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Jack E Dixon - One of the best experts on this subject based on the ideXlab platform.
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Chapter 13 Localization and Function of the 2Fe‐2S Outer Mitochondrial Membrane Protein mitoNEET
Methods in enzymology, 2009Co-Authors: Sandra E Wiley, Matthew J. Rardin, Jack E DixonAbstract:MitoNEET is an integral protein of the Outer Mitochondrial Membrane and is the flagship of a small family of proteins whose hallmark is the presence of a CDGSH domain. Initially annotated as a zinc finger, the CDGSH domain actually binds a redox-active 2Fe-2S cluster, giving mitoNEET the distinction of being the first 2Fe-2S protein identified in the Outer Membrane of mitochondria. This chapter describes methods for isolating Mitochondrial Membrane fractions that are enriched in mitoNEET, generating constructs for the expression of recombinant mitoNEET protein and analyzing the 2Fe-2S cluster of mitoNEET in vitro.
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mitoneet is a uniquely folded 2fe 2s Outer Mitochondrial Membrane protein stabilized by pioglitazone
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Mark L Paddock, Sandra E Wiley, Edward C Abresch, Rachel Nechushtai, Anne N Murphy, Herbert L Axelrod, Aina E Cohen, Melinda Roy, Dominique T Capraro, Jack E DixonAbstract:Iron–sulfur (Fe–S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified Outer Mitochondrial Membrane Fe–S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the ≈650 reported Fe–S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a β-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe–2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe–2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe–S cluster transfer.
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MitoNEET is a uniquely folded 2Fe–2S Outer Mitochondrial Membrane protein stabilized by pioglitazone
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Mark L Paddock, Sandra E Wiley, Edward C Abresch, Rachel Nechushtai, Anne N Murphy, Herbert L Axelrod, Aina E Cohen, Melinda Roy, Dominique T Capraro, Jack E DixonAbstract:Iron–sulfur (Fe–S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified Outer Mitochondrial Membrane Fe–S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the ≈650 reported Fe–S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a β-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe–2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe–2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe–S cluster transfer.
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the Outer Mitochondrial Membrane protein mitoneet contains a novel redox active 2fe 2s cluster
Journal of Biological Chemistry, 2007Co-Authors: Sandra E Wiley, Mark L Paddock, Edward C Abresch, Larry A Gross, Peter Van Der Geer, Rachel Nechushtai, Anne N Murphy, Patricia A Jennings, Jack E DixonAbstract:The Outer Mitochondrial Membrane protein mitoNEET was discovered as a binding target of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class used to treat type 2 diabetes (Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M., Bannow, C. A., Lund, E. T., and Mathews, W. R. (2004) Am. J. Physiol. 286, E252-E260). We have shown that mitoNEET is a member of a small family of proteins containing a 39-amino-acid CDGSH domain. Although the CDGSH domain is annotated as a zinc finger motif, mitoNEET was shown to contain iron (Wiley, S. E., Murphy, A. N., Ross, S. A., van der Geer, P., and Dixon, J. E. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 5318-5323). Optical and electron paramagnetic resonance spectroscopy showed that it contained a redox-active pH-labile Fe-S cluster. Mass spectrometry showed the loss of 2Fe and 2S upon cofactor extrusion. Spectroscopic studies of recombinant proteins showed that the 2Fe-2S cluster was coordinated by Cys-3 and His-1. The His ligand was shown to be involved in the observed pH lability of the cluster, indicating that loss of this ligand via protonation triggered release of the cluster. mitoNEET is the first identified 2Fe-2S-containing protein located in the Outer Mitochondrial Membrane. Based on the biophysical data and domain fusion analysis, mitoNEET may function in Fe-S cluster shuttling and/or in redox reactions.
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mitoneet is an iron containing Outer Mitochondrial Membrane protein that regulates oxidative capacity
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Sandra E Wiley, Peter Van Der Geer, Anne N Murphy, Stuart A Ross, Jack E DixonAbstract:Members of the thiazolidinedione (TZD) class of insulin-sensitizing drugs are extensively used in the treatment of type 2 diabetes. Pioglitazone, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252–E260]. Bioinformatic analysis reveals that mitoNEET is a member of a small family of proteins containing a domain annotated as a CDGSH-type zinc finger. Although annotated as a zinc finger protein, mitoNEET contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in iron binding. Localization studies demonstrate that mitoNEET is an integral protein present in the Outer Mitochondrial Membrane. An amino-terminal anchor sequence tethers the protein to the Outer Membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mito- NEET is an important iron-containing protein involved in the control of maximal Mitochondrial respiratory rates.
Marco Colombini - One of the best experts on this subject based on the ideXlab platform.
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bcl xl promotes the open configuration of the voltage dependent anion channel and metabolite passage through the Outer Mitochondrial Membrane
Journal of Biological Chemistry, 2001Co-Authors: E Gottleib, R B Hill, C B Thompson, Marco ColombiniAbstract:The diffusion of metabolites across the Outer Mitochondrial Membrane is essential for coupled cellular respiration. The Outer Membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-x(L) restores metabolite exchange across the Outer Membrane without inducing the loss of cytochrome c from the interMembrane space. Restoration of Outer Membrane permeability to anionic metabolites does not occur directly through Bcl-x(L) ion channels. Instead, recombinant Bcl-x(L) maintains the Outer Mitochondrial Membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous beta-NADH, recombinant Bcl-x(L) can sustain Outer Mitochondrial Membrane permeability to ADP. beta-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the Outer Mitochondrial Membrane by inhibiting VDAC closure.
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bcl x l promotes the open configuration of the voltage dependent anion channel and metabolite passage through the Outer Mitochondrial Membrane
Journal of Biological Chemistry, 2001Co-Authors: Matthew Vander G Heiden, Xiao Xian Li, Craig B Thompson, E Gottleib, Blake R Hill, Marco ColombiniAbstract:Abstract The diffusion of metabolites across the Outer Mitochondrial Membrane is essential for coupled cellular respiration. The Outer Membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-xL restores metabolite exchange across the Outer Membrane without inducing the loss of cytochrome c from the interMembrane space. Restoration of Outer Membrane permeability to anionic metabolites does not occur directly through Bcl-xLion channels. Instead, recombinant Bcl-xL maintains the Outer Mitochondrial Membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous β-NADH, recombinant Bcl-xL can sustain Outer Mitochondrial Membrane permeability to ADP. β-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-xL can maintain metabolite exchange across the Outer Mitochondrial Membrane by inhibiting VDAC closure.
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Outer Mitochondrial Membrane permeability can regulate coupled respiration and cell survival
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Matthew Vander G Heiden, Marco Colombini, Navdeep S Chandel, Xiao Xian Li, Paul T Schumacker, Craig B ThompsonAbstract:Coupled cellular respiration requires that ATP and ADP be efficiently exchanged between the cytosol and the Mitochondrial matrix. When growth factors are withdrawn from dependent cells, metabolism is disrupted by a defect in ATP/ADP exchange across the Mitochondrial Membranes. Unexpectedly, we find that this defect results from loss of Outer Mitochondrial Membrane permeability to metabolic anions. This decrease in anion permeability correlates with the changes in conductance properties that accompany closure of the voltage-dependent anion channel (also known as Mitochondrial porin). Loss of Outer Membrane permeability (i) results in the accumulation of stored metabolic energy within the interMembrane space in the form of creatine phosphate, (ii) is prevented by the Outer Mitochondrial Membrane proteins Bcl-xL and Bcl-2, and (iii) can be reversed by growth factor readdition. If Outer Membrane impermeability persists, the disruption of Mitochondrial homeostasis culminates in loss of Outer Mitochondrial Membrane integrity, cytochrome c redistribution, and apoptosis. The recognition that Outer Membrane permeability is regulated under physiological conditions has important implications for the understanding of bioenergetics and cell survival.
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the Outer Mitochondrial Membrane channel vdac is modulated by a protein localized in the interMembrane space
Biochimica et Biophysica Acta, 1993Co-Authors: Marcia J Holden, Marco ColombiniAbstract:The Mitochondrial Outer Membrane channel, VDAC, provides a pathway for the flux of metabolites between the cytoplasm and mitochondrion. VDAC is voltage-dependent and occupies states of differing conductivity and ion selectivity that are dependent on transMembrane potential. A protein, derived from preparations of mitochondria, has been shown to increase the voltage dependence of VDAC and is called the VDAC modulator. Both VDAC and the VDAC modulator have been extensively characterized by reconstitution into planar lipid bilayers. In order for the VDAC modulator to have physiological significance it must have physical access to VDAC in the cell. This constraint dictates that the modulator be an extrinsic Outer Mitochondrial Membrane protein, occupy the Mitochondrial interMembrane space, or be a cytoplasmic constituent. To address the question of subcellular localization, purified mitochondria were selectively lysed with digitonin or treated with trypsin while resuspended in hypo-osmotic or iso-osmotic medium. Marker enzymes and modulator activity were monitored during the various treatments. Results indicate that the integrity of the Outer Membrane was necessary to prevent modulator release or protection from trypsin digestion. Outer Membrane lysis, under conditions where the inner Membrane remained intact, resulted in modulator release or inactivation by trypsin. These results suggest an interMembrane space location for the VDAC modulator in the mitochondrion.
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cloning and functional expression in yeast of two human isoforms of the Outer Mitochondrial Membrane channel the voltage dependent anion channel
Journal of Biological Chemistry, 1993Co-Authors: E Blachlydyson, Marco Colombini, E B Zambronicz, V Adams, E R Mccabe, John P Adelman, Michael ForteAbstract:Abstract The voltage-dependent anion channel (VDAC) of the Outer Mitochondrial Membrane is a small abundant protein found in all eukaryotic kingdoms which forms a voltage-gated pore when incorporated into planar lipid bilayers. VDAC is also the site of binding of the metabolic enzymes hexokinase and glycerol kinase to the mitochondrion in what may be a significant metabolic regulatory interaction. Recently, there has been speculation that there may be multiple forms of VDAC in mammals which differ in their localization in the Outer Mitochondrial Membrane and in their physiological function. In this report, we describe the identification and characterization of two human cDNAs encoding VDAC homologs (HVDAC1 and HVDAC2). To confirm VDAC function, each human protein has been expressed in yeast lacking the endogenous VDAC gene. Human proteins isolated from yeast mitochondria formed channels with the characteristics expected of VDAC when incorporated into planar lipid bilayers. In addition, expression of the human proteins in such strains can complement phenotypic defects associated with elimination of the endogenous yeast VDAC gene. Since VDAC is the site of binding of hexokinase to the Outer Mitochondrial Membrane, the binding capacity of each VDAC isoform expressed in yeast mitochondria was assessed. When compared with the binding of hexokinase to mitochondria lacking VDAC, the results show that mitochondria expressing HVDAC1 are capable of specifically binding hexokinase, whereas mitochondria expressing HVDAC2 only bind hexokinase at background levels. The expression of each human cDNA has been assessed by Northern blot and polymerase chain reaction techniques. With one exception, each is expressed in all human cell lines and tissues examined.
Sandra E Wiley - One of the best experts on this subject based on the ideXlab platform.
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Chapter 13 Localization and Function of the 2Fe‐2S Outer Mitochondrial Membrane Protein mitoNEET
Methods in enzymology, 2009Co-Authors: Sandra E Wiley, Matthew J. Rardin, Jack E DixonAbstract:MitoNEET is an integral protein of the Outer Mitochondrial Membrane and is the flagship of a small family of proteins whose hallmark is the presence of a CDGSH domain. Initially annotated as a zinc finger, the CDGSH domain actually binds a redox-active 2Fe-2S cluster, giving mitoNEET the distinction of being the first 2Fe-2S protein identified in the Outer Membrane of mitochondria. This chapter describes methods for isolating Mitochondrial Membrane fractions that are enriched in mitoNEET, generating constructs for the expression of recombinant mitoNEET protein and analyzing the 2Fe-2S cluster of mitoNEET in vitro.
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mitoneet is a uniquely folded 2fe 2s Outer Mitochondrial Membrane protein stabilized by pioglitazone
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Mark L Paddock, Sandra E Wiley, Edward C Abresch, Rachel Nechushtai, Anne N Murphy, Herbert L Axelrod, Aina E Cohen, Melinda Roy, Dominique T Capraro, Jack E DixonAbstract:Iron–sulfur (Fe–S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified Outer Mitochondrial Membrane Fe–S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the ≈650 reported Fe–S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a β-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe–2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe–2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe–S cluster transfer.
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MitoNEET is a uniquely folded 2Fe–2S Outer Mitochondrial Membrane protein stabilized by pioglitazone
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Mark L Paddock, Sandra E Wiley, Edward C Abresch, Rachel Nechushtai, Anne N Murphy, Herbert L Axelrod, Aina E Cohen, Melinda Roy, Dominique T Capraro, Jack E DixonAbstract:Iron–sulfur (Fe–S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified Outer Mitochondrial Membrane Fe–S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the ≈650 reported Fe–S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a β-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe–2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe–2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe–S cluster transfer.
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the Outer Mitochondrial Membrane protein mitoneet contains a novel redox active 2fe 2s cluster
Journal of Biological Chemistry, 2007Co-Authors: Sandra E Wiley, Mark L Paddock, Edward C Abresch, Larry A Gross, Peter Van Der Geer, Rachel Nechushtai, Anne N Murphy, Patricia A Jennings, Jack E DixonAbstract:The Outer Mitochondrial Membrane protein mitoNEET was discovered as a binding target of pioglitazone, an insulin-sensitizing drug of the thiazolidinedione class used to treat type 2 diabetes (Colca, J. R., McDonald, W. G., Waldon, D. J., Leone, J. W., Lull, J. M., Bannow, C. A., Lund, E. T., and Mathews, W. R. (2004) Am. J. Physiol. 286, E252-E260). We have shown that mitoNEET is a member of a small family of proteins containing a 39-amino-acid CDGSH domain. Although the CDGSH domain is annotated as a zinc finger motif, mitoNEET was shown to contain iron (Wiley, S. E., Murphy, A. N., Ross, S. A., van der Geer, P., and Dixon, J. E. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 5318-5323). Optical and electron paramagnetic resonance spectroscopy showed that it contained a redox-active pH-labile Fe-S cluster. Mass spectrometry showed the loss of 2Fe and 2S upon cofactor extrusion. Spectroscopic studies of recombinant proteins showed that the 2Fe-2S cluster was coordinated by Cys-3 and His-1. The His ligand was shown to be involved in the observed pH lability of the cluster, indicating that loss of this ligand via protonation triggered release of the cluster. mitoNEET is the first identified 2Fe-2S-containing protein located in the Outer Mitochondrial Membrane. Based on the biophysical data and domain fusion analysis, mitoNEET may function in Fe-S cluster shuttling and/or in redox reactions.
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mitoneet is an iron containing Outer Mitochondrial Membrane protein that regulates oxidative capacity
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Sandra E Wiley, Peter Van Der Geer, Anne N Murphy, Stuart A Ross, Jack E DixonAbstract:Members of the thiazolidinedione (TZD) class of insulin-sensitizing drugs are extensively used in the treatment of type 2 diabetes. Pioglitazone, a member of the TZD family, has been shown to bind specifically to a protein named mitoNEET [Colca JR, McDonald WG, Waldon DJ, Leone JW, Lull JM, Bannow CA, Lund ET, Mathews WR (2004) Am J Physiol 286:E252–E260]. Bioinformatic analysis reveals that mitoNEET is a member of a small family of proteins containing a domain annotated as a CDGSH-type zinc finger. Although annotated as a zinc finger protein, mitoNEET contains no zinc, but instead contains 1.6 mol of Fe per mole of protein. The conserved sequence C-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in iron binding. Localization studies demonstrate that mitoNEET is an integral protein present in the Outer Mitochondrial Membrane. An amino-terminal anchor sequence tethers the protein to the Outer Membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mito- NEET is an important iron-containing protein involved in the control of maximal Mitochondrial respiratory rates.
Cecile Martel - One of the best experts on this subject based on the ideXlab platform.
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glycogen synthase kinase 3 mediated voltage dependent anion channel phosphorylation controls Outer Mitochondrial Membrane permeability during lipid accumulation
Hepatology, 2013Co-Authors: Cecile Martel, Maya Allouche, Degli D Esposti, Elena Fanelli, Celine Boursier, Celine Henry, Joel Chopineau, Giuseppe CalamitaAbstract:Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early Mitochondrial impairment and late hepatocyte cell death. However, Mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high-fat diet, and in vitro models of lipotoxicity, we show that Outer Mitochondrial Membrane permeability is altered and identified a posttranslational modification of voltage-dependent anion channel (VDAC), a Membrane channel and NADH oxidase, as a cause of early Mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. Conclusion: VDAC acts as an early sensor of lipid toxicity and its GSK3-mediated phosphorylation status controls Outer Mitochondrial Membrane permeabilization in hepatosteatosis. (HEPATOLOGY 2013)
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glycogen synthase kinase 3 mediated voltage dependent anion channel phosphorylation controls Outer Mitochondrial Membrane permeability during lipid accumulation
Hepatology, 2013Co-Authors: Cecile Martel, Maya Allouche, Degli D Esposti, Elena Fanelli, Celine Boursier, Celine Henry, Joel Chopineau, Giuseppe CalamitaAbstract:UNLABELLED: Nonalcoholic steatosis is a liver pathology characterized by fat accumulation and severe metabolic alterations involving early Mitochondrial impairment and late hepatocyte cell death. However, Mitochondrial dysfunction mechanisms remain elusive. Using four models of nonalcoholic steatosis, i.e., livers from patients with fatty liver disease, ob/ob mice, mice fed a high-fat diet, and in vitro models of lipotoxicity, we show that Outer Mitochondrial Membrane permeability is altered and identified a posttranslational modification of voltage-dependent anion channel (VDAC), a Membrane channel and NADH oxidase, as a cause of early Mitochondrial dysfunction. Thus, in nonalcoholic steatosis VDAC exhibits reduced threonine phosphorylation, which increases the influx of water and calcium into mitochondria, sensitizes the organelle to matrix swelling, depolarization, and cytochrome c release without inducing cell death. This also amplifies VDAC enzymatic and channel activities regulation by calcium and modifies its interaction with proteic partners. Moreover, lipid accumulation triggers a rapid lack of VDAC phosphorylation by glycogen synthase kinase 3 (GSK3). Pharmacological and genetic manipulations proved GSK3 to be responsible for VDAC phosphorylation in normal cells. Notably, VDAC phosphorylation level correlated with steatosis severity in patients. CONCLUSION: VDAC acts as an early sensor of lipid toxicity and its GSK3-mediated phosphorylation status controls Outer Mitochondrial Membrane permeabilization in hepatosteatosis.
Ralf Kölling - One of the best experts on this subject based on the ideXlab platform.
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The yeast deubiquitinating enzyme Ubp16 is anchored to the Outer Mitochondrial Membrane.
FEBS letters, 2003Co-Authors: Andrea Kinner, Ralf KöllingAbstract:We looked for Membrane-associated Dubs (deubiquitinating enzymes) among the 16 yeast members of the ubiquitin-specific processing protease (Ubp) family to identify potential regulators of ubiquitin-dependent processes at Membranes. For each of the Ubps examined, a certain fraction was found to be Membrane associated. This fraction was only small for most Ubps but quite substantial for some Ubps. For Ubp4/Doa4 almost 40% of the protein was found in the Membrane fraction suggesting that this protein performs a major function at Membranes, probably at endosomes. Among the proteins tested, only one protein (Ubp16) was exclusively Membrane associated. By cell fractionation and immunofluorescence experiments, we could show that Ubp16 is localized to mitochondria. Ubp16 contains an N-terminal hydrophobic domain that is similar to N-terminal sequences of other yeast Outer Mitochondrial Membrane proteins. The presence of this putative signal sequence and the result of protease protection experiments suggest that Ubp16 is an integral Membrane protein of the Outer Mitochondrial Membrane with an N(in)-C(out) orientation. Phenotypic characterization of the Deltaubp16 mutant and overexpression studies further suggest that Ubp16 is probably not important for the general functioning of mitochondria, but that it rather performs a more specialized function at mitochondria.
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The yeast deubiquitinating enzyme Ubp16 is anchored to the Outer Mitochondrial Membrane
FEBS Letters, 2003Co-Authors: Andrea Kinner, Ralf KöllingAbstract:We looked for Membrane-associated Dubs (deubiquitinating enzymes) among the 16 yeast members of the ubiquitin-specific processing protease (Ubp) family to identify potential regulators of ubiquitin-dependent processes at Membranes. For each of the Ubps examined, a certain fraction was found to be Membrane associated. This fraction was only small for most Ubps but quite substantial for some Ubps. For Ubp4/Doa4 almost 40% of the protein was found in the Membrane fraction suggesting that this protein performs a major function at Membranes, probably at endosomes. Among the proteins tested, only one protein (Ubp16) was exclusively Membrane associated. By cell fractionation and immunofluorescence experiments, we could show that Ubp16 is localized to mitochondria. Ubp16 contains an N-terminal hydrophobic domain that is similar to N-terminal sequences of other yeast Outer Mitochondrial Membrane proteins. The presence of this putative signal sequence and the result of protease protection experiments suggest that Ubp16 is an integral Membrane protein of the Outer Mitochondrial Membrane with an Nin–Cout orientation. Phenotypic characterization of the Δubp16 mutant and overexpression studies further suggest that Ubp16 is probably not important for the general functioning of mitochondria, but that it rather performs a more specialized function at mitochondria.
