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USP14) in patients with HCC using quantitative PCR and immunohistochemical techniques. The expression of USP14 in tumor tissues of patients with HCC was significantly higher than that in adjacent non-cancerous and normal liver tissues. It was also determined whether the expression profile of USP14 was associated with the clinical characteristics of HCC. Increased USP14 expression was associated with some clinicopatho-logical variables, such as advancing tumor stage. A Kaplan-Meier curve analysis demonstrated that patients with HCC having a high USP14 expression had a significantly poorer prognosis after surgery than patients with lower USP14 expression levels. Knockdown of USP14 with the lentiviral vector delivery of shRNA in human hepatocarcinoma SMMC7721 cells suppressed cell proliferation, altered the cell cycle and induced cell apoptosis. Additionally, the Wnt/beta-catenin pathway was activated in HCC patients with USP14 overexpression. These findings strongly suggested that USP14 activation plays an oncogenic role in promoting tumor progression in HCC. Thus, our findings suggested that USP14 is involved in the progression of HCC and may be a useful therapeutic target in HCC. These findings likely reflect the key role that USP14 plays in the pathogenesis of HCC. Therefore, the identification of USP14 and USP14-driven genes may promote the investigation of its functional role to develop more effective therapies for HCC, especially advanced HCC.

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preference for ubiquitin-cyclin B conjugates that carry more than one ubiquitin modification or chain. This specificity is conserved from yeast to humans and is independent of chain linkage type. USP14 has been thought to cleave single ubiquitin groups from the distal tip of a chain, but we find that it removes chains from cyclin B en bloc, proceeding until a single chain remains. The suppression of degradation by USP14's catalytic activity reflects its capacity to act on a millisecond time scale, before the proteasome can initiate degradation of the substrate. In addition, single-molecule studies showed that the dwell time of ubiquitin conjugates at the proteasome was reduced by USP14-dependent deubiquitination. In summary, the specificity of the proteasome can be regulated by rapid ubiquitin chain removal, which resolves substrates based on a novel aspect of ubiquitin conjugate architecture.

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ated the mechanism by which phosphorylation of TH affected the proteasome pathway. The inhibition of proteasomes by MG-132 increased the percentage of TH molecules phosphorylated at their Ser19, Ser31 and/or Ser40 among the total TH proteins to about 70% in PC12D cells over a 24-hr period; although the percentage of phosphorylated TH molecules was about 20% under basal conditions. Moreover, the inhibition of proteasomes by epoxomicin with high specificity increased primarily the quantity of TH molecules phosphorylated at their Ser19. The phosphorylation of Ser19 potentiated Ser40 phosphorylation in cells by a process known as hierarchical phosphorylation. Therefore, the proteasome inhibition might result in an increase in the levels of all 3 phosphorylated TH forms, thus complicating interpretation of data. Conversely, activation of proteasome degradation by IU-1, which is an inhibitor for the deubiquitinating activity of USP14, decreased only the quantity of TH molecules phosphorylated at their Ser19, although it did not decrease that of TH phosphorylated at its Ser31 and Ser40 or that of TH molecules. These results suggest that the phosphorylation of Ser19 in the N-terminal portion of TH is critical as a trigger for the degradation of this enzyme by the ubiquitin-proteasome pathway.