RESUMO
Uncoupling protein 1 (UCP1) conducts protons through the inner mitochondrial membrane to uncouple mitochondrial respiration from ATP production, thereby converting the electrochemical gradient of protons into heat1,2. The activity of UCP1 is activated by endogenous fatty acids and synthetic small molecules, such as 2,4-dinitrophenol (DNP), and is inhibited by purine nucleotides, such as ATP3-5. However, the mechanism by which UCP1 binds to these ligands remains unknown. Here we present the structures of human UCP1 in the nucleotide-free state, the DNP-bound state and the ATP-bound state. The structures show that the central cavity of UCP1 is open to the cytosolic side. DNP binds inside the cavity, making contact with transmembrane helix 2 (TM2) and TM6. ATP binds in the same cavity and induces conformational changes in TM2, together with the inward bending of TM1, TM4, TM5 and TM6 of UCP1, resulting in a more compact structure of UCP1. The binding site of ATP overlaps with that of DNP, suggesting that ATP competitively blocks the functional engagement of DNP, resulting in the inhibition of the proton-conducting activity of UCP1.
Assuntos
2,4-Dinitrofenol , Trifosfato de Adenosina , Proteína Desacopladora 1 , Humanos , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Prótons , Proteína Desacopladora 1/química , Proteína Desacopladora 1/metabolismo , Ácidos Graxos/metabolismo , 2,4-Dinitrofenol/química , 2,4-Dinitrofenol/metabolismo , Conformação Proteica , Membrana Celular/metabolismo , Citosol/metabolismoRESUMO
Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocytes. In the presence of long-chain fatty acids (LCFAs), UCP1 increases the proton conductance, which, in turn, increases fatty acid oxidation and energy release as heat. Atomic models of UCP1 and UCP2 have been generated based on the NMR backbone structure of UCP2 in dodecylphosphocholine (DPC), a detergent known to inactivate UCP1. Based on NMR titration experiments on UCP1 with LCFA, it has been proposed that K56 and K269 are crucial for LCFA binding and UCP1 activation. Given the numerous controversies on the use of DPC for structure-function analyses of membrane proteins, we revisited those UCP1 mutants in a more physiological context by expressing them in the mitochondria of Saccharomyces cerevisiae. Mitochondrial respiration, assayed on permeabilized spheroplasts, enables the determination of UCP1 activation and inhibition. The K56S, K269S, and K56S/K269S mutants did not display any default in activation, which shows that the NMR titration experiments in DPC detergent are not relevant to UCP1 function.
Assuntos
Adipócitos Marrons/ultraestrutura , Proteínas de Desacoplamento Mitocondrial/ultraestrutura , Conformação Proteica , Proteína Desacopladora 1/ultraestrutura , Adipócitos Marrons/metabolismo , Animais , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Humanos , Canais Iônicos/genética , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/ultraestrutura , Proteínas de Desacoplamento Mitocondrial/química , Modelos Estruturais , Consumo de Oxigênio/genética , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Prótons , Ratos , Saccharomyces cerevisiae , Relação Estrutura-Atividade , Proteína Desacopladora 1/química , Proteína Desacopladora 1/genéticaRESUMO
Small molecule mitochondrial uncouplers have recently garnered great interest for their potential in treating nonalcoholic steatohepatitis (NASH). In this study, we report the structure-activity relationship profiling of a 6-amino[1,2,5]oxadiazolo[3,4-b]pyrazin-5-ol core, which utilizes the hydroxy moiety as the proton transporter across the mitochondrial inner membrane. We demonstrate that a wide array of substituents is tolerated with this novel scaffold that increased cellular metabolic rates in vitro using changes in oxygen consumption rate as a readout. In particular, compound SHS4121705 (12i) displayed an EC50 of 4.3 µM in L6 myoblast cells and excellent oral bioavailability and liver exposure in mice. In the STAM mouse model of NASH, administration of 12i at 25 mg kg-1 day-1 lowered liver triglyceride levels and improved liver markers such as alanine aminotransferase, NAFLD activity score, and fibrosis. Importantly, no changes in body temperature or food intake were observed. As potential treatment of NASH, mitochondrial uncouplers show promise for future development.
Assuntos
Pirazinas/química , Alanina Transaminase/metabolismo , Compostos de Anilina/química , Animais , Linhagem Celular , Dieta Hiperlipídica , Modelos Animais de Doenças , Meia-Vida , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Mitocôndrias/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/patologia , Consumo de Oxigênio/efeitos dos fármacos , Pirazinas/farmacocinética , Pirazinas/farmacologia , Pirazinas/uso terapêutico , Ratos , Relação Estrutura-Atividade , Triglicerídeos/metabolismo , Proteína Desacopladora 1/química , Proteína Desacopladora 1/metabolismoRESUMO
Fucoxanthin (Fx), a major carotenoid found in brown seaweed, is known to show a unique and wide variety of biological activities. Upon absorption, Fx is metabolized to fucoxanthinol and amarouciaxanthin, and these metabolites mainly accumulate in visceral white adipose tissue (WAT). As seen in other carotenoids, Fx can quench singlet oxygen and scavenge a wide range of free radicals. The antioxidant activity is related to the neuroprotective, photoprotective, and hepatoprotective effects of Fx. Fx is also reported to show anti-cancer activity through the regulation of several biomolecules and signaling pathways that are involved in either cell cycle arrest, apoptosis, or metastasis suppression. Among the biological activities of Fx, anti-obesity is the most well-studied and most promising effect. This effect is primarily based on the upregulation of thermogenesis by uncoupling protein 1 expression and the increase in the metabolic rate induced by mitochondrial activation. In addition, Fx shows anti-diabetic effects by improving insulin resistance and promoting glucose utilization in skeletal muscle.
Assuntos
Suplementos Nutricionais/análise , Alga Marinha/química , Xantofilas/química , Xantofilas/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/metabolismo , Animais , Fármacos Antiobesidade/química , Fármacos Antiobesidade/metabolismo , Antioxidantes/química , Antioxidantes/metabolismo , Descoberta de Drogas , Radicais Livres/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/metabolismo , Resistência à Insulina , Fígado/metabolismo , Estrutura Molecular , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Depuradores/metabolismo , Oxigênio Singlete/metabolismo , Proteína Desacopladora 1/química , Proteína Desacopladora 1/metabolismo , Xantofilas/efeitos adversos , beta Caroteno/análogos & derivados , beta Caroteno/químicaRESUMO
The acquisition of UCP1-mediated non-shivering thermogenesis (NST) was an important event during the evolution of mammals. Here, we assessed the thermogenic neofunctionalization that occurred in the mammalian UCP1, by performing detailed comparative evolutionary genomics analyses (including phylogenetic and selection analyses) of the UCP family members across all major vertebrate classes. Heterogeneously distributed positive selection signatures were found in several UCPs, being preferably located in the mitochondrial matrix domains. Additionally, comparisons with non-mammalian orthologs showed increased evolutionary rates of the mammalian UCP1, not observable in the phylogenetically related UCP2 and UCP3 paralogs. Also, parallel signatures of episodic positive selection (ω > 1) were found in the ancestral branches of both Glires (rodents and lagomorphs) and Afroinsectivores (afrosoricids and macroscelids), underlining the importance of the UCP1 thermogenic activity in these mammalian groups. Finally, we hypothesize that the independent positive selection events that occurred in these two lineages resulted in two UCP1-mediated NST approaches, namely the cold acute response in the Glires and the reproduction success enhancement in the Afroinsectivores.
Assuntos
Termogênese/genética , Proteína Desacopladora 1/genética , Animais , Códon , Evolução Molecular , Genômica , Humanos , Mamíferos/genética , Camundongos , Modelos Moleculares , Filogenia , Conformação Proteica , Seleção Genética , Proteína Desacopladora 1/química , Proteína Desacopladora 1/classificação , Vertebrados/genéticaRESUMO
Recent studies suggested that activation of Uncoupling Protein 1 (UCP1) has become an appealing therapeutic strategy against obesity and diabetes. In our research, the 3D structure of UCP1 was constructed through homology modelling, refined through molecular dynamics simulation, and evaluated by Ramachandran plot, the molecular docking of UCP1 activators brought about the proposal of an interaction mode inside the UCP1 active site. Remarkably, Reside Lys126 formed hydrogen bond; residues Pro121, Val125, Tyr146, Tyr149 and Arg150 formed hydrophobic interaction, which are key amino acids within UCP1 site. Then a pharmacophore model was generated consisting of three hydrophobic groups, a negative center and an additional hydrophobic group. Pharmacophore-based virutal screening of Specs database yield 5 hits. In vitro assay indicated ZINC 04660290 significantly increased the protein expression of UCP1 and decreased the fat droplet in a dose-dependent manner. Besides, pharmacokinetic properties were predicted for those five compounds through ADME/T prediction. All of these will guide us to design new UCP1 activators for the treatment of obesity and diabetes.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Proteína Desacopladora 1/agonistas , Algoritmos , Sítios de Ligação/efeitos dos fármacos , Desenho de Fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Moleculares , Estrutura Molecular , Proteína Desacopladora 1/químicaRESUMO
Oxidative stress causes selective oxidation of cardiolipin (CL), a four-tail lipid specific for the inner mitochondrial membrane. Interaction with oxidized CL transforms cytochrome c into peroxidase capable of oxidizing even more CL molecules. Ultimately, this chain of events leads to the pore formation in the outer mitochondrial membrane and release of mitochondrial proteins, including cytochrome c, into the cytoplasm. In the cytoplasm, cytochrome c promotes apoptosome assembly that triggers apoptosis (programmed cell death). Because of this amplification cascade, even an occasional oxidation of a single CL molecule by endogenously formed reactive oxygen species (ROS) might cause cell death, unless the same CL oxidation triggers a separate chain of antiapoptotic reactions that would prevent the CL-mediated apoptotic cascade. Here, we argue that the key function of CL in mitochondria and other coupling membranes is to prevent proton leak along the interface of interacting membrane proteins. Therefore, CL oxidation should increase proton permeability through the CL-rich clusters of membrane proteins (CL islands) and cause a drop in the mitochondrial membrane potential (MMP). On one hand, the MMP drop should hinder ROS generation and further CL oxidation in the entire mitochondrion. On the other hand, it is known to cause rapid fission of the mitochondrial network and formation of many small mitochondria, only some of which would contain oxidized CL islands. The fission of mitochondrial network would hinder apoptosome formation by preventing cytochrome c release from healthy mitochondria, so that slowly working protein quality control mechanisms would have enough time to eliminate mitochondria with the oxidized CL. Because of these two oppositely directed regulatory pathways, both triggered by CL oxidation, the fate of the cell appears to be determined by the balance between the CL-mediated proapoptotic and antiapoptotic reactions. Since this balance depends on the extent of CL oxidation, mitochondria-targeted antioxidants might be able to ensure cell survival in many pathologies by preventing CL oxidation.
Assuntos
Apoptose , Cardiolipinas/química , Mitocôndrias/metabolismo , Sequência de Aminoácidos , Animais , Antioxidantes/química , Cardiolipinas/metabolismo , Citocromos c/metabolismo , Humanos , Potencial da Membrana Mitocondrial , Camundongos , Mitofagia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Alinhamento de Sequência , Proteína Desacopladora 1/química , Proteína Desacopladora 1/metabolismoRESUMO
In mammals, uncoupling protein 1 (UCP1) is well known for its thermogenic role in brown adipose tissue (BAT). However, the UCP1 physiological roles are still unclear in fish, although several teleost ucp1 genes have been identified. The aim of this study is to investigate the potential roles of fish UCP1 involved in food intake regulation and energy homeostasis. We herein report on the molecular cloning, tissue distribution and the effect of fasting and refeeding on the expression of ucp1 in Channa argus. UCP1 consisted of a 921â¯bp open reading frame predicted to encode 306 amino acids. Sequence analysis revealed that snakehead UCP1 was highly conserved (>80%) with teleost UCP1, but shared a lower identity (60-72%) with mammals. Phylogenetic analysis supported that snakehead UCP1 was closely related to piscine UCP1. In addition, ucp1 was found to extensively expressed in all detected tissues, with the highest level in liver. Futhermore, the hepatic ucp1 was found to significantly increased after short-term and long-term food deprivation, and dramatically increased following refeeding. These findings suggested that snakehead UCP1 might play important roles in food intake regulation and fatty acid metabolism in snakehead fish, and it could be as a potential target locus to improve commercial production of this kind of fish.
Assuntos
Jejum , Comportamento Alimentar , Proteínas de Peixes/metabolismo , Perciformes/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Perciformes/fisiologia , Filogenia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Proteína Desacopladora 1/químicaRESUMO
The mitochondrial uncoupling protein 1 (UCP1) generates heat by causing proton leak across the mitochondrial inner membrane that requires fatty acid (FA). The mechanism by which UCP1 uses FA to conduct proton remains unsolved, and it is also unclear whether a direct physical interaction between UCP1 and FA exists. Here, we have shown using nuclear magnetic resonance that FA can directly bind UCP1 at a helix-helix interface site composed of residues from the transmembrane helices H1 and H6. According to the paramagnetic relaxation enhancement data and molecular dynamics simulation, the FA acyl chain appears to fit into the groove between H1 and H6 while the FA carboxylate group interacts with the basic residues near the matrix side of UCP1. Functional mutagenesis showed that mutating the observed FA binding site severely reduced UCP1-mediated proton flux. Our study identifies a functionally important FA-UCP1 interaction that is potentially useful for mechanistic understanding of UCP1-mediated thermogenesis.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Proteína Desacopladora 1/química , Proteína Desacopladora 1/metabolismo , Sítios de Ligação , Humanos , Hidrogênio/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Termogênese , Proteína Desacopladora 1/genéticaRESUMO
Brown adipose tissue (BAT) mitochondria are distinct from their counterparts in other tissues in that ATP production is not their primary physiologic role. BAT mitochondria are equipped with a specialized protein known as uncoupling protein 1 (UCP1). UCP1 short-circuits the electron transport chain, allowing mitochondrial membrane potential to be transduced to heat, making BAT a tissue capable of altering energy expenditure and fuel metabolism in mammals without increasing physical activity. The recent discovery that adult humans have metabolically active BAT has rekindled an interest in this intriguing tissue, with the overarching aim of manipulating BAT function to augment energy expenditure as a countermeasure for obesity and the metabolic abnormalities it incurs. Subsequently, there has been heightened interest in quantifying BAT function and more specifically, determining UCP1-mediated thermogenesis in BAT specimens - including in those obtained from humans. In this article, BAT mitochondrial bioenergetics will be described and compared with more conventional mitochondria in other tissues. The biochemical methods typically used to quantify BAT mitochondrial function will also be discussed in terms of their specificity for assaying UCP1 mediated thermogenesis. Finally, recent data concerning BAT UCP1 function in humans will be described and discussed.
Assuntos
Tecido Adiposo Marrom/metabolismo , Termogênese/fisiologia , Proteína Desacopladora 1/metabolismo , Adipócitos Marrons/fisiologia , Tecido Adiposo/metabolismo , Animais , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Proteína Desacopladora 1/química , Proteína Desacopladora 1/genéticaRESUMO
Uncoupling protein 1 (UCP1) is an integral membrane protein found in the mitochondrial inner membrane of brown adipose tissue, and facilitates the process of non-shivering thermogenesis in mammals. Its activation by fatty acids, which overcomes its inhibition by purine nucleotides, leads to an increase in the proton conductance of the inner mitochondrial membrane, short-circuiting the mitochondrion to produce heat rather than ATP. Despite 40 years of intense research, the underlying molecular mechanism of UCP1 is still under debate. The protein belongs to the mitochondrial carrier family of transporters, which have recently been shown to utilise a domain-based alternating-access mechanism, cycling between a cytoplasmic and matrix state to transport metabolites across the inner membrane. Here, we review the protein properties of UCP1 and compare them to those of mitochondrial carriers. UCP1 has the same structural fold as other mitochondrial carriers and, in contrast to past claims, is a monomer, binding one purine nucleotide and three cardiolipin molecules tightly. The protein has a single substrate binding site, which is similar to those of the dicarboxylate and oxoglutarate carriers, but also contains a proton binding site and several hydrophobic residues. As found in other mitochondrial carriers, UCP1 has two conserved salt bridge networks on either side of the central cavity, which regulate access to the substrate binding site in an alternating way. The conserved domain structures and mobile inter-domain interfaces are consistent with an alternating access mechanism too. In conclusion, UCP1 has retained all of the key features of mitochondrial carriers, indicating that it operates by a conventional carrier-like mechanism.
Assuntos
Cardiolipinas/metabolismo , Ácidos Graxos/metabolismo , Mitocôndrias/metabolismo , Prótons , Termogênese/fisiologia , Proteína Desacopladora 1/química , Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Metabolismo Energético/fisiologia , Regulação da Expressão Gênica , Humanos , Transporte de Íons , Mitocôndrias/genética , Modelos Moleculares , Nucleotídeos de Purina/metabolismo , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
The uncoupling protein UCP1 from brown adipose tissue is a mitochondrial carrier which allows dissipation of metabolic energy as heat. We have characterized the human UCP1 (HsUCP1) recombinantly expressed in Saccharomyces cerevisiae and we demonstrate that HsUCP1 is activated by fatty acids and retinoids in a nucleotide sensitive manner just as its rodent orthologs. However, in the absence of regulators, rodent UCP1 presents a high ohmic proton conductance that cannot be detected in HsUCP1. Since the human protein can be activated in a nucleotide sensitive manner, we conclude that it must have lost selectively the basal proton conductance.