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.