RESUMEN
Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is an adaptor protein composed of two homophilic protein-protein interaction domains, a PYRIN domain (PYD) and a caspase recruitment domain. PYD-dependent oligomerization of ASC is thought to play a crucial role in formation of a molecular platform, the inflammasome, which activates caspase-1. When expressed in cells, the PYD of ASC was shown to form cytoplasmic filaments through self-association. Over 70 single point mutants were analyzed for filament formation in cells expressing the mutant proteins. The set of mutations comprised every single amino acid residue with a charged side chain (Arg, Lys, Asp, and Glu) and a large hydrophobic side chain (Ile, Leu, Met, Phe, Pro, and Val). Filament formation of the ASC PYD was prevented by mutation of Lys21, Leu25, Lys26, Pro40, Arg41, Asp48, and Asp51 of helices 2, 3, and 4. These data identify a coherent interaction surface, establishing a molecular model of PYD-PYD complexes with an important role for charge-charge interactions.
Asunto(s)
Apoptosis , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Apoptosis/genética , Proteínas Adaptadoras de Señalización CARD , Células COS , Proteínas Portadoras/genética , Chlorocebus aethiops , Proteínas del Citoesqueleto/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Espectroscopía de Resonancia Magnética , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Mutación Puntual , Estructura Terciaria de Proteína/genética , Proteínas/genética , Pirina , Electricidad EstáticaRESUMEN
It is thought that the nuclear nonchromatin structures, such as the nuclear matrix and lamina, play regulatory roles in gene expression. In this study, we identified an insoluble protein that was associated with the chromatin-depleted nuclear structure of proliferating human leukemia HL-60 cells. Preparation of the chromatin-depleted nuclear structure, referred to as the nuclear matrix-intermediate filament scaffold (Fey, E., Krochmalnic, G., and Penman, S. (1986) J. Cell. Biol. 102, 1654-1665), involved cell extraction using a series of buffers containing Triton X-100, DNase I, and 2 M NaCl. A yeast two-hybrid assay revealed that this protein bound to the catalytic subunit of protein phosphatase-1 (PP1). Furthermore, it inhibited PP1 activity in vitro. We therefore named it scapinin (scaffold-associated PP1 inhibiting protein). cDNA cloning revealed that scapinin had two splicing variants of 448 amino acids (scapinin-S) and 518 amino acids (scapinin-L). Scapinin was down-regulated by differentiation in HL-60 cells. These results suggest that scapinin is a putative regulatory subunit of PP1 and is involved in transformed or immature phenotypes of HL-60 cells. We also describe the presence of scapinin family proteins from worm to human.
