3.4 | DSG induced UHRF1 protein degradation through the ubiquitin-proteasome system pathway
Next, we investigated the impact of DSG on UHRF1 protein stability by testing the half-time of UHRF1 protein. The protein synthesis was inhibited by cycloheximide (CHX), and then UHRF1 protein was assessed at continuous time points. DSG reduced the half-life of UHRF1 protein in C4-2 and DU145 cells (Fig. 4a-b). suggesting that DSG promoted the protein degradation of UHRF1. It is reported that the ubiquitin-proteasome pathway is the major reason of UHRF1 protein degradation (Ma, et al., 2012). We treated PCa cells with DSG for 40h, and then followed by the treatment of a ubiquitination-proteasome pathway inhibitor MG132 for additional 8h. The data showed that DSG induced the protein degradation of UHRF1, while the protein degradation was reversed by MG132 (Fig. 4c-d). The results were further validated by the in vivo ubiquitination assay. The plasmids of HA-ubiquitin and UHRF1-His were co-transfected into C4-2 or PC3 cells, and then treated with different doses of DSG. UHRF1 protein was immunoprecipitated with anti-His antibody, and poly-ubiquitinated UHRF1 was assessed with anti-HA antibody. The results showed that the ubiquitination level of UHRF1 protein increased with DSG concentrations (Fig. 4e-f). Taken together, these results validated that DSG promoted the protein degradation of UHRF1 through the ubiquitin-proteasome system.