RESUMO
Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin (Htt) protein. N-terminal Htt peptides with polyglutamine tracts in the pathological range (51-122 glutamines) form high-molecular-weight protein aggregates with fibrillar morphology in vitro, and they form discrete inclusion bodies in a cell-culture model. However, in some studies, formation of discrete Htt inclusions does not correlate well with cell death. We coexpressed N-terminal Htt fragments containing 91 glutamines fused to different affinity tags in HEK293 cells, and we isolated small aggregates by double sequential-affinity chromatography to assure the isolation of multimeric molecules. Transmission electron microscopy and atomic force microscopy revealed the isolated aggregates as globules or clusters of globules 4-50 nm in diameter without any detectable fibrillar species. Because small nonfibrillar oligomers, not mature fibrils, recently have been suggested to be the principal cytotoxic species in neurodegenerative disease, these Htt globular aggregates formed in cells may represent the pathogenic form of mutant Htt.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Marcadores de Afinidade , Linhagem Celular , Cromatografia de Afinidade , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Humanos , Proteína Huntingtina , Microscopia de Força Atômica , Microscopia Eletrônica , Complexos Multiproteicos , Mutação , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/ultraestrutura , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/ultraestrutura , TransfecçãoRESUMO
PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway.
Assuntos
NF-kappa B/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Regulação para Baixo , Genes Reporter/genética , Vetores Genéticos/genética , Humanos , Luciferases/metabolismo , Dados de Sequência Molecular , NF-kappa B/metabolismo , Testes de Precipitina , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas/genética , Proteínas/metabolismo , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Fator 2 Associado a Receptor de TNF , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genéticaRESUMO
PKNalpha is a fatty acid- and Rho-activated serine/threonine protein kinase having a catalytic domain homologous to members of the protein kinase C family. Recently it was reported that PKNalpha is involved in the p38 mitogen-activated protein kinase (MAPK) signaling pathway. To date, however, how PKNalpha regulates the p38gamma MAPK signaling pathway is unclear. Here we demonstrate that PKNalpha efficiently phosphorylates MLTKalpha (MLK-like mitogen-activated protein triple kinase), which was recently identified as a MAPK kinase kinase (MAPKKK) for the p38 MAPK cascade. Phosphorylation of MLTKalpha by PKNalpha enhances its kinase activity in vitro. Expression of the kinase-negative mutant of PKNalpha inhibited the mobility shift of MLTKalpha caused by osmotic shock in SDS-PAGE. Furthermore, PKNalpha associates with each member of the p38gamma MAPK signaling pathway (p38gamma, MKK6, and MLTKalpha). These results suggest that PKNalpha functions as not only an upstream activator of MLTKalpha but also a putative scaffold protein for the p38gamma MAPK signaling pathway.