RESUMO
In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the ß1d-α6 loop (W123F, M126I, and Y127F) and ß1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.
Assuntos
Etanolaminas/química , Proteínas de Helminto/química , Metiltransferases/química , Nematoides/enzimologia , Pinus/parasitologia , Doenças das Plantas/parasitologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Etanolaminas/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Cinética , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Nematoides/genética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , TermodinâmicaRESUMO
Mutations in the microfibrillar protein fibrillin-1 or the absence of its binding partner microfibril-associated glycoprotein (MAGP1) lead to increased TGFß signaling due to an inability to sequester latent or active forms of TGFß, respectively. Mouse models of excess TGFß signaling display increased adiposity and predisposition to type-2 diabetes. It is therefore interesting that individuals with Marfan syndrome, a disease in which fibrillin-1 mutation leads to aberrant TGFß signaling, typically present with extreme fat hypoplasia. The goal of this project was to characterize multiple fibrillin-1 mutant mouse strains to understand how fibrillin-1 contributes to metabolic health. The results of this study demonstrate that fibrillin-1 contributes little to lipid storage and metabolic homeostasis, which is in contrast to the obesity and metabolic changes associated with MAGP1 deficiency. MAGP1 but not fibrillin-1 mutant mice had elevated TGFß signaling in their adipose tissue, which is consistent with the difference in obesity phenotypes. However, fibrillin-1 mutant strains and MAGP1-deficient mice all exhibit increased bone length and reduced bone mineralization which are characteristic of Marfan syndrome. Our findings suggest that Marfan-associated adipocyte hypoplasia is likely not due to microfibril-associated changes in adipose tissue, and provide evidence that MAGP1 may function independently of fibrillin in some tissues.
Assuntos
Fibrilina-1/genética , Metabolismo dos Lipídeos , Tecido Adiposo Marrom/patologia , Animais , Composição Corporal , Calcificação Fisiológica , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Fibrilina-1/metabolismo , Masculino , Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Síndrome de Marfan/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microfibrilas/metabolismo , Tamanho do Órgão , Especificidade de Órgãos , Fatores de Processamento de RNA , Transdução de Sinais , Gordura Subcutânea/patologia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
Williams-Beuren syndrome is the consequence of a large contiguous-gene deletion on the seventh human chromosome that includes the elastin gene. Elastin is an extracellular matrix protein responsible for the cardiovascular abnormalities associated with Williams's syndrome, including hypertension and aortic stenosis. A high percentage of individuals with Williams's syndrome also have impaired glucose tolerance, independent of traditional risk factors for diabetes. Here, we show that murine adipose tissue does assemble elastic fibers; however, isolated elastin insufficiency (Eln+/-) in mice does not independently influence glucose metabolism or tissue lipid accumulation. Similarly, isolated ApoE deficiency (ApoE-/-), a model of hyperlipidemia and atherosclerosis, does not impair insulin sensitivity. However, Eln+/-; ApoE-/- double mutant mice exhibit notable hyperglycemia, adipocyte hypertrophy, inflammation of adipose tissue, and ectopic lipid accumulation in liver tissue. Further, Eln+/-; ApoE-/- mutants have significant impairment of insulin sensitivity by insulin tolerance testing, independent of body weight or diet, suggesting that elastin insufficiency predisposes to metabolic disease in susceptible individuals.