ABSTRACT
Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27 provides a powerful route for enforcing normal progression through the mammalian cell cycle. According to a current model, the ubiquitination of p27 during S-phase progression is mediated by SCF(Skp2) E3 ligase that captures Thr187-phosphorylated p27 by means of the F-box protein Skp2, which in turn couples the bound substrate via Skp1 to a catalytic core complex composed of Cul1 and the Rbx/Roc RING finger protein. Here we identify Skp2 as a component of an Skp1-cullin-F-box complex that is based on a Cul1-Ro52 RING finger B-box coiled-coil motif family protein catalytic core. Ro52-containing complexes display E3 ligase activity and promote the ubiquitination of Thr187-phosphorylated p27 in a RING-dependent manner in vitro. The knockdown of Ro52 expression in human cells with small interfering RNAs causes the accumulation of p27 and the failure of cells to enter S phase. Importantly, these effects are abrogated by the simultaneous removal of p27. Taken together, these data suggest a key role for Ro52 RING finger protein in the regulation of p27 degradation and S-phase progression in mammalian cells and provide evidence for the existence of a Cul1-based catalytic core that utilizes Ro52 RING protein to promote ubiquitination.
Subject(s)
Cyclin-Dependent Kinase Inhibitor p27/metabolism , Protein Processing, Post-Translational , Ribonucleoproteins/chemistry , Ribonucleoproteins/metabolism , S Phase , Amino Acid Motifs , Cell Cycle Proteins/metabolism , Cullin Proteins/metabolism , Down-Regulation/genetics , HeLa Cells , Humans , Phosphothreonine/metabolism , Protein Binding , Ribonucleoproteins/isolation & purification , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/metabolism , beta-Transducin Repeat-Containing Proteins/metabolismABSTRACT
There is evidence for an inhibition of interferon-alpha antiviral activity by the hepatitis C viral protein, NS5A. To identify the mechanisms through which NS5A blocks interferon activity, we compared the gene expression profile of interferon-treated Huh7 cells, stably expressing NS5A with control, using microarrays. Following interferon treatment, 50 genes were up-regulated by at least twofold in control clones, whereas induction of 9 of the 50 genes was significantly reduced in NS5A-expressing clones. The strongest effect of NS5A on interferon response was observed for the OAS-p69 gene. Remarkably, Huh7 cells expressing NS5A showed an up-regulation of interleukin-8. Up-regulation of interleukin-8 was also observed upon transient expression of NS5A mutants isolated from patients responsive or resistant to interferon therapy. Addition of interleukin-8 to Huh7 cells inhibited the antiviral activity of interferon and, similarly to NS5A, reduced the induction by interferon-alpha of selective genes including OAS-p69. Our findings provide a mechanism for NS5A-mediated interferon resistance.
Subject(s)
Gene Expression Profiling , Hepacivirus/metabolism , Hepatocytes/physiology , Interferon-gamma/pharmacology , Interleukin-8/metabolism , Oligonucleotide Array Sequence Analysis , Viral Nonstructural Proteins/physiology , Gene Expression Regulation , Hepacivirus/genetics , Hepatocytes/drug effects , Humans , Interleukin-8/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Cells, Cultured , Up-Regulation , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
Liver steatosis, which involves accumulation of intracytoplasmic lipid droplets, is characteristic of hepatitis C virus (HCV) infection. By use of an in vivo transgenic murine model, we demonstrate that hepatic overexpression of HCV core protein interferes with the hepatic assembly and secretion of triglyceride-rich very low density lipoproteins (VLDL). Core expression led to reduction in microsomal triglyceride transfer protein (MTP) activity and in the particle size of nascent hepatic VLDL without affecting accumulation of MTP and protein disulfide isomerase. Hepatic human apolipoprotein AII (apo AII) expression in double-core/apo AII transgenic mice diminished intrahepatic core protein accumulation and abrogated its effects on VLDL production. Apo AII and HCV core colocalized in human HCV-infected liver biopsies, thus testifying to the relevance of this interaction in productive HCV infection. Our results lead us to propose a new pathophysiological animal model for induction of viral-related steatosis whereby the core protein of HCV targets microsomal triglyceride transfer protein activity and modifies hepatic VLDL assembly and secretion.
