Upregulation of TRPC1 protects against high glucose-induced HUVECs dysfunction by inhibiting oxidative stress.

-To explore the effect of TRPC1 on endothelial cell function damage under a high glucose environment and its downstream molecular mechanism, and provide new theory and strategy for improving diabetic endothelial cell function and promoting vascular injury repair. In vitro, we use high glucose to treat human umbilical vein endothelial cells (HUVECs) and upregulated TRPC1 with adenovirus infection. HUVECs were split into 4 groups: (i) NG Group: Treated with normal glucose; (ii) HG Group: Treated with high glucose; (iii) HG + adGFP Group: High glucose + the control adenovirus (adGFP); (iv) HG + adTRPC1 Group: High glucose + recombinant adenovirus encoding TRPC1. We found that high glucose significantly decreased the expression level of TRPC1 protein, and impaired the proliferation and migration of HUVECs, which could be reversed by overexpression of TRPC1. In addition, high glucose induced an increase in ROS and MDA and a decrease in SOD activity, whereas TRPC1 overexpression could inhibit the growth of oxidative stress level. These findings suggest that overexpression of TRPC1 prevents HUVECs proliferation and migration dysfunction induced by high glucose via inhibiting oxidative stress injuries.

HERC5 E3 ligase mediates ISGylation of hepatitis B virus X protein to promote viral replication.

Ubiquitin and ubiquitin-like protein modification play important roles in modulating the functions of viral proteins in many viruses. Here we demonstrate that hepatitis B virus (HBV) X protein (HBx) is modified by ISG15, which is a type I IFN-inducible, ubiquitin-like protein; this modification is called ISGylation. Immunoblot analyses revealed that HBx proteins derived from four different HBV genotypes accepted ISGylation in cultured cells. Site-directed mutagenesis revealed that three lysine residues (K91, K95 and K140) on the HBx protein, which are well conserved among all the HBV genotypes, are involved in acceptance of ISGylation. Using expression plasmids encoding three known E3 ligases involved in the ISGylation to different substrates, we found that HERC5 functions as an E3 ligase for HBx-ISGylation. Treatment with type I and type III IFNs resulted in the limited suppression of HBV replication in Hep38.7-Tet cells. When cells were treated with IFN-α, silencing of ISG15 resulted in a marked reduction of HBV replication in Hep38.7-Tet cells, suggesting a role of ISG15 in the resistance to IFN-α. In contrast, the silencing of USP18 (an ISG15 de-conjugating enzyme) increased the HBV replication in Hep38.7-Tet cells. Taken together, these results suggest that the HERC5-mediated ISGylation of HBx protein confers pro-viral functions on HBV replication and participates in the resistance to IFN-α-mediated antiviral activity.