Collaborator of alternative reading frame protein (CARF) regulates early processing of pre-ribosomal RNA by retaining XRN2 (5'-3' exoribonuclease) in the nucleoplasm.

Collaborator of alternative reading frame protein (CARF) associates directly with ARF, p53, and/or human double minute 2 protein (HDM2), a ubiquitin-protein ligase, without cofactors and regulates cell proliferation by forming a negative feedback loop. Although ARF, p53, and HDM2 also participate in the regulation of ribosome biogenesis, the involvement of CARF in this process remains unexplored. In this study, we demonstrate that CARF associates with 5'-3' exoribonuclease 2 (XRN2), which plays a major role in both the maturation of rRNA and the degradation of a variety of discarded pre-rRNA species. We show that overexpression of CARF increases the localization of XRN2 in the nucleoplasm and a concomitant suppression of pre-rRNA processing that leads to accumulation of the 5' extended from of 45S/47S pre-rRNA and 5'-01, A0-1 and E-2 fragments of pre-rRNA transcript in the nucleolus. This was also observed upon XRN2 knockdown. Knockdown of CARF increased the amount of XRN2 in the nucleolar fraction as determined by cell fractionation and by immnocytochemical analysis. These observations suggest that CARF regulates early steps of pre-rRNA processing during ribosome biogenesis by controlling spatial distribution of XRN2 between the nucleoplasm and nucleolus.

Feasibility and safety of intracoronary nicorandil infusion as a novel hyperemic agent for fractional flow reserve measurements.

Fractional flow reserve (FFR) is a useful modality to assess the functional significance of coronary stenoses. Although adenosine triphosphate (ATP) is generally used as the hyperemic stimulus, we sometimes encounter adverse events like hypotension during FFR measurement. Nicorandil, an ATP-sensitive potassium channel opener, recognized as an epicardial and resistance vessel dilator, has not been fully evaluated as a possible alternative hyperemic agent. The aim of this study was to evaluate the feasibility and safety of intracoronary nicorandil infusion compared to intravenous ATP for FFR measurement in patients with coronary artery disease. A total of 102 patients with 124 intermediate lesions (diameter stenosis >40 and <70% by visual assessment) were enrolled. All vessels underwent FFR measurements with both ATP (150 µg/kg/min) and nicorandil (2.0 mg) stimulus. FFR, hemodynamic values, and periprocedural adverse events between the two groups were evaluated. A strong correlation was observed between FFR with ATP and FFR with nicorandil (r = 0.954, p < 0.001). The agreement between the two sets of measurements was also high, with a mean difference of 0.01 ± 0.03. The mean aortic pressure drop during pharmacological stimulus was significantly larger with ATP compared to nicorandil (9.6 ± 9.6 vs. 5.5 ± 5.8 mmHg, p < 0.001). During FFR measurement, transient atrioventricular block was frequently observed with ATP compared to nicorandil (4.0 vs. 0%, p = 0.024). This study suggests that intracoronary nicorandil infusion is associated with clinical utility and safety compared to ATP as an alternative hyperemic agent for FFR measurement.

Cellular vimentin content regulates the protein level of hepatitis C virus core protein and the hepatitis C virus production in cultured cells.

Hepatitis C virus (HCV) core protein is essential for virus particle formation. Using HCV core-expressing and non-expressing Huh7 cell lines, Uc39-6 and Uc321, respectively, we performed comparative proteomic studies of proteins in the 0.5% Triton X-100-insoluble fractions of cells, and found that core-expressing Uc39-6 cells had much lower vimentin content than Uc321 cells. In experiments using vimentin-overexpressing and vimentin-knocked-down cells, we demonstrated that core protein levels were affected by cellular vimentin content. When vimentin expression was knocked-down, there was no difference in mRNA level of core protein; but proteasome-dependent degradation of the core protein was strongly reduced. These findings suggest that the turnover rate of core protein is regulated by cellular vimentin content. HCV production was also affected by cellular vimentin content. Our findings together suggest that modulation of hepatic vimentin expression might enable the control of HCV production.

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

Enhancement of de novo fatty acid biosynthesis in hepatic cell line Huh7 expressing hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein plays important roles in the pathogeneses of liver steatosis as well as hepatocellular carcinomas due to HCV infection. In this study, we examined de novo fatty acid biosynthesis in hepatic cell line Huh7 cells expressing HCV core protein. The rate of metabolic labeling of cellular fatty acids with [(3)H]acetate in core-expressing (Uc39-6) cells was ca. 1.5-fold higher than that in non-expressing (Uc321) cells. The enzyme activities responsible for fatty acid biosynthesis were assayed in vitro. Cytosolic acetyl-CoA carboxylase activity in Uc39-6 cells was ca. 1.6-fold higher than that in Uc321 cells. On the other hand, cytosolic fatty acid synthase activity in Uc39-6 cells was only slightly higher than that in Uc321 cells. Immunoblot analysis of acetyl-CoA carboxylase 1 (ACC1), which is a rate-limiting enzyme for fatty acid biosynthesis, revealed a higher expression level of the protein in Uc39-6 cells than in Uc321 cells. The ACC1 mRNA content in Uc39-6 cells was 1.4-fold higher than that in Uc321 cells. These results strongly suggest that enhancement of fatty acid biosynthesis in core-expressing cells is caused by increased expression of fatty acid biosynthetic enzymes, especially ACC1. Up-regulation of de novo fatty acid biosynthesis by HCV core protein may affect cellular lipid metabolism, resulting in neutral lipid accumulation in HCV-infected cells.

Proteomic profiling of lipid droplet proteins in hepatoma cell lines expressing hepatitis C virus core protein.

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.