The Experts below are selected from a list of 23736 Experts worldwide ranked by ideXlab platform
Daniel Ricquier - One of the best experts on this subject based on the ideXlab platform.
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Avian UCP: The Killjoy in the Evolution of the Mitochondrial Uncoupling Proteins
Journal of Molecular Evolution, 2007Co-Authors: Yalin Emre, Daniel Ricquier, Frédéric Bouillaud, Corinne Hurtaud, Joseph Hughes, François CriscuoloAbstract:The understanding of mitochondrial functioning is of prime importance since it combines the production of energy as adenosine triphosphate (ATP) with an efficient chain of redox reactions, but also with the unavoidable production of reactive oxygen species (ROS) involved in aging. Mitochondrial respiration may be uncoupled from ATP synthesis by a proton leak induced by the thermogenic uncoupling protein 1 (UCP1). Mild uncoupling activity, as proposed for UCP2, UCP3, and avian UCP could theoretically control ROS production, but the nature of their transport activities is far from being definitively understood. The recent discovery of a UCP1 gene in fish has balanced the evolutionary view of uncoupling protein history. The thermogenic proton transport of mammalian UCP1 seems now to be a late evolutionary characteristic and the hypothesis that ancestral UCPs may carry other substrates is tempting. Using in silico genome analyses among taxa and a biochemical approach, we present a detailed phylogenetic analysis of UCPs and investigate whether avian UCP is a good candidate for pleiotropic mitochondrial activities, knowing that only one UCP has been characterized in the avian genome, unlike all other vertebrates. We show, here, that the avian class seems to be the only vertebrate lineage lacking two of the UCP1/2/3 homologues present in fish and mammals. We suggest, based on phylogenetic evidence and synteny of the UCP genes, that birds have lost UCP1 and UCP2. The phylogeny also supports the history of two rounds of duplication during vertebrate evolution. The avian uncoupling protein then represents a unique opportunity to explore how UCPs’ activities are controlled, but also to understand why birds exhibit such a particular relationship between high metabolism and slow rate of aging.
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UCP2 is a mitochondrial transporter with an unusual very short half-life.
FEBS Letters, 2007Co-Authors: Sophie Rousset, Daniel Ricquier, Julien Mozo, Sandrine Masscheleyn, Yalin Emre, Geneviève Dujardin, Anne-marie Cassard-doulcierAbstract:This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30min, compared to 30h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.
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UCP2 is a mitochondrial transporter with an unusual very short half‐life
FEBS letters, 2007Co-Authors: Sophie Rousset, Daniel Ricquier, Julien Mozo, Sandrine Masscheleyn, Yalin Emre, Geneviève Dujardin, Anne-marie Cassard-doulcierAbstract:This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30 min, compared to 30 h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.
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Thermoregulation: What Role for UCPs in Mammals and Birds?
Bioscience Reports, 2005Co-Authors: Julien Mozo, Daniel Ricquier, Frédéric Bouillaud, Yalin Emre, François CriscuoloAbstract:Mammals and birds are endotherms and respond to cold exposure by the means of regulatory thermogenesis, either shivering or non-shivering. In this latter case, waste of cell energy as heat can be achieved by uncoupling of mitochondrial respiration. Uncoupling proteins, which belong to the mitochondrial carrier family, are able to transport protons and thus may assume a thermogenic function. The mammalian UCP1 physiological function is now well understood and gives to the brown adipose tissue the capacity for heat generation. But is it really the case for its more recently discovered isoforms UCP2 and UCP3? Additionally, whereas more and more evidence suggests that non-shivering also exists in birds, is the avian UCP also involved in response to cold exposure? In this review, we consider the latest advances in the field of UCP biology and present putative functions for UCP1 homologues.
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The biology of mitochondrial uncoupling proteins.
Diabetes, 2004Co-Authors: Sophie Rousset, Frédéric Bouillaud, Marie-clotilde Alves-guerra, Julien Mozo, Bruno Miroux, Anne-marie Cassard-doulcier, Daniel RicquierAbstract:Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They are found in all mammals and in plants. They belong to the family of anion mitochondrial carriers including adenine nucleotide transporters. The term "uncoupling protein" was originally used for UCP1, which is uniquely present in mitochondria of brown adipocytes, the thermogenic cells that maintain body temperature in small rodents. In these cells, UCP1 acts as a proton carrier activated by free fatty acids and creates a shunt between complexes of the respiratory chain and ATP synthase. Activation of UCP1 enhances respiration, and the uncoupling process results in a futile cycle and dissipation of oxidation energy as heat. UCP2 is ubiquitous and highly expressed in the lymphoid system, macrophages, and pancreatic islets. UCP3 is mainly expressed in skeletal muscles. In comparison to the established uncoupling and thermogenic activities of UCP1, UCP2 and UCP3 appear to be involved in the limitation of free radical levels in cells rather than in physiological uncoupling and thermogenesis. Moreover, UCP2 is a regulator of insulin secretion and UCP3 is involved in fatty acid metabolism.
Frédéric Bouillaud - One of the best experts on this subject based on the ideXlab platform.
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Glutathionylation Acts as a Control Switch for Uncoupling Proteins UCP2 and UCP3
The Journal of biological chemistry, 2011Co-Authors: Ryan J Mailloux, Frédéric Bouillaud, Sheila Collins, Erin L. Seifert, Céline Aguer, Mary-ellen HarperAbstract:The mitochondrial uncoupling proteins 2 and 3 (UCP2 and -3) are known to curtail oxidative stress and participate in a wide array of cellular functions, including insulin secretion and the regulation of satiety. However, the molecular control mechanism(s) governing these proteins remains elusive. Here we reveal that UCP2 and UCP3 contain reactive cysteine residues that can be conjugated to glutathione. We further demonstrate that this modification controls UCP2 and UCP3 function. Both reactive oxygen species and glutathionylation were found to activate and deactivate UCP3-dependent increases in non-phosphorylating respiration. We identified both Cys(25) and Cys(259) as the major glutathionylation sites on UCP3. Additional experiments in thymocytes from wild-type and UCP2 null mice demonstrated that glutathionylation similarly diminishes non-phosphorylating respiration. Our results illustrate that UCP2- and UCP3-mediated state 4 respiration is controlled by reversible glutathionylation. Altogether, these findings advance our understanding of the roles UCP2 and UCP3 play in modulating metabolic efficiency, cell signaling, and oxidative stress processes.
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UCP2, not a physiologically relevant uncoupler but a glucose sparing switch impacting ROS production and glucose sensing
Biochimica et biophysica acta, 2009Co-Authors: Frédéric BouillaudAbstract:In mammals the two proteins UCP2 and UCP3 are highly similar to the mitochondrial uncoupling protein found in the brown adipose tissue (UCP1). Accordingly, it was proposed that UCP2 and UCP3 are also uncoupling proteins i.e. protonophores with impact on mitochondrial ROS production and glucose signaling. However, it appears now impossible to explain the physiological relevance of the new UCPs uniquely by their uncoupling activity as observed in vitro. Therefore, we propose a metabolic hypothesis in which UCP2 acts through a transport distinct of the proton transport. A consequence of this transport activity would be a decrease of the mitochondrial oxidation of the pyruvate originating from glucose. This would put UCP2 and UCP3 in a crucial position to influence cellular metabolism. The tight control exerted on UCP2 expression appears consistent with it. In this hypothesis, UCP2/3 would allow a cell to remain glycolytic within an aerobic organism. This tallies with the high expression level of UCP2 or UCP3 in glycolytic cells. The metabolic hypothesis would explain the spectacular modifications associated with UCP2 manipulation as well as the uncoupling activity usually called for and which in fact remains elusive in vivo.
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Avian UCP: The Killjoy in the Evolution of the Mitochondrial Uncoupling Proteins
Journal of Molecular Evolution, 2007Co-Authors: Yalin Emre, Daniel Ricquier, Frédéric Bouillaud, Corinne Hurtaud, Joseph Hughes, François CriscuoloAbstract:The understanding of mitochondrial functioning is of prime importance since it combines the production of energy as adenosine triphosphate (ATP) with an efficient chain of redox reactions, but also with the unavoidable production of reactive oxygen species (ROS) involved in aging. Mitochondrial respiration may be uncoupled from ATP synthesis by a proton leak induced by the thermogenic uncoupling protein 1 (UCP1). Mild uncoupling activity, as proposed for UCP2, UCP3, and avian UCP could theoretically control ROS production, but the nature of their transport activities is far from being definitively understood. The recent discovery of a UCP1 gene in fish has balanced the evolutionary view of uncoupling protein history. The thermogenic proton transport of mammalian UCP1 seems now to be a late evolutionary characteristic and the hypothesis that ancestral UCPs may carry other substrates is tempting. Using in silico genome analyses among taxa and a biochemical approach, we present a detailed phylogenetic analysis of UCPs and investigate whether avian UCP is a good candidate for pleiotropic mitochondrial activities, knowing that only one UCP has been characterized in the avian genome, unlike all other vertebrates. We show, here, that the avian class seems to be the only vertebrate lineage lacking two of the UCP1/2/3 homologues present in fish and mammals. We suggest, based on phylogenetic evidence and synteny of the UCP genes, that birds have lost UCP1 and UCP2. The phylogeny also supports the history of two rounds of duplication during vertebrate evolution. The avian uncoupling protein then represents a unique opportunity to explore how UCPs’ activities are controlled, but also to understand why birds exhibit such a particular relationship between high metabolism and slow rate of aging.
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UCP2, UCP3, avUCP, what do they do when proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism.
BBA - Biochimica et Biophysica Acta, 2006Co-Authors: François Criscuolo, Julien Mozo, Corinne Hurtaud, Tobias Nübel, Frédéric BouillaudAbstract:Uncoupling proteins (UCPs) are specialized members of the mitochondrial transporter family. They allow passive proton transport through the mitochondrial inner membrane. This activity leads to uncoupling of mitochondrial respiration and to energy waste, which is well documented with UCP1 in brown adipose tissue. The uncoupling activity of the new UCPs (discovered after 1997), such as UCP2 and UCP3 in mammals or avUCP in birds, is more difficult to characterize. However, extensive data support the idea that the new UCPs are involved in the control of reactive oxygen species (ROS) generation. This fits with the hypothesis that mild uncoupling caused by the UCPs prevents ROS production. Activators and inhibitors regulate the proton transport activity of the UCPs. In the absence of activators of proton transport, the UCP allows the permeation of other ions. We suggest that this activity has physiological significance and, for example, UCP3 expressed in glycolytic muscle fibres may be a passive pyruvate transporter ensuring equilibrium between glycolysis and oxidative phosphorylation. Induction of UCP2 expression by glutamine strengthens the proposal that new UCPs could act to determine the choice of mitochondrial substrate. This would obviously have an impact on mitochondrial bioenergetics and ROS production.
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Assessment of a high-throughput screening methodology for the measurement of purified UCP1 uncoupling activity.
Analytical Biochemistry, 2006Co-Authors: Julien Mozo, Bruno Miroux, Gilles Ferry, Jean A Boutin, Sandrine Masscheleyn, Frédéric BouillaudAbstract:Three mitochondrial uncoupling proteins (UCP1, 2, 3) have been described. The proton transport activity of UCP1 triggers mitochondrial uncoupling and thermogenesis but the roles of UCP2 and UCP3 remain debated. Accordingly, compounds able to finely control the proton permeability of the mitochondrial inner membrane where and when needed may have enormous practical consequences. Using purified hamster brown adipose tissue UCP1 reconstituted in liposomes, we describe herein a robust assay allowing the measurement of this artificial membrane conductance to protons in a format compatible with high-throughput screening. The assay was initially developed with a known chemical protonophore in an aproteic system. Then, using the proteolipid reconstituted UCP1 preparation, we assessed the assay with known modulators of UCP1, particularly retinoic acid and guanosine 5'-triphosphate. The system was developed for a 96-well plate format. We then exemplified its use by generating primary data on a set of compounds screened in this system. These primary data will open new routes for the search of candidate compounds that will help biochemical studies on UCPs.
Martin D. Brand - One of the best experts on this subject based on the ideXlab platform.
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The regulation and turnover of mitochondrial uncoupling proteins
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2010Co-Authors: Vian Azzu, Martin Jastroch, Ajit S. Divakaruni, Martin D. BrandAbstract:AbstractUncoupling proteins (UCP1, UCP2 and UCP3) are important in regulating cellular fuel metabolism and as attenuators of reactive oxygen species production through strong or mild uncoupling. The generic function and broad tissue distribution of the uncoupling protein family means that they are increasingly implicated in a range of pathophysiological processes including obesity, insulin resistance and diabetes mellitus, neurodegeneration, cardiovascular disease, immunity and cancer. The significant recent progress describing the turnover of novel uncoupling proteins, as well as current views on the physiological roles and regulation of UCPs, is outlined
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The on-off switches of the mitochondrial uncoupling proteins
Trends in biochemical sciences, 2009Co-Authors: Vian Azzu, Martin D. BrandAbstract:Mitochondrial uncoupling proteins disengage substrate oxidation from ADP phosphorylation by dissipating the proton electrochemical gradient that is required for ATP synthesis. In doing this, the archetypal uncoupling protein, UCP1, mediates adaptive thermogenesis. By contrast, its paralogues UCP2 and UCP3 are not thought to mediate whole body thermogenesis in mammals. Instead, they have been implicated in a variety of physiological and pathological processes, including protection from oxidative stress, negative regulation of glucose sensing systems and the adaptation of fatty acid oxidation capacity to starving. Although much work has been devoted to how these proteins are activated, little is known of the mechanisms that reverse this activation.
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Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential
Bioscience Reports, 2008Co-Authors: Nadeene Parker, Antonio J Vidal-puig, Martin D. BrandAbstract:Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of reactive oxygen species. This proton leak can be induced by lipid peroxidation products of reactive oxygen species, such as 4-hydroxynonenal (HNE). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and the adenine nucleotide translocase, thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We find that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent upon incubation with respiratory substrate. In the presence of HNE, mitochondria energised with succinate become progressively more leaky to protons over time compared to mitochondria in the absence of either HNE or succinate. Energised mitochondria must attain a high membrane potential to allow HNE to activate uncoupling; a drop of 10 – 20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), adenine nucleotide translocase (64%) and other pathways (25%). Our findings show that exogenous HNE only activates uncoupling at high membrane potential. They suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production
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The reactions catalysed by the mitochondrial uncoupling proteins UCP2 and UCP3
Biochimica et biophysica acta, 2005Co-Authors: Telma C. Esteves, Martin D. BrandAbstract:The mitochondrial uncoupling proteins UCP2 and UCP3 may be important in attenuating mitochondrial production of reactive oxygen species, in insulin signalling (UCP2), and perhaps in thermogenesis and other processes. To understand their physiological roles, it is necessary to know what reactions they are able to catalyse. We critically examine the evidence for proton transport and anion transport by UCP2 and UCP3. There is good evidence that they increase mitochondrial proton conductance when activated by superoxide, reactive oxygen species derivatives such as hydroxynonenal, and other alkenals or their analogues. However, they do not catalyse proton leak in the absence of such acute activation. They can also catalyse export of fatty acid and other anions, although the relationship of anion transport to proton transport remains controversial.
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Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3
Cell metabolism, 2005Co-Authors: Martin D. Brand, Telma C. EstevesAbstract:Evidence for the physiological functions of UCP2 and UCP3 is critically reviewed. They do not mediate adaptive thermogenesis, but they may be significantly thermogenic under specific pharmacological conditions. There is strong evidence that the mild regulated uncoupling they cause attenuates mitochondrial ROS production, protects against cellular damage, and diminishes insulin secretion. Evidence that they export fatty acids physiologically is weak. UCP2 and UCP3 are important potential targets for treatment of aging, degenerative diseases, diabetes, and perhaps obesity.
Bradford B. Lowell - One of the best experts on this subject based on the ideXlab platform.
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the mitochondrial uncoupling protein homologues
Nature Reviews Molecular Cell Biology, 2005Co-Authors: Stefan Krauss, Chen-yu Zhang, Bradford B. LowellAbstract:Uncoupling protein(UCP)1 is an integral membrane protein that is located in the mitochondrial inner membrane of brown adipocytes. Its physiological role is to mediate a regulated, thermogenic proton leak. UCP2 and UCP3 are recently identified UCP1 homologues. They also mediate regulated proton leak, and might function to control the production of superoxide and other downstream reactive oxygen species. However, their role in normal physiology remains unknown. Recent studies have shown that UCP2 has an important part in the pathogenesis of type-2 diabetes. The obscure roles of the UCP homologues in normal physiology, together with their emerging role in pathophysiology, provide exciting potential for further investigation.
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Uncoupling protein-3 (UCP3): a mitochondrial carrier in search of a function.
International Journal of Obesity, 1999Co-Authors: Bradford B. LowellAbstract:UCP3 is a mitochondrial protein with high homology to the established uncoupling protein, UCP1. Its high degree of homology to UCP1 suggests that UCP3 may be a true uncoupling protein. Preliminary biochemical studies are consistent with UCP3 having uncoupling activity. However, detailed functional studies are required to understand the true biochemical and physiological purpose of UCP3. These efforts should be aided by identification of humans with inactivating mutations and/or the generation of gene knockout mice lacking UCP3.
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the human uncoupling protein 3 gene genomic structure chromosomal localization and genetic basis for short and long form transcripts
Journal of Biological Chemistry, 1997Co-Authors: Gemma Solanes, Antonio Vidalpuig, Danica Grujic, Jeffrey S Flier, Bradford B. LowellAbstract:Abstract Uncoupling protein-3 (UCP3) is a recently identified candidate mediator of adaptive thermogenesis in humans. Unlike UCP1 and UCP2, UCP3is expressed preferentially and at high levels in human skeletal muscle and exists as short and long form transcripts,UCP3 S and UCP3 L.UCP3 S is predicted to encode a protein which lacks the last 37 C-terminal residues of UCP3 L. In the present study, we have defined the intron-exon structure for the human UCP3 gene and determined thatUCP3 S is generated when a cleavage and polyadenylation signal (AATAAA) located in the last intron prematurely terminates message elongation. In addition we have mappedUCP3 to the distal segment of human chromosome 11q13 (between framework markers D11S916 and D11S911), adjacent toUCP2. Of note, UCP2 and UCP3 in both mice and humans colocalize in P1 and BAC genomic clones indicating that these two UCPs are located within 75–150 kilobases of each other and most likely resulted from a gene duplication event. Previous studies have noted that mouse UCP2 maps to a region of chromosome 7 which is coincident with three independently mapped quantitative trait loci for obesity. Our study shows thatUCP3 is also coincident with these quantitative trait loci raising the possibility that abnormalities in UCP3 are responsible for obesity in these models.
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ucp3 an uncoupling protein homologue expressed preferentially and abundantly in skeletal muscle and brown adipose tissue
Biochemical and Biophysical Research Communications, 1997Co-Authors: Antonio Vidalpuig, Gemma Solanes, Danica Grujic, Jeffrey S Flier, Bradford B. LowellAbstract:Abstract Uncoupling proteins (UCPs) are inner mitochondrial membrane transporters which dissipate the proton gradient, releasing stored energy as heat. UCP1 is expressed exclusively in brown adipocytes while UCP2 is expressed widely. We now report the molecular cloning of a third uncoupling protein homologue, designated UCP3. At the amino acid level, hUCP3 is 71% identical to hUCP2 and 57% identical to hUCP1. UCP3 is distinguished from UCP1 and UCP2 by its abundant and preferential expression in skeletal muscle in humans, and brown adipose tissue and skeletal muscle in rodents. Since skeletal muscle and brown adipose tissue are believed to be important sites for regulated energy expenditure in humans and rodents, respectively, UCP3 may be an important mediator of adaptive thermogenesis. Since UCP3 is minimally expressed in human heart and other critical organs, it is a promising target for anti-obesity drug development aimed at increasing thermogenesis.
Julien Mozo - One of the best experts on this subject based on the ideXlab platform.
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UCP2 is a mitochondrial transporter with an unusual very short half-life.
FEBS Letters, 2007Co-Authors: Sophie Rousset, Daniel Ricquier, Julien Mozo, Sandrine Masscheleyn, Yalin Emre, Geneviève Dujardin, Anne-marie Cassard-doulcierAbstract:This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30min, compared to 30h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.
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UCP2 is a mitochondrial transporter with an unusual very short half‐life
FEBS letters, 2007Co-Authors: Sophie Rousset, Daniel Ricquier, Julien Mozo, Sandrine Masscheleyn, Yalin Emre, Geneviève Dujardin, Anne-marie Cassard-doulcierAbstract:This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30 min, compared to 30 h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.
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UCP2, UCP3, avUCP, what do they do when proton transport is not stimulated? Possible relevance to pyruvate and glutamine metabolism.
BBA - Biochimica et Biophysica Acta, 2006Co-Authors: François Criscuolo, Julien Mozo, Corinne Hurtaud, Tobias Nübel, Frédéric BouillaudAbstract:Uncoupling proteins (UCPs) are specialized members of the mitochondrial transporter family. They allow passive proton transport through the mitochondrial inner membrane. This activity leads to uncoupling of mitochondrial respiration and to energy waste, which is well documented with UCP1 in brown adipose tissue. The uncoupling activity of the new UCPs (discovered after 1997), such as UCP2 and UCP3 in mammals or avUCP in birds, is more difficult to characterize. However, extensive data support the idea that the new UCPs are involved in the control of reactive oxygen species (ROS) generation. This fits with the hypothesis that mild uncoupling caused by the UCPs prevents ROS production. Activators and inhibitors regulate the proton transport activity of the UCPs. In the absence of activators of proton transport, the UCP allows the permeation of other ions. We suggest that this activity has physiological significance and, for example, UCP3 expressed in glycolytic muscle fibres may be a passive pyruvate transporter ensuring equilibrium between glycolysis and oxidative phosphorylation. Induction of UCP2 expression by glutamine strengthens the proposal that new UCPs could act to determine the choice of mitochondrial substrate. This would obviously have an impact on mitochondrial bioenergetics and ROS production.
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Assessment of a high-throughput screening methodology for the measurement of purified UCP1 uncoupling activity.
Analytical Biochemistry, 2006Co-Authors: Julien Mozo, Bruno Miroux, Gilles Ferry, Jean A Boutin, Sandrine Masscheleyn, Frédéric BouillaudAbstract:Three mitochondrial uncoupling proteins (UCP1, 2, 3) have been described. The proton transport activity of UCP1 triggers mitochondrial uncoupling and thermogenesis but the roles of UCP2 and UCP3 remain debated. Accordingly, compounds able to finely control the proton permeability of the mitochondrial inner membrane where and when needed may have enormous practical consequences. Using purified hamster brown adipose tissue UCP1 reconstituted in liposomes, we describe herein a robust assay allowing the measurement of this artificial membrane conductance to protons in a format compatible with high-throughput screening. The assay was initially developed with a known chemical protonophore in an aproteic system. Then, using the proteolipid reconstituted UCP1 preparation, we assessed the assay with known modulators of UCP1, particularly retinoic acid and guanosine 5'-triphosphate. The system was developed for a 96-well plate format. We then exemplified its use by generating primary data on a set of compounds screened in this system. These primary data will open new routes for the search of candidate compounds that will help biochemical studies on UCPs.
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Expression of UCP3 in CHO cells does not cause uncoupling, but controls mitochondrial activity in the presence of glucose.
Biochemical Journal, 2006Co-Authors: Julien Mozo, Claire Pecqueur, Gilles Ferry, Aurélie Studeny, Marianne Rodriguez, Jean A Boutin, Frédéric BouillaudAbstract:The proton-transport activity of UCP1 (uncoupling protein 1) triggers mitochondrial uncoupling and thermogenesis. The exact role of its close homologues, UCP2 and UCP3, is unclear. Mounting evidence associates them with the control of mitochondrial superoxide production. Using CHO (Chinese-hamster ovary) cells stably expressing UCP3 or UCP1, we found no evidence for respiration uncoupling. The explanation lies in the absence of an appropriate activator of UCP protonophoric function. Accordingly, the addition of retinoic acid uncouples the respiration of the UCP1-expressing clone, but not that of the UCP3-expressing ones. In a glucose-containing medium, the extent of the hyperpolarization of mitochondria by oligomycin was close to 22 mV in the five UCP3-expressing clones, contrasting with the variable values observed with the 15 controls. Our observations suggest that, when glycolysis and mitochondria generate ATP, and in the absence of appropriate activators of proton transport, UCPs do not transport protons (uncoupling), but rather other ions of physiological relevance that control mitochondrial activity. A model is proposed using the known passive transport of pyruvate by UCP1.
