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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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Cloning and functional characterization of an Uncoupling Protein homolog in hummingbirds.
Physiological genomics, 2001Co-Authors: Claudia R. Vianna, Thilo Hagen, Chen-yu Zhang, Bradford B. Lowell, Eric S. Bachman, Olivier Boss, Balázs Gereben, Anselmo Sigari Moriscot, José Eduardo P. W. Bicudo, Antonio C. BiancoAbstract:The cDNA of an Uncoupling Protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird Uncoupling Protein (HmUCP) cDNA was amplified from pectoral...
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Assessment of Uncoupling activity of the human Uncoupling Protein 3 short form and three mutants of the Uncoupling Protein gene using a yeast heterologous expression system.
FEBS letters, 1999Co-Authors: Thilo Hagen, Chen-yu Zhang, Lawrence J. Slieker, Wendy K. Chung, Rudolph L. Leibel, Bradford B. LowellAbstract:The human Uncoupling Protein 3 gene generates two mRNA transcripts, Uncoupling Protein 3L and Uncoupling Protein 3S, which are predicted to encode long and short forms of the Uncoupling Protein 3 Protein, respectively. While Uncoupling Protein 3L is similar in length to the other known Uncoupling Proteins, Uncoupling Protein 3S lacks the last 37 C-terminal residues. A splice site mutation in the human Uncoupling Protein 3 gene, resulting in the exclusive expression of Uncoupling Protein 3S, and a number of point mutations in the Uncoupling Protein 3 gene have been described. This study compares the biochemical activity of Uncoupling Protein 3S as well as three mutants of the Uncoupling Protein 3 gene (V9M, V1021, R282C) with that of Uncoupling Protein 3L utilizing a yeast expression system. All Proteins were expressed at similar levels and had qualitatively similar effects on parameters related to the Uncoupling function. Both Uncoupling Protein 3S and Uncoupling Protein 3L decreased the yeast growth rate by 3S and 52%, increased the whole yeast basal O2 consumption by 26 and 48%, respectively, and decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodide. In isolated mitochondria, Uncoupling Protein 3S and Uncoupling Protein 3L caused a similar (33 and 35%, respectively) increase in state 4 respiration, which was relatively small compared to Uncoupling Protein 1 (102% increase). A truncated version of Uncoupling Protein 3S, lacking the last three C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif that is highly conserved among all mitochondrial carriers, had a greatly reduced Uncoupling activity. The two naturally occurring Uncoupling Protein 3 mutants, V9M and V1021, were similar to Uncoupling Protein 3L with respect to effects on the yeast growth and whole yeast O2 consumption. The R282C mutant had a reduced effect compared to Uncoupling Protein 3L. In summary, Uncoupling Protein 3S and the three mutants of Uncoupling Protein 3 appear to be functional Proteins with biochemical activities similar to Uncoupling Protein 3L, although Uncoupling Protein 3S and the R282C mutant have a modestly reduced function.
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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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Assessment of Uncoupling activity of Uncoupling Protein 3 using a yeast heterologous expression system.
FEBS letters, 1999Co-Authors: Chen-yu Zhang, Thilo Hagen, Lawrence J. Slieker, V K Mootha, Bradford B. LowellAbstract:Uncoupling Protein 3L, Uncoupling Protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both Uncoupling Protein 3L and Uncoupling Protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, Uncoupling Protein 1 increased the state 4 respiration by 1.8-fold, while Uncoupling Protein 3L increased the state 4 respiration by 1.2-fold. Interestingly, mutant Uncoupling Protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of Uncoupling Protein 1 when assessed using a proteoliposome system (Bienengraeber et al. (1998) Biochem. 37, 3-8), uncoupled the mitochondrial respiration to almost the same degree as wild-type Uncoupling Protein 1. Thus, absence of this histidine pair in Uncoupling Protein 2 and Uncoupling Protein 3 does not by itself rule out the possibility that these carriers have an Uncoupling function. The oxoglutarate carrier had no effect on any of the studied parameters. In summary, a discordance exists between the magnitude of effects of Uncoupling Protein 3L and Uncoupling Protein 1 in whole yeast versus isolated mitochondria, with Uncoupling Protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria. These findings suggest that Uncoupling Protein 3L, like Uncoupling Protein 1, has an Uncoupling activity. However, the mechanism of action and/or regulation of the activity of Uncoupling Protein 3L is likely to be different.
Martin Klingenspor - One of the best experts on this subject based on the ideXlab platform.
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Uncoupling Protein 1 and the capacity for nonshivering thermogenesis are components of the glucose homeostatic system
American Journal of Physiology-Endocrinology and Metabolism, 2020Co-Authors: Stefanie Maurer, Tobias Fromme, Sabine Mocek, Anika Zimmermann, Martin KlingensporAbstract:Uncoupling Protein 1 (Ucp1) provides nonshivering thermogenesis (NST) fueled by the dissipation of energy from macronutrients in brown and brite adipocytes. The availability of thermogenic fuels is...
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Fibroblast growth factor 8b induces Uncoupling Protein 1 expression in epididymal white preadipocytes
Scientific Reports, 2019Co-Authors: Sören Westphal, Martin Klingenspor, Thomas Gantert, Caroline Kless, Kristina Hüttinger, Tobias FrommeAbstract:The number of brown adipocytes residing within murine white fat depots (brite adipocytes) varies a lot by depot, strain and physiological condition. Several endocrine fibroblast growth factors are implicated in the regulation of brite adipocyte abundance. The family of fibroblast growth factors can be categorized by their site of action into endocrine, paracrine and intracellular peptides. We here screened paracrine fibroblast growth factors for their potential to drive brite adipogenesis in differentiating epididymal white adipocytes and identified fibroblast growth factor 8b to induce Uncoupling Protein 1 expression, but at the same time to interfere in adipogenesis. In an in vivo trial, fibroblast growth factor 8b released into the epididymal fat depot failed to robustly increase the number of brite adipocytes. The specific action of fibroblast growth factor 8b on the Uncoupling Protein 1 promoter in cultured epididymal adipocytes provides a model system to dissect specific gene regulatory networks.
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Uncoupling Protein 1 expression and high fat diets
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2011Co-Authors: Tobias Fromme, Martin KlingensporAbstract:Uncoupling Protein 1 (Ucp1) is the key component of β-adrenergically controlled nonshivering thermogenesis in brown adipocytes. This process combusts stored and nutrient energy as heat. Cold exposure not only activates Ucp1-mediated thermogenesis to maintain normothermia but also results in adaptive thermogenesis, i.e., the recruitment of thermogenic capacity in brown adipose tissue. As a hallmark of adaptive thermogenesis, Ucp1 synthesis is increased proportionally to temperature and duration of exposure. Beyond this classical thermoregulatory function, it has been suggested that Ucp1-mediated thermogenesis can also be employed for metabolic thermogenesis to prevent the development of obesity. Accordingly, in times of excess caloric intake, one may expect a positive regulation of Ucp1. The general impression from an overview of the present literature is, indeed, an increased brown adipose tissue Ucp1 mRNA and Protein content after feeding a high-fat diet (HFD) to mice and rats. The reported increases are very variable in magnitude, and the effect size seems to be independent of dietary fat content and duration of the feeding trial. In white adipose tissue depots Ucp1 mRNA is generally downregulated by HFD, indicating a decline in the number of interspersed brown adipocytes.
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Brown adipose tissue specific lack of Uncoupling Protein 3 is associated with impaired cold tolerance and reduced transcript levels of metabolic genes
Journal of Comparative Physiology B, 2008Co-Authors: Tobias Fromme, Gerhard Heldmaier, Carola W. Meyer, Christa Praun, Martin KlingensporAbstract:Uncoupling Protein 3 (Ucp3) is located within the mitochondrial inner membrane of brown adipose tissue and skeletal muscle. It is thought to be implicated in lipid metabolism and defense against reactive oxygen species. We previously reported on a mutation in our breeding colony of Djungarian hamsters ( Phodopus sungorus ) that leads to brown adipose tissue specific lack of Ucp3 expression. In this study we compared wildtype with mutant hamsters on a broad genetic background. Hamsters lacking Ucp3 in brown adipose tissue displayed a reduced cold tolerance due to impaired nonshivering thermogenesis. This phenotype is associated with a global decrease in expression of metabolic genes but not of Uncoupling Protein 1. These data implicate that Ucp3 is necessary to sustain high metabolic rates in brown adipose tissue.
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Uncoupling Protein 2 and 3 in marsupials identification phylogeny and gene expression in response to cold and fasting in antechinus flavipes
Physiological Genomics, 2004Co-Authors: Kerry Withers, Martin KlingensporAbstract:We searched for the presence of Uncoupling Protein genes so far unknown in marsupials and monotremes and identified Uncoupling Protein 2 (UCP2) and UCP3 full-length cDNAs in libraries constructed f...
Daniel Ricquier - One of the best experts on this subject based on the ideXlab platform.
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The Uncoupling Protein 2 modulates the cytokine balance in innate immunity.
Cytokine, 2006Co-Authors: Sophie Rousset, Daniel Ricquier, Yalin Emre, Olivier Join-lambert, Corinne Hurtaud, Anne-marie Cassard-doulcierAbstract:The Uncoupling Protein 2 (UCP2) is located in the inner mitochondrial membrane and downregulates the production of reactive oxygen species (ROS). Recent data suggested a role for UCP2 in the immune response. We analyzed further this hypothesis during acute Listeria monocytogenes infection in mice. Death of infected Ucp2(-/-) mice was delayed in comparison with Ucp2(+/+), suggesting a role of UCP2 in the early step of the immune response. In vitro, the higher resistance of Ucp2(-/-) mice was not associated with a better control of bacterial growth by macrophages. In vivo, a significant increase of recruited phagocytes was observed in the spleen of Ucp2(-/-) mice. This was associated with a higher level of ROS in the spleen. Upregulation of pro-inflammatory cytokines IFNgamma, IL6, and IL1beta and of the chemokine MCP1 was observed in Ucp2(-/-) mice 4 days after infection, preceded by a decrease of the anti-inflammatory cytokine IL10 production. Present data highlight that, in an acute model of infection, UCP2 modulates innate immunity, via the modulation of ROS production, cytokine and chemokine production and consequently phagocyte recruitment.
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protective role of Uncoupling Protein 2 in atherosclerosis
Circulation, 2003Co-Authors: Jeanjacques Blanc, Daniel Ricquier, Bruno Miroux, Marieclotilde Alvesguerra, Bruno Esposito, Sophie Rousset, Pierre Gourdy, Alain Tedgui, Ziad MallatAbstract:Background— Uncoupling Protein 2 (UCP2) regulates the production of reactive oxygen species in macrophages. However, its role in atherosclerosis is unknown. Methods and Results— Irradiated low-density lipoProtein receptor deficient mice (LDLR-/-) were transplanted with bone marrow from either UCP2 deficient mice (Ucp2-/-) or wild type mice (Ucp2+/+). Mice were fed an atherogenic diet for 7 weeks. Engraftment of bone marrow cells was confirmed by the presence of UCP2 Protein expression in spleen cell mitochondria of Ucp2+/+ transplanted mice and its absence in Ucp2-/- transplanted mice. Leukocyte counts and plasma cholesterol levels were comparable in both groups. We found a marked increase in atherosclerotic lesion size in the thoracic aorta of Ucp2-/- transplanted mice compared with control Ucp2+/+ transplanted mice (8.3±0.9% versus 4.3±0.4%, respectively; P<0.005), as well as in the aortic sinus (150 066±12 388 μm2 versus 105 689±9 727 μm2, respectively; P<0.05). This was associated with increased nitro...
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An Uncoupling Protein homologue putatively involved in facultative muscle thermogenesis in birds
Biochemical Journal, 2001Co-Authors: Serge Raimbault, Frédéric Bouillaud, Elodie Couplan, Sami Dridi, Frédérique Denjean, Joël Lachuer, Andre Bordas, Claude Duchamp, Mohamed Taouis, Daniel RicquierAbstract:The cDNA of an Uncoupling Protein (UCP) homologue was obtained by screening a chicken skeletal-muscle library. The predicted 307-amino-acid sequence of avian UCP (avUCP) is 55, 70, 70 and 46% identical with mammalian UCP1, UCP2 and UCP3 and plant UCP respectively. avUCP mRNA expression is restricted to skeletal muscle and its abundance was increased 1.3-fold in a chicken line showing diet-induced thermogenesis, and 3.6- and 2.6-fold in cold-acclimated and glucagon-treated ducklings developing muscle non-shivering thermogenesis respectively. The present data support the implication of avUCP in avian energy expenditure.
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Distribution of the Uncoupling Protein 2 mRNA in the mouse brain.
The Journal of comparative neurology, 1998Co-Authors: Denis Richard, Frédéric Bouillaud, Odette Champigny, Robert Rivest, Qingling Huang, Daniel Sanchis, Daniel RicquierAbstract:The present study was conducted to investigate the brain distribution of the recently cloned Uncoupling Protein 2 (UCP2). Northern blot analyses were first carried out to confirm the presence of UCP2 in the brain. These analyses revealed the brain presence of UCP2 mRNA and the absence of the mRNAs encoding Uncoupling Protein 1 and Uncoupling Protein 3. They also demonstrate that UCP2 mRNA expression was abundant in the hypothalamus and not affected by cold acclimation. In situ hybridization histochemistry was used to determine the brain distribution of the mRNA encoding UCP2. A markedly intense hybridization signal was found in the hypothalamus, the ventral septal region, the caudal hindbrain (medulla), the ventricular region, and the cerebellum. A very highly intense hybridization signal was apparent in the suprachiasmatic nucleus, the medial parvicellular part of the paraventricular hypothalamic nucleus, the arcuate nucleus, the dorsal motor nucleus of the vagus nerve, and the choroid plexus. The specifically localized expression of UCP2 mRNA suggests that this mRNA has a neuronal localization. Neuronal expression was particularly manifest in the nucleus of the horizontal limb of the diagonal band, the submedius thalamic nucleus and the dorsal motor nucleus of the vagus nerve, where agglomerations of the silver grains delineated individual cells. The role played by UCP2 in the brain has yet to be fully described, but the pattern of distribution of the transcript suggests that this mitochondrial Protein is part of neuronal circuitries controlling neuroendocrine functions, autonomic responses, and the general arousal of the brain. Given the involvement of the Proteins from the Uncoupling Protein's family in the Uncoupling of cellular respiration, it can be argued that UCP2 contributes to the metabolic rate and thermoregulation of these circuitries. In addition, by promoting oxygen consumption in the brain, UCP2 could control the production of reactive oxygen species and thereby influence the process of neural degeneration. J. Comp. Neurol. 397:549–560, 1998. © 1998 Wiley-Liss, Inc.
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a plant cold induced Uncoupling Protein
Nature, 1997Co-Authors: Maryse Laloi, Frédéric Bouillaud, Mathieu Klein, Jorg W Riesmeier, Bernd Mullerrober, Christophe Fleury, Daniel RicquierAbstract:In mammals, body temperature can be raised by the action of Uncoupling Proteins (UCPs), which dissipate the proton electrochemical gradient across the inner mitochondrial membrane to produce heat rather than synthesize ATP1. Any similar mechanism of thermogenesis in plants is not so well understood. We have now identifiedcomplementary DNA from potatoes (Solanum tuberosum) that encodes a plant Uncoupling Protein, named StUCP, which is induced by cold treatment and so may be involved in thermoregulation in plants.
Tobias Fromme - One of the best experts on this subject based on the ideXlab platform.
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Uncoupling Protein 1 and the capacity for nonshivering thermogenesis are components of the glucose homeostatic system
American Journal of Physiology-Endocrinology and Metabolism, 2020Co-Authors: Stefanie Maurer, Tobias Fromme, Sabine Mocek, Anika Zimmermann, Martin KlingensporAbstract:Uncoupling Protein 1 (Ucp1) provides nonshivering thermogenesis (NST) fueled by the dissipation of energy from macronutrients in brown and brite adipocytes. The availability of thermogenic fuels is...
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Fibroblast growth factor 8b induces Uncoupling Protein 1 expression in epididymal white preadipocytes
Scientific Reports, 2019Co-Authors: Sören Westphal, Martin Klingenspor, Thomas Gantert, Caroline Kless, Kristina Hüttinger, Tobias FrommeAbstract:The number of brown adipocytes residing within murine white fat depots (brite adipocytes) varies a lot by depot, strain and physiological condition. Several endocrine fibroblast growth factors are implicated in the regulation of brite adipocyte abundance. The family of fibroblast growth factors can be categorized by their site of action into endocrine, paracrine and intracellular peptides. We here screened paracrine fibroblast growth factors for their potential to drive brite adipogenesis in differentiating epididymal white adipocytes and identified fibroblast growth factor 8b to induce Uncoupling Protein 1 expression, but at the same time to interfere in adipogenesis. In an in vivo trial, fibroblast growth factor 8b released into the epididymal fat depot failed to robustly increase the number of brite adipocytes. The specific action of fibroblast growth factor 8b on the Uncoupling Protein 1 promoter in cultured epididymal adipocytes provides a model system to dissect specific gene regulatory networks.
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degradation of brown adipocyte purine nucleotides regulates Uncoupling Protein 1 activity
Molecular metabolism, 2017Co-Authors: Tobias Fromme, Karin Kleigrewe, Andreas Dunkel, Angelika Retzler, Stefanie Maurer, Natascha Fischer, Rolf Diezko, Timo Kanzleiter, Yongguo Li, Verena HirschbergAbstract:Abstract Objective Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic Uncoupling Protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in Uncoupling Protein 1 activation. Methods With mass spectrometry, purine nucleotide metabolites were quantified in cellular homogenates and supernatants of cultured primary brown adipocytes. We also determined oxygen consumption in response to a β-adrenergic agonist. Results Upon adrenergic activation, brown adipocytes decreased the intracellular concentration of inhibitory nucleotides (ATP, ADP, GTP and GDP) and released the respective degradation products. At the same time, an increase in cellular calcium occurred. None of these phenomena occurred in white adipocytes or myotubes. The brown adipocyte expression of enzymes implicated in purine metabolic remodeling is altered upon cold exposure. Pharmacological and genetic interference of purine metabolism altered Uncoupling Protein 1 mediated uncoupled respiration. Conclusion Adrenergic stimulation of brown adipocytes lowers the intracellular concentration of purine nucleotides, thereby contributing to Uncoupling Protein 1 activation.
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Uncoupling Protein 1 expression and high fat diets
American Journal of Physiology-regulatory Integrative and Comparative Physiology, 2011Co-Authors: Tobias Fromme, Martin KlingensporAbstract:Uncoupling Protein 1 (Ucp1) is the key component of β-adrenergically controlled nonshivering thermogenesis in brown adipocytes. This process combusts stored and nutrient energy as heat. Cold exposure not only activates Ucp1-mediated thermogenesis to maintain normothermia but also results in adaptive thermogenesis, i.e., the recruitment of thermogenic capacity in brown adipose tissue. As a hallmark of adaptive thermogenesis, Ucp1 synthesis is increased proportionally to temperature and duration of exposure. Beyond this classical thermoregulatory function, it has been suggested that Ucp1-mediated thermogenesis can also be employed for metabolic thermogenesis to prevent the development of obesity. Accordingly, in times of excess caloric intake, one may expect a positive regulation of Ucp1. The general impression from an overview of the present literature is, indeed, an increased brown adipose tissue Ucp1 mRNA and Protein content after feeding a high-fat diet (HFD) to mice and rats. The reported increases are very variable in magnitude, and the effect size seems to be independent of dietary fat content and duration of the feeding trial. In white adipose tissue depots Ucp1 mRNA is generally downregulated by HFD, indicating a decline in the number of interspersed brown adipocytes.
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Brown adipose tissue specific lack of Uncoupling Protein 3 is associated with impaired cold tolerance and reduced transcript levels of metabolic genes
Journal of Comparative Physiology B, 2008Co-Authors: Tobias Fromme, Gerhard Heldmaier, Carola W. Meyer, Christa Praun, Martin KlingensporAbstract:Uncoupling Protein 3 (Ucp3) is located within the mitochondrial inner membrane of brown adipose tissue and skeletal muscle. It is thought to be implicated in lipid metabolism and defense against reactive oxygen species. We previously reported on a mutation in our breeding colony of Djungarian hamsters ( Phodopus sungorus ) that leads to brown adipose tissue specific lack of Ucp3 expression. In this study we compared wildtype with mutant hamsters on a broad genetic background. Hamsters lacking Ucp3 in brown adipose tissue displayed a reduced cold tolerance due to impaired nonshivering thermogenesis. This phenotype is associated with a global decrease in expression of metabolic genes but not of Uncoupling Protein 1. These data implicate that Ucp3 is necessary to sustain high metabolic rates in brown adipose tissue.
Chen-yu Zhang - 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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Cloning and functional characterization of an Uncoupling Protein homolog in hummingbirds.
Physiological genomics, 2001Co-Authors: Claudia R. Vianna, Thilo Hagen, Chen-yu Zhang, Bradford B. Lowell, Eric S. Bachman, Olivier Boss, Balázs Gereben, Anselmo Sigari Moriscot, José Eduardo P. W. Bicudo, Antonio C. BiancoAbstract:The cDNA of an Uncoupling Protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird Uncoupling Protein (HmUCP) cDNA was amplified from pectoral...
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Assessment of Uncoupling activity of the human Uncoupling Protein 3 short form and three mutants of the Uncoupling Protein gene using a yeast heterologous expression system.
FEBS letters, 1999Co-Authors: Thilo Hagen, Chen-yu Zhang, Lawrence J. Slieker, Wendy K. Chung, Rudolph L. Leibel, Bradford B. LowellAbstract:The human Uncoupling Protein 3 gene generates two mRNA transcripts, Uncoupling Protein 3L and Uncoupling Protein 3S, which are predicted to encode long and short forms of the Uncoupling Protein 3 Protein, respectively. While Uncoupling Protein 3L is similar in length to the other known Uncoupling Proteins, Uncoupling Protein 3S lacks the last 37 C-terminal residues. A splice site mutation in the human Uncoupling Protein 3 gene, resulting in the exclusive expression of Uncoupling Protein 3S, and a number of point mutations in the Uncoupling Protein 3 gene have been described. This study compares the biochemical activity of Uncoupling Protein 3S as well as three mutants of the Uncoupling Protein 3 gene (V9M, V1021, R282C) with that of Uncoupling Protein 3L utilizing a yeast expression system. All Proteins were expressed at similar levels and had qualitatively similar effects on parameters related to the Uncoupling function. Both Uncoupling Protein 3S and Uncoupling Protein 3L decreased the yeast growth rate by 3S and 52%, increased the whole yeast basal O2 consumption by 26 and 48%, respectively, and decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3'3-dihexyloxacarbocyanine iodide. In isolated mitochondria, Uncoupling Protein 3S and Uncoupling Protein 3L caused a similar (33 and 35%, respectively) increase in state 4 respiration, which was relatively small compared to Uncoupling Protein 1 (102% increase). A truncated version of Uncoupling Protein 3S, lacking the last three C-terminal residues, Tyr, Lys and Gly, that are part of a carrier motif that is highly conserved among all mitochondrial carriers, had a greatly reduced Uncoupling activity. The two naturally occurring Uncoupling Protein 3 mutants, V9M and V1021, were similar to Uncoupling Protein 3L with respect to effects on the yeast growth and whole yeast O2 consumption. The R282C mutant had a reduced effect compared to Uncoupling Protein 3L. In summary, Uncoupling Protein 3S and the three mutants of Uncoupling Protein 3 appear to be functional Proteins with biochemical activities similar to Uncoupling Protein 3L, although Uncoupling Protein 3S and the R282C mutant have a modestly reduced function.
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Assessment of Uncoupling activity of Uncoupling Protein 3 using a yeast heterologous expression system.
FEBS letters, 1999Co-Authors: Chen-yu Zhang, Thilo Hagen, Lawrence J. Slieker, V K Mootha, Bradford B. LowellAbstract:Uncoupling Protein 3L, Uncoupling Protein 1 and the mitochondrial oxoglutarate carrier were expressed in Saccharomyces cerevisae. Effects on different parameters related to the energy expenditure were studied. Both Uncoupling Protein 3L and Uncoupling Protein 1 reduced the growth rate by 49% and 32% and increased the whole yeast O2 consumption by 31% and 19%, respectively. In isolated mitochondria, Uncoupling Protein 1 increased the state 4 respiration by 1.8-fold, while Uncoupling Protein 3L increased the state 4 respiration by 1.2-fold. Interestingly, mutant Uncoupling Protein 1 carrying the H145Q and H147N mutations, previously shown to markedly decrease the H+ transport activity of Uncoupling Protein 1 when assessed using a proteoliposome system (Bienengraeber et al. (1998) Biochem. 37, 3-8), uncoupled the mitochondrial respiration to almost the same degree as wild-type Uncoupling Protein 1. Thus, absence of this histidine pair in Uncoupling Protein 2 and Uncoupling Protein 3 does not by itself rule out the possibility that these carriers have an Uncoupling function. The oxoglutarate carrier had no effect on any of the studied parameters. In summary, a discordance exists between the magnitude of effects of Uncoupling Protein 3L and Uncoupling Protein 1 in whole yeast versus isolated mitochondria, with Uncoupling Protein 3L having greater effects in whole yeast and a smaller effect on the state 4 respiration in isolated mitochondria. These findings suggest that Uncoupling Protein 3L, like Uncoupling Protein 1, has an Uncoupling activity. However, the mechanism of action and/or regulation of the activity of Uncoupling Protein 3L is likely to be different.
