- Email: [email protected]
Site-specific cysteine oxidation regulates 26S proteasomes
S100
D. Rossin et al. / Free Radical Biology and Medicine 108 (2017) S18–S107
Keywords: lipotoxicity; ubiquitin-proteasome system; thiol
P-236
Oxidative Stress Markers of Alzheimer's Disease in Peripheral Cell Mitochondria Christian Schoneich 1, Asha Hewarathna 1, Ranu Pal 2, Lei Jiang 2, Elias Michaelis 2 1
University of Kansas, Pharmaceutical Chemistry, United States of America 2 University of Kansas, Pharmacology and Toxicology, USA Keywords: Alzheimer's Disease; oxidative stress; protein oxidation; mitochondria
Alzheimer's disease (AD) represents a neurodegenerative disease leading to progressive dementia in the elderly. AD has been associated with increased oxidative stress and mitochondrial dysfunction. Hence, markers for oxidative stress are expected to accumulate not only in brain mitochondria, but also in mitochondria of peripheral cells. We have developed and optimized a method for the rapid and selective fluorogenic derivatization and enrichment of specific protein oxidation products, 3-nitrotyrosine and 3,4-dihydroxyphenylalanine (DOPA), in order to quantitatively compare protein oxidation in the mitochondria of peripheral, white blood cells obtained from healthy donors, patients with mild cognitive impairment (MCI), and AD patients. Double blind studies reveal a ca. two-fold increase of protein nitration/oxidation between mitochondrial proteins derived from healthy volunteers and MCI patients, with no further increase between MCI and AD patients. Proteomic studies will show whether specific proteins are target for nitration/oxidation during the pathogenesis of MCI and AD. Mitochondrial protein nitration/oxidation may be used for early screening of patients with the genetic risk for the development of AD or as companion diagnostic for pharmacological interventions.
The main machinery responsible for cellular protein maintenance is the ubiquitin-proteasomal system. The main task of the system is a fast and efficient degradation of proteins not needed anymore in cellular metabolism. It is accepted that upon oxidative stress, the proteasome suffers a series of functional alterations, including a reversible, oxidation-triggered 26 S proteasome disassembly into its catalytic (CP) and regulatory (RP) particles. Formation of low fluxes of oxidants were stimulated with antimycin or palmitic acid in pancreatic MIN6 cells, inducing a partial increase of dimerization of mitochondrial and citosolic peroxiredoxins. Under this conditions, no significant increase of total protein thiol oxidation was detected. However, ATP-stimulation of 26 S proteasomes was inhibited while the particle remained fully assemblied. Discrete cysteine residues of both CP and RP were identified oxidized by a cysteine-targeted proteomics approach. Such results may rely on differential reactivity of cysteines in the subunits and suggest specific regulation of particles by discrete cysteine oxidation. Further studies will elucidate the type of modification on each cysteine residue and the impact. Our results suggest a novel mechanism of redox regulation of 26 S proteasome that precedes oxidation-driven 26 S particle disassembly. E-mail address: [email protected] (M. Hugo) http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.322
P-238
Mitochondrial adaptive response in a model of CoQ10 deprived human dermal fibroblasts Fabio Marcheggiani 3, Ilenia Cirilli 3, Patrick Orlando 1, Anja Knott 2, Julia Weise 2, Luca Tiano 1
E-mail address: [email protected] (C. Schoneich) 1
Department of Life and Environmental Sciences, Polytechnic University of Marche, Italy 2 Research and Development, Beiersdorf AG, Hamburg, Germany 3 Department of Clinical and Dental Sciences, Polytechnic University of Marche, Italy
Acknowledgements
NIH. http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.321
P-237
Site-specific cysteine proteasomes
oxidation
regulates
26S
Martín Hugo 1, Ioana Korovila 1, Leticia Prates Roma 6, Antonio Martínez-Ruiz 2, Anabel Marina 3, Carlos Garcia 3, Tilman Grune 1,4,5 1
German Institute of Human Nutrition, Germany Immunology Department, Hospital of La Princesa, Research Institute Princesa (IP), Autonomous University of Madrid, Madrid 28009, Spain 3 Centro de Biología Molecular Severo Ochoa CBM-SO (CSIC-UAM), Cantoblanco , Spain 4 German Center for Diabetes Research (DZD), 85764 MünchenNeuherberg, Germany 5 German Center for Cardiovascular Research (DZHK), 10117 Berlin 6 Division of Redox Regulation, DKFZ-ZMBH (German Cancer Research Center-Center for Molecular Biology of the University of Heidelberg) Alliance, DKFZ, 69120 Heidelberg, Germany 2
CoQ10 is a ubiquitous isoprenilated quinone with a key role in cellular bioenergetic and as antioxidant in membranes. The organism produces adequate amount to support physiologic demand although biosynthesis could be influenced by genetic background, nutrition and lifestyle. Moreover, ageing and of HMG-CoA reductase inhibitors may play an important role in further lowering synthesis. At skin level CoQ10 decrease is known to be associated with senescence manifestation. In the present study we developed a model of CoQ10 deprivation in human dermal fibroblast to mimic processes associated with ageing in order to elucidate the role of this cofactor in mitochondrial remodeling and adaptive response. Flow cytometric analysis of mitochondrial function and ROS production was associated with respirometry profiling and molecular markers of mitochondrial biogenesis in cells exposed for 72hrs to a log range of simvastatin concentrations. Analysis outlined a series of complex adaptative response where statin dose dependently affected CoQ10 levels and oxidative status steering either hormetic responses or mitochondrial PTP opening associated to selective elimination of defective mitochondria or overt cellular toxicity stressing the central role of this molecule in the biochemistry of cell ageing
D. Rossin et al. / Free Radical Biology and Medicine 108 (2017) S18–S107
Keywords: lipotoxicity; ubiquitin-proteasome system; thiol
P-236
Oxidative Stress Markers of Alzheimer's Disease in Peripheral Cell Mitochondria Christian Schoneich 1, Asha Hewarathna 1, Ranu Pal 2, Lei Jiang 2, Elias Michaelis 2 1
University of Kansas, Pharmaceutical Chemistry, United States of America 2 University of Kansas, Pharmacology and Toxicology, USA Keywords: Alzheimer's Disease; oxidative stress; protein oxidation; mitochondria
Alzheimer's disease (AD) represents a neurodegenerative disease leading to progressive dementia in the elderly. AD has been associated with increased oxidative stress and mitochondrial dysfunction. Hence, markers for oxidative stress are expected to accumulate not only in brain mitochondria, but also in mitochondria of peripheral cells. We have developed and optimized a method for the rapid and selective fluorogenic derivatization and enrichment of specific protein oxidation products, 3-nitrotyrosine and 3,4-dihydroxyphenylalanine (DOPA), in order to quantitatively compare protein oxidation in the mitochondria of peripheral, white blood cells obtained from healthy donors, patients with mild cognitive impairment (MCI), and AD patients. Double blind studies reveal a ca. two-fold increase of protein nitration/oxidation between mitochondrial proteins derived from healthy volunteers and MCI patients, with no further increase between MCI and AD patients. Proteomic studies will show whether specific proteins are target for nitration/oxidation during the pathogenesis of MCI and AD. Mitochondrial protein nitration/oxidation may be used for early screening of patients with the genetic risk for the development of AD or as companion diagnostic for pharmacological interventions.
The main machinery responsible for cellular protein maintenance is the ubiquitin-proteasomal system. The main task of the system is a fast and efficient degradation of proteins not needed anymore in cellular metabolism. It is accepted that upon oxidative stress, the proteasome suffers a series of functional alterations, including a reversible, oxidation-triggered 26 S proteasome disassembly into its catalytic (CP) and regulatory (RP) particles. Formation of low fluxes of oxidants were stimulated with antimycin or palmitic acid in pancreatic MIN6 cells, inducing a partial increase of dimerization of mitochondrial and citosolic peroxiredoxins. Under this conditions, no significant increase of total protein thiol oxidation was detected. However, ATP-stimulation of 26 S proteasomes was inhibited while the particle remained fully assemblied. Discrete cysteine residues of both CP and RP were identified oxidized by a cysteine-targeted proteomics approach. Such results may rely on differential reactivity of cysteines in the subunits and suggest specific regulation of particles by discrete cysteine oxidation. Further studies will elucidate the type of modification on each cysteine residue and the impact. Our results suggest a novel mechanism of redox regulation of 26 S proteasome that precedes oxidation-driven 26 S particle disassembly. E-mail address: [email protected] (M. Hugo) http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.322
P-238
Mitochondrial adaptive response in a model of CoQ10 deprived human dermal fibroblasts Fabio Marcheggiani 3, Ilenia Cirilli 3, Patrick Orlando 1, Anja Knott 2, Julia Weise 2, Luca Tiano 1
E-mail address: [email protected] (C. Schoneich) 1
Department of Life and Environmental Sciences, Polytechnic University of Marche, Italy 2 Research and Development, Beiersdorf AG, Hamburg, Germany 3 Department of Clinical and Dental Sciences, Polytechnic University of Marche, Italy
Acknowledgements
NIH. http://dx.doi.org/10.1016/j.freeradbiomed.2017.04.321
P-237
Site-specific cysteine proteasomes
oxidation
regulates
26S
Martín Hugo 1, Ioana Korovila 1, Leticia Prates Roma 6, Antonio Martínez-Ruiz 2, Anabel Marina 3, Carlos Garcia 3, Tilman Grune 1,4,5 1
German Institute of Human Nutrition, Germany Immunology Department, Hospital of La Princesa, Research Institute Princesa (IP), Autonomous University of Madrid, Madrid 28009, Spain 3 Centro de Biología Molecular Severo Ochoa CBM-SO (CSIC-UAM), Cantoblanco , Spain 4 German Center for Diabetes Research (DZD), 85764 MünchenNeuherberg, Germany 5 German Center for Cardiovascular Research (DZHK), 10117 Berlin 6 Division of Redox Regulation, DKFZ-ZMBH (German Cancer Research Center-Center for Molecular Biology of the University of Heidelberg) Alliance, DKFZ, 69120 Heidelberg, Germany 2
CoQ10 is a ubiquitous isoprenilated quinone with a key role in cellular bioenergetic and as antioxidant in membranes. The organism produces adequate amount to support physiologic demand although biosynthesis could be influenced by genetic background, nutrition and lifestyle. Moreover, ageing and of HMG-CoA reductase inhibitors may play an important role in further lowering synthesis. At skin level CoQ10 decrease is known to be associated with senescence manifestation. In the present study we developed a model of CoQ10 deprivation in human dermal fibroblast to mimic processes associated with ageing in order to elucidate the role of this cofactor in mitochondrial remodeling and adaptive response. Flow cytometric analysis of mitochondrial function and ROS production was associated with respirometry profiling and molecular markers of mitochondrial biogenesis in cells exposed for 72hrs to a log range of simvastatin concentrations. Analysis outlined a series of complex adaptative response where statin dose dependently affected CoQ10 levels and oxidative status steering either hormetic responses or mitochondrial PTP opening associated to selective elimination of defective mitochondria or overt cellular toxicity stressing the central role of this molecule in the biochemistry of cell ageing