived reactive species formed on proteins induce changes in protein and lipid turnover Michael Davies University of Copenhagen (Panum Institute), Department of Biomedical Sciences, Denmark
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
C.G. Fraga, P. Oteiza / Free Radical Biology and Medicine 75 (2014) S3–S12
S3-3
Oxysterols in the orchestra of liver cell metabolism Gaetano Serviddio, Francesco Bellanti, Gianluigi Vendemiale Centro Universitario per la Ricerca e la Cura delle Malattie Epatiche (C.U.R.E.), Institute of Internal Medicine, University of Foggia, Italy Non-alcoholic fatty liver disease (NAFLD) is a chronic hepatic disorder affecting up to 25% of the general population. Several intracellular events leading to NAFLD and progression to non-alcoholic steatohepatitis (NASH) have been identified, including lipid accumulation, mitochondrial dysfunction and oxidative stress. Emerging evidence has suggested the cooperation of both hepatic free fatty acids (FFAs) and cholesterol (FC) accumulation in NAFLD development. Oxysterols, oxidized derivatives of cholesterol were reported as activating ligands of Liver X Receptors. Interestingly, serum levels of agonist oxysterols such as 25-HC and 27-HC are significantly increased in NAFLD patients. By contrast, 22-s- HC is considered an antagonist ligand of LXRα. 22-s-Hc down-regulated expression of the FAS gene through an LXRE located in the promoter and abolished the effect of the synthetic LXRα agonist. In addition it has been reported that 22-s-HC attenuated hepatic steatogenesis in a mouse model of high-fat-induced fatty liver. Very recently, it has been demonstrated that the inhibition of LXRα by 22-s-HC dramatically represses steatosis and HIF-1 mediated activation of MCP-1 in ethanol-induced fatty liver injury in hepatocytes as well as in Kupferr cells. Our data demonstrated that High fat diet (HF) caused liver steatosis while an atherogenic diet (ATH) diet induced hepatocellular ballooning, but only the Athþ HF diet resulted in steatohepatitis with associated mitochondrial dysfunction and impaired mitochondriogenesis. We identified in the the oxysterol cholestane-3beta,5alpha,6beta-triol (Triol) the molecule able to impair mitochondrial respiration and mitochondriogenesis by down-regulation of PGC1-alpha, mTFA and NRF1 signal. It seems that the role of oxysterols in the pathogenesis of human diseases, particularly in fat induced injury, should take into account the possible disruption of the balance between activation and inhibition of LXR signalling. Development of LXRα that specifically control inflammation and hepatic lipogenesis may provide novel therapeutics that block the development and progression of NAFLD.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.838
S3-4
NASH (Nonalcoholic steatohepatitis): A case of multiorganelle failure Stephen Caldwell University of Virginia (Medical Center), Hepatology, Charlottesville, USA The clinical term ‘multiorgan failure’ lends itself, modified to ‘multiorganelle failure’, to the cascading events in cellular systems leading to hepatocyte injury, cell death, inflammation and fibrosis and ultimately to cirrhosis in NASH (non-alcoholic steatohepatitis). NASH is one of the most common forms of liver disease and constitutes the severe form of NAFLD (non-alcoholic fatty liver disease). The key features that distinguish potentially progressive NASH from relatively stable non-NASH fatty liver (NNFL, often referred to as simple steatosis) are cellular ballooning, inflammation and fibrosis. These findings, together with steatosis or accumulation of greater than normal hepatic lipid, usually constitute histological NASH seen on liver biopsy or in laboratory samples. Cellular ballooning is not specific to NASH but it is perhaps the most emblematic finding on histological samples. The ballooned hepatocyte has evidence of cytoskeletal injury (depletion and
condensation as Mallory-Denk bodies), accumulation of partially oxidized small fat droplets, mitochondrial morphological changes presumably related to organelle dysfunction, dilated endoplasmic reticulum and autophagosomes - an attempt at cellular repair. Ballooning itself likely results from a combination of cytoskeletal injury resulting in loss of normal cell shape and from accumulation of injured and somewhat derelict small fat droplets and dilated endoplasmic reticulum. Cellular injury in NASH and especially cellular ballooning can be viewed as a process of ‘multi-organelle failure’ beginning with generation of super oxide and failure to contain the subsequent oxidative injury and by-products in an environment rich in lipid fuel. These events lead to activation of imunologic pathways. Dysfunction of the small fat droplet appears to be a central mechanism and the oxidative injury can be viewed as the process of rancidification – the chemical decomposition of oils, lipids and fats.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.839
Symposium 4: Proteolysis, mitochondria and ageing
S4-1
What Goes Wrong with Lon in Ageing? Kelvin J.A. Davies University of Southern California, Andrus Gerontology Center of the Davis School of Gerontology, and Division of Molecular & Computational Biology, College of Letters, Arts & Sciences, USA
We have shown previously that the product of the human lon gene, the Lon protease, selectively degrades oxidized mitochondrial proteins, thus preventing their aggregation and cross-linking. We have also shown that lon is a stress-responsive gene, whose protease product is a stress-responsive protein that is induced by multiple stressors, including heat shock, serum starvation, and oxidative stress. Lon induction, by pre-treatment with lowlevel stress, protects against oxidative protein damage, diminished mitochondrial function, and loss of cell proliferation, induced by toxic levels of hydrogen peroxide. Blocking Lon induction, with lon siRNA, also blocks this induced protection. All of these results were obtained in young, healthy cells. In senescent cells, and in older primary cells, however, Lon activity declines, and adaptational responses become sluggish or even ineffectual. Studies in Drosophila melanogaster flies and in mice now suggest that declining Lon activity and declining responsiveness to stress, may contribute to the ageing process, and to various age-associated diseases. We propose that Lon is a generalized stress-protective enzyme whose decline may contribute to the increased levels of protein damage and mitochondrial dysfunction observed in ageing and various age-related diseases. We further propose that impaired ability to induce lon, and other stress-responsive genes, now needs to be included in any Free Radical Theory of Ageing.
http://dx.doi.org/10.1016/j.freeradbiomed.2014.10.840
S4-2
Long-lived reactive species formed on proteins induce changes in protein and lipid turnover Michael Davies University of Copenhagen (Panum Institute), Department of Biomedical Sciences, Denmark
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