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1.
J Biol Chem ; 284(16): 10923-34, 2009 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-19201756

RESUMO

Srv2/cyclase-associated protein is expressed in virtually all plant, animal, and fungal organisms and has a conserved role in promoting actin depolymerizing factor/cofilin-mediated actin turnover. This is achieved by the abilities of Srv2 to recycle cofilin from ADP-actin monomers and to promote nucleotide exchange (ATP for ADP) on actin monomers. Despite this important and universal role in facilitating actin turnover, the mechanism underlying Srv2 function has remained elusive. Previous studies have demonstrated a critical functional role for the G-actin-binding C-terminal half of Srv2. Here we describe an equally important role in vivo for the N-terminal half of Srv2 in driving actin turnover. We pinpoint this activity to a conserved patch of surface residues on the N-terminal dimeric helical folded domain of Srv2, and we show that this functional site interacts with cofilin-actin complexes. Furthermore, we show that this site is essential for Srv2 acceleration of cofilin-mediated actin turnover in vitro. A cognate Srv2-binding site is identified on a conserved surface of cofilin, suggesting that this function likely extends to other organisms. In addition, our analyses reveal that higher order oligomerization of Srv2 depends on its N-terminal predicted coiled coil domain and that oligomerization optimizes Srv2 function in vitro and in vivo. Based on these data, we present a revised model for the mechanism by which Srv2 promotes actin turnover, in which coordinated activities of its N- and C-terminal halves catalyze sequential steps in recycling cofilin and actin monomers.


Assuntos
Fatores de Despolimerização de Actina/química , Fatores de Despolimerização de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Difosfato de Adenosina/análogos & derivados , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Despolimerização de Actina/genética , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal , Difosfato de Adenosina/química , Difosfato de Adenosina/genética , Difosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
2.
Diabetes Metab Res Rev ; 24(7): 554-62, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18615850

RESUMO

BACKGROUND: Adiponectin acts as an insulin sensitizer in rodent models. The direct effect of adiponectin in intact type 2 diabetic muscle is unknown. We examined whether adiponectin stimulates glucose transport in isolated skeletal muscle strips from type 2 diabetic men. METHODS: We obtained open muscle biopsies from 12 men with type 2 diabetes (56 +/- 1 years, 30.5 +/- 1.1 kg/m(2)), and from 15 non-diabetic men (59 +/- 1 years, 28.0 +/- 1.0 kg/m(2)). Skeletal muscle strips were isolated and exposed to globular adiponectin (2.5 microg/mL), insulin (120 nM) and/or AICAR (1 mM) in vitro for 1 h. Glucose transport was analysed by accumulation of intracellular 3-O-methyl [(3)H] glucose, phosphorylation of Akt-Ser(473) and Akt-Thr(308) was determined using phosphospecific antibodies, and adiponectin receptor 1 and 2 content was measured using specific antibodies. RESULTS: Globular adiponectin increased glucose transport rate by 1.3-fold (P < 0.01) in type 2 diabetic, but not in non-diabetic muscle. Insulin-stimulated glucose transport rate was unaltered by exposure to globular adiponectin in either group. AICAR increased glucose transport and enhanced insulin-stimulated glucose transport in type 2 diabetic and non-diabetic muscles. Insulin-stimulated phosphorylation of Akt-Ser(473) or Akt-Thr(308) was comparable in type 2 diabetic and non-diabetic muscles, and unaltered by the addition of globular adiponectin in either group. Adiponectin receptor expression was similar in skeletal muscle from type 2 diabetic and non-diabetic men. CONCLUSIONS: Globular adiponectin directly increases glucose transport in skeletal muscle from type 2 diabetic patients. This may occur via Akt-independent signalling routes.


Assuntos
Adiponectina/fisiologia , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Músculo Esquelético/metabolismo , Adenilato Quinase/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Estudos de Coortes , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Insulina/farmacologia , Masculino , Pessoa de Meia-Idade , Músculo Esquelético/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Adiponectina/biossíntese , Ribonucleotídeos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
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