RESUMO
A histidine-tagged recombinant N-terminal fragment of type-1 mouse liver diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), MmDGAT1(1-95)His6, was expressed in Escherichia coli, and used to investigate possible acyl-CoA-binding properties. Analysis of the purified fragment by MALDI-TOF mass spectrometry revealed a polypeptide with molecular mass of about 11 kDa which was consistent with the calculated molecular mass based on the deduced amino acid sequence. Lipidex-1000 binding assays indicated that MmDGAT1(1-95)His(6) interacted with long chain fatty acyl-CoAs similar to observations on DGAT1 from oilseed rape (Brassica napus). Binding, as a function of acyl-CoA concentration, differed for palmitoyl (16:0), stearoyl (18:0), and erucoyl (cisDelta(13)22:1)-CoA. Binding of stearoyl- or erucoyl-CoA to MmDGAT1(1-95)His(6) as a function of acyl-CoA concentration, however, was sigmoid and displayed positive cooperativity suggesting that MmDGAT1 may be subject to allosteric modulation by acyl-CoAs. An intra-polypeptide segment within the N-terminal region of MmDGAT1 contained remnants of an acyl-CoA-binding signature initially identified in plant DGAT1. The acyl-CoA-binding site in mammalian DGAT1 could represent a potential target for therapeutic interventions for disorders such as type-2 diabetes and obesity.
Assuntos
Acil Coenzima A/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Acil Coenzima A/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Brassica napus/enzimologia , Brassica napus/genética , Sequência Conservada , Dextranos/química , Diacilglicerol O-Aciltransferase/química , Diacilglicerol O-Aciltransferase/genética , Escherichia coli/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to generate triacylglycerol and CoA. The deduced amino acid sequence of cDNAs encoding DGAT1 from plants and mammals exhibit a hydrophilic N-terminal region followed by a number of potential membrane-spanning segments, which is consistent with the membrane-bound nature of this enzyme family. In order to gain insight into the structure/function properties of DGAT1 from Brassica napus (BnDGAT1), we produced and partially characterized a recombinant polyHis-tagged N-terminal fragment of the enzyme, BnDGAT1(1-116)His6, with calculated molecular mass of 13,278 Da. RESULTS: BnDGAT1(1-116)His6 was highly purified from bacterial lysate and plate-like monoclinic crystals were grown using this preparation. Lipidex-1000 binding assays and gel electrophoresis indicated that BnDGAT1(1-116)His6 interacts with long chain acyl-CoA. The enzyme fragment displayed enhanced affinity for erucoyl (22:1cisDelta13)-CoA over oleoyl (18:1cisDelta9)-CoA, and the binding process displayed positive cooperativity. Gel filtration chromatography and cross-linking studies indicated that BnDGAT1(1-116)His6 self-associated to form a tetramer. Polyclonal antibodies raised against a peptide of 15 amino acid residues representing a segment of BnDGAT1(1-116)His6 failed to react with protein in microsomal vesicles following treatment with proteinase K, suggesting that the N-terminal fragment of BnDGAT1 was localized to the cytosolic side of the ER. CONCLUSION: Collectively, these results suggest that BnDGAT1 may be allosterically modulated by acyl-CoA through the N-terminal region and that the enzyme self-associates via interactions on the cytosolic side of the ER.
Assuntos
Brassica napus/enzimologia , Diacilglicerol O-Aciltransferase/química , Proteínas de Plantas/química , Acil Coenzima A/metabolismo , DNA Complementar , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/isolamento & purificação , Diacilglicerol O-Aciltransferase/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
The commonly occurring E23K and I337V Kir6.2 polymorphisms in the ATP-sensitive potassium (KATP) channel are more frequent in Caucasian type 2 diabetic populations. However, the underlying cellular mechanisms contributing to the pathogenesis of type 2 diabetes remain uncharacterized. Chronic elevation of plasma free fatty acids observed in obese and type 2 diabetic subjects leads to cytosolic accumulation of long-chain acyl CoAs (LC-CoAs) in pancreatic beta-cells. We postulated that the documented stimulatory effects of LC-CoAs on KATP channels might be enhanced in polymorphic KATP channels. Patch-clamp experiments were performed on inside-out patches containing recombinant KATP channels (Kir6.2/SUR1) to record macroscopic currents. KATP channels containing Kir6.2 (E23K/I337V) showed significantly increased activity in response to physiological palmitoyl-CoA concentrations (100-1,000 nmol/l) compared with wild-type KATP channels. At physiological intracellular ATP concentrations (mmol/l), E23K/I337V polymorphic KATP channels demonstrated significantly enhanced activity in response to palmitoyl-CoA. The observed increase in KATP channel activity may result in multiple defects in glucose homeostasis, including impaired insulin and glucagon-like peptide-1 secretion and increased glucagon release. In summary, these results suggest that the E23K/I337V polymorphism may have a diabetogenic effect via increased KATP channel activity in response to endogenous levels of LC-CoAs in tissues involved in the maintenance of glucose homeostasis.
