Domain Analysis Reveals That a Deubiquitinating Enzyme USP13 Performs Non-Activating Catalysis for Lys63-Linked Polyubiquitin

Deubiquitination is a reverse process of cellular ubiquitination important for many biological events. Ubiquitin (Ub)-specific protease 13 (USP13) is an ortholog of USP5 implicated in catalyzing hydrolysis of various Ub chains, but its enzymatic properties and catalytic regulation remain to be explored. Here we report studies of the roles of the Ub-binding domains of USP13 in regulatory catalysis by biochemical and NMR structural approaches. Our data demonstrate that USP13, distinct from USP5, exhibits a weak deubiquitinating activity preferring to Lys63-linked polyubiquitin (K63-polyUb) in a non-activation manner. The zinc finger (ZnF) domain of USP13 shares a similar fold with that of USP5, but it cannot bind with Ub, so that USP13 has lost its ability to be activated by free Ub. Substitution of the ZnF domain with that of USP5 confers USP13 the property of catalytic activation. The tandem Ub-associated (UBA) domains of USP13 can bind with different types of diUb but preferentially with K63-linked, providing a possible explanation for the weak activity preferring to K63-polyUb. USP13 can also regulate the protein level of CD3δ in cells, probably depending on its weak deubiquitinating activity and the Ub-binding properties of the UBA domains. Thus, the non-activating catalysis of USP13 for K63-polyUb chains implies that it may function differently from USP5 in cellular deubiquitination processes

Gene Expression Profiling of Liver Cancer Stem Cells by RNA-Sequencing

Background: Accumulating evidence supports that tumor growth and cancer relapse are driven by cancer stem cells. Our previous work has demonstrated the existence of CD90 + liver cancer stem cells (CSCs) in hepatocellular carcinoma (HCC). Nevertheless, the characteristics of these cells are still poorly understood. In this study, we employed a more sensitive RNA-sequencing (RNA-Seq) to compare the gene expression profiling of CD90 + cells sorted from tumor (CD90 +CSCs) with parallel non-tumorous liver tissues (CD90 +NTSCs) and elucidate the roles of putative target genes in hepatocarcinogenesis. Methodology/Principal Findings: CD90 + cells were sorted respectively from tumor and adjacent non-tumorous human liver tissues using fluorescence-activated cell sorting. The amplified RNAs of CD90 + cells from 3 HCC patients were subjected to RNA-Seq analysis. A differential gene expression profile was established between CD90 +CSCs and CD90 +NTSCs, and validated by quantitative real-time PCR (qRT-PCR) on the same set of amplified RNAs, and further confirmed in an independent cohort of 12 HCC patients. Five hundred genes were differentially expressed (119 up-regulated and 381 down-regulated genes) between CD90 +CSCs and CD90 +NTSCs. Gene ontology analysis indicated that the over-expressed genes in CD90 +CSCs were associated with inflammation, drug resistance and lipid metabolism. Among the differentially expressed genes, glypican-3 (GPC3), a member of glypican family, was markedly elevated in CD90 +CSCs compared to CD90 +NTSCs. Immunohistochemistry demonstrated that GPC3 was highly expressed in forty-two human liver tumor tissues but absent in adjacent non-tumorous liver tissues. Flow cytometry indicated that GPC3 was highly expressed in liver CD90 +CSCs and mature cancer cells in liver cancer cell lines and human liver tumor tissues. Furthermore, GPC3 expression was positively correlated with the number of CD90 +CSCs in liver tumor tissues. Conclusions/Significance: The identified genes, such as GPC3 that are distinctly expressed in liver CD90 +CSCs, may be promising gene candidates for HCC therapy without inducing damages to normal liver stem cells. © 2012 Ho et al.published_or_final_versio