RESUMEN
TNF receptor-1 (TNF-R1) signal transduction is mediated through the assembly of scaffolding proteins, adaptors, and kinases. TNF receptor ubiquitous scaffolding and signaling protein (TRUSS), a 90.1 kDa TNF-R1-associated scaffolding protein, also interacts with TRAF2 and IKK and contributes to TNF-alpha-induced nuclear factor-kappaB (NF-kappaB) and c-Jun-NH(2)-terminal kinase (JNK) activation. Little is known about the mechanism of interaction among TRUSS, TNF-R1, and TRAF2. To address this issue, we used deletional and site-directed mutagenesis approaches to systematically investigate (i) the regions of TRUSS that interact with TNF-R1 and TRAF2 and (ii) the ability of TRUSS to self-associate to form higher-order complexes. Here we show that sequences located in the N-terminal (residues 1-248) and central (residues 249-440) regions of TRUSS are required to form a docking interface that supports binding to both TNF-R1 and TRAF2. While the C-terminal region (residues 441-797) did not directly interact with TNF-R1 or TRAF2, sequences located in this region were capable of self-association. Collectively, these data suggest that (i) the interaction between TNF-R1 and TRAF2 requires sequences located in the entire N-terminal half (residues 1-440) of TRUSS, (ii) the binding interface for TNF-R1 is closely linked with the TRAF2 binding interface, and (iii) the assembly of homomeric TRUSS complexes may contribute to its role in TNF-R1 signaling.
Asunto(s)
Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Factor 2 Asociado a Receptor de TNF/metabolismo , Canales Catiónicos TRPC/química , Secuencia de Aminoácidos , Sitios de Unión , Células Cultivadas , Humanos , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Receptores Tipo I de Factores de Necrosis Tumoral/química , Factor 2 Asociado a Receptor de TNF/química , Canales Catiónicos TRPC/metabolismoRESUMEN
Cervical cancer is known to be highly associated with viral oncogene E6 and E7 of human papilloma virus. Down-regulation of oncogene expression by antisense-based gene therapy has been extensively studied. To investigate the effect of HPV 16 E6 antisense nucleic acid (AS) on cervical cancer cells, human cervical cancer cell lines, CaSki and SiHa cells harboring HPV 16 genome were transfected with plasmid containing E6(AS). The decreased viability and the apoptotic morphology were observed in E6(AS)-transfected cervical cancer cell lines. By 6 h after transfection, inhibition of E6 splicing, rapid upregulations of p53 and a p53-responsive protein, GADD45, were displayed in E6(AS)-transfected CaSki cells. Furthermore, E6(AS) induced loss of mitochondrial transmembrane potential, release of mitochondrial cytochrome c into the cytoplasm, and subsequent activation of caspase-9 and caspase-3. These results indicate that HPV 16 E6(AS) induces apoptosis in CaSki cells via upregulation of p53 and release of cytochrome c into cytoplasm, consequently activating procaspase-9 and procaspase-3.