Peroxisome Proliferator Activated Receptor gamma 의 유비퀴틴화와 탈유비퀴틴화 효소에 관한 연구

Abstract

The peroxisome proliferater-activated receptor gamma (PPARg) as a key regulator of critical metabolic processes is a ligand-activated transcription factor and subject to ubiquitination. However, to date, no ubiquitinating or deubiqutinating enzyme of PPARg has been reported. First, I tried to identify the ubiquitinating enzyme, E3 ligase, for PPARg. In these studies, interacting proteins with PPARg were screened using immunoprecipitation for PPARg and mass-spectrometry analysis. As E3 ligase, two candidates, F-box protein 9 (FBXO9) and HECT domain containing 2 (HECTD2), were isolated. Co-immunoprecipitation assay showed the interaction between PPARg and FBXO9 or HECTD2. However, PPARg protein levels were decreased only by FBXO9. FBXO9 decreased the protein stability of PPARg through ubiquitin-proteasome pathway. In adipocytes, overexpression of FBXO9 reduced endogenous PPARg protein levels, but knockdown of FBXO9 did not affect. Taken together, these results indicate that FBXO9 is an E3 ligase of PPARg in adipocytes although physiological roles of FBXO9 in the regulation of PPARg stability are not investigated Next, I studied to identify the deubiquitinating enzyme for PPARg. In the present study, GST-pull down assay was performed to isolate PPARgregulatory proteins.Mass analysis using isolated proteins showed that PPARg could bind with a Herpesvirus-Associated Ubiquitin-Specific Protease (HAUSP/USP7). Co-immunoprecipitation analysis revealed that both endogenous and exogenous PPARg could associate with HAUSP. In addition, the DNA binding and ligand binding domains of PPARg were responsible for the interaction with HAUSP. HAUSP, but not the HAUSP C223S mutant, increased the stability of both endogenous and exogenous PPARg through its deubiquitinating activity. Site directed mutagenesis experiments showed that the K462 residue of PPARg2 is critical for ubiqutination. HBX 41,108, a specific inhibitor of HAUSP, abolished the increase of PPARg stability induced by HASUP. HAUSP enhanced the transcriptional activity of PPARg in luciferase activity assays. Quantitative RT-PCR analysis showed that HAUSP increased the transcript levels of the PPARg target genes in HepG2 cells, resulting in the enhanced uptake of glucose and fatty acid. Taken together, these results demonstrate that the stability and activity of PPARg is modulated by the deubiquitinating activity of HAUSP. In these studies, I identified the ubiquitinating and the deubiquitinating enzyme of PPARg for the first time. These enzymes, FBXO9 and HAUSP, could be targets for the metabolic regulation through PPARg although it needs the further study for physiological roles and conditions of these enzymes in the regulation of PPARg.