Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells.

We show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes' expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC

Hepatitis C Virus NS5A Protein Promotes the Lysosomal Degradation of Hepatocyte Nuclear Factor 1α via Chaperone-Mediated Autophagy

Hepatitis C virus (HCV) infection is closely associated with type 2 diabetes. We reported that HCV infection induces the lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) via interaction with HCV nonstructural protein 5A (NS5A) protein, thereby suppressing GLUT2 gene expression. The molecular mechanisms of selective degradation of HNF-1α caused by NS5A are largely unknown. Chaperone-mediated autophagy (CMA) is a selective lysosomal degradation pathway. Here, we investigated whether CMA is involved in the selective degradation of HNF-1α in HCV-infected cells and observed that the pentapeptide spanning from amino acid (aa) 130 to aa 134 of HNF-1α matches the rule for the CMA-targeting motif, also known as KFERQ motif. A cytosolic chaperone protein, heat shock cognate protein of 70 kDa (HSC70), and a lysosomal membrane protein, lysosome-associated membrane protein type 2A (LAMP-2A), are key components of CMA. Immunoprecipitation analysis revealed that HNF-1α was coimmunoprecipitated with HSC70, whereas the Q130A mutation (mutation of Q to A at position 130) of HNF-1α disrupted the interaction with HSC70, indicating that the CMA-targeting motif of HNF-1α is important for the association with HSC70. Immunoprecipitation analysis revealed that increasing amounts of NS5A enhanced the association of HNF-1α with HSC70. To determine whether LAMP-2A plays a role in the degradation of HNF-1α protein, we knocked down LAMP-2A mRNA by RNA interference; this knockdown by small interfering RNA (siRNA) recovered the level of HNF-1α protein in HCV J6/JFH1-infected cells. This result suggests that LAMP-2A is required for the degradation of HNF-1α. Immunofluorescence study revealed colocalization of NS5A and HNF-1α in the lysosome. Based on our findings, we propose that HCV NS5A interacts with HSC70 and recruits HSC70 to HNF-1α, thereby promoting the lysosomal degradation of HNF-1α via CMA. IMPORTANCE Many viruses use a protein degradation system, such as the ubiquitin-proteasome pathway or the autophagy pathway, for facilitating viral propagation and viral pathogenesis. We investigated the mechanistic details of the selective lysosomal degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) induced by hepatitis C virus (HCV) NS5A protein. Using site-directed mutagenesis, we demonstrated that HNF-1α contains a pentapeptide chaperone-mediated autophagy (CMA)-targeting motif within the POU-specific domain of HNF-1α. The CMA-targeting motif is important for the association with HSC70. LAMP-2A is required for degradation of HNF-1α caused by NS5A. We propose that HCV NS5A interacts with HSC70, a key component of the CMA machinery, and recruits HSC70 to HNF-1α to target HNF-1α for CMA-mediated lysosomal degradation, thereby facilitating HCV pathogenesis. We discovered a role of HCV NS5A in CMA-dependent degradation of HNF-1α. Our results may lead to a better understanding of the role of CMA in the pathogenesis of HCV

Unconjugated interferon-stimulated gene 15 specifically interacts with the hepatitis C virus NS5A protein via domain I

Interferon-stimulated gene 15 (ISG15), a ubiquitin-like protein, is induced by type I INF. Although several groups have reported ISGylation of the HCV NS5A protein, it is still unclear whether ISGylation of NS5A has anti- or pro-viral effects in hepatitis C virus (HCV) infection. In the present study, the role of ISGylation-independent, unconjugated ISG15 in HCV infection was examined. Immunoprecipitation analyses revealed that ISG15 interacts specifically with NS5A domain I. ISG15 mutants lacking the C-terminal glycine residue that is essential for ISGylation still interacted with NS5A protein. Taken together, these results suggest that unconjugated ISG15 affects the functions of HCV NS5A through protein-protein interaction

Low Cell-Matrix Adhesion Reveals Two Subtypes of Human Pluripotent Stem Cells.

We show that a human pluripotent stem cell (hPSC) population cultured on a low-adhesion substrate developed two hPSC subtypes with different colony morphologies: flat and domed. Notably, the dome-like cells showed higher active proliferation capacity and increased several pluripotent genes' expression compared with the flat monolayer cells. We further demonstrated that cell-matrix adhesion mediates the interaction between cell morphology and expression of KLF4 and KLF5 through a serum response factor (SRF)-based regulatory double loop. Our results provide a mechanistic view on the coupling among adhesion, stem cell morphology, and pluripotency, shedding light on the critical role of cell-matrix adhesion in the induction and maintenance of hPSC