bTrCP泛素连接酶在肝特异性转录因子CREB-H的泛素化及降解中作用的研究

Liver-enriched transcription factor CREB-H has important regulatory functions in glucose metabolism, lipid metabolism as well as hepatocyte growth. CREB-H is typically located in the endoplasmic reticulum and activated by regulated intramembrane proteolysis to remove the C-terminus and release the active form CREB-HdTC which then translocate to the nucleus to activate the expression of its target genes. Recent studies have found that over-expression of CREB-HdTC in mouse liver showed alleviating effects on obesity and diabetes. From our study on CREB-H structure and function for over a decade, we found that CREB-HdTC is rapidly degraded inside cells but its cause and mechanism is not known. After some extensive preliminary work, we have discovered a novel mechanism in which CREB-HdTC is rapidly degraded through ubiquitin ligase bTrCP-mediated Ubiquitination-proteasome pathway. Basing on this finding, we will further investigate the roles, mechanisms and biological significance of bTrCP on CREB-HdTC Ubiquitination and degradation. We will make use of over-expression, RNA interference and CRISPR/Cas9 techniques to confirm the actual effect of bTrCP on CREB-H and CREB-HdTC degradation. Using site-directed mutagenesis approach, the degron of CREB-HdTC can be identified and the stimulatory effect on degradation by degron phosphorylation can be clarified. Furthermore, studying the influence of bTrCP on CREB-H’s target gene expression can help reveal the molecular basis on how bTrCP regulate CREB-HdTC degradation as well as the biological significance of bTrCP in glucose metabolism and lipid metabolism. Our work will not only clarify the molecular mechanism on regulating CREB-H degradation but will also provide important inspiration for drug development to fight against obesity and diabetes.

肝特异性转录因子CREB-H对糖代谢及脂代谢均有重要调节作用。CREB-H通常位于内质网，经膜内蛋白酶解释放出去除C端的CREB-HdTC活性形式，进入核内激活其靶基因表达。在小鼠肝脏中过表达CREB-HdTC对肥胖及糖尿病均有缓解作用。我们对CREB-H结构与功能进行了十多年研究，发现CREB-HdTC在细胞内很快被降解，但原因与机制不明。我的大量前期研究表明CREB-HdTC可通过泛素连接酶bTrCP介导的泛素化-蛋白酶体通路而被快速降解，本项目将在此基础上作更深入研究。我们将通过过表达及RNA干扰与CRISPR技术确定bTrCP对CREB-H降解的影响，通过定点诱变阐明CREB-H的降解子及其磷酸化对降解的刺激作用，并通过研究bTrCP对CREB-H靶基因表达的影响揭示前者调节CREB-H降解的分子基础及其在糖代谢和脂代谢中的生物学意义。本项目将对抗肥胖和抗糖尿的新药研发有重要启示。

肝特异性转录因子CREB-H对糖代谢、脂代谢及肝细胞生长均有重要调节作用。CREB-H通常位于内质网，经膜内蛋白酶解释放出去除C端的CREB-HΔTC活性形式，进入核内激活其靶基因表达。我们过往的研究已发现CREB-HΔTC在细胞内很快被降解，但原因与机制不明。在本项目中，我们发现并深入研究了CREB-HΔTC可通过泛素连接酶βTrCP介导的泛素化-蛋白酶体通路而被快速降解的新机制。我们通过过表达及RNA干扰技术确定βTrCP对CREB-H降解的影响，通过定点诱变阐明CREB-H的降解子及其磷酸化对降解的刺激作用，并通过研究βTrCP对CREB-H靶基因表达的影响揭示前者调节CREB-H降解的分子基础及其在糖代谢和脂代谢中的生物学意义。本项目对抗肥胖和抗糖尿的新药研发有重要启示。