GGPP结合FBP1调控糖脂代谢参与肝细胞癌发生发展的机制研究

Long-term disorders of glucose and lipid metabolism lead to chronic liver diseases and even induce hepatocellular carcinoma (HCC). HCC is the most frequent liver cancer and one of the most common causes of cancer-related death. Rates of incidence and mortality of HCC are increasing worldwide. Thus, it is imperative to find new targets and strategies for HCC treatment. Expression of geranylgeranyl pyrophosphate synthase 1 (GGPPS1) is positively correlated with deterioration in HCC patients. Phenotype analysis of liver-specific Ggpps1 knockout mice also suggests that GGPP plays an important role in the regulation of glucose and lipid metabolism in liver. By affinity purification coupled with mass spectrometry analysis, we successfully identified a series of glycolipid metabolism-related GGPP binding proteins from primary hepatocytes, including Fructose-1,6-bisphosphatase (FBP1), which is a rate-limiting enzyme of gluconeogenesis and also a tumor suppressor. Further studies have shown that GGPP directly binds to FBP1 and markedly increases its enzyme activity. Therefore, in this study we will confirm the binding mode, key sites and biological functions of the binding event between GGPP and FBP1, clarify how the direct binding of GGPP and FBP1 regulates glycolipid metabolism, and ultimately causes the initiation and development of HCC.

长期的糖脂代谢紊乱导致慢性肝病的发生，甚至诱发肝细胞癌。肝细胞癌的发病率和死亡率在全球范围内位居恶性肿瘤前列，而且缺乏有效的治疗药物，因此亟须寻找新的治疗靶点和方案。香叶基香叶基焦磷酸合成酶1（GGPPS1）的表达量与肝细胞癌病人的恶性程度呈正相关，对肝脏特异性Ggpps1敲除小鼠的表型分析也提示GGPP在肝脏的糖脂代谢调控中具有重要作用。通过以GGPP为探针的亲和纯化结合质谱分析，我们成功从肝脏中筛选到多个参与糖脂代谢调控的GGPP直接结合蛋白，其中糖异生的关键限速酶、抑癌蛋白FBP1尤其值得关注。进一步研究表明，GGPP通过与FBP1直接结合上调其活性。本研究中，我们将确认GGPP与FBP1结合的模式、关键位点以及生物学功能，阐明GGPP通过与其靶点蛋白FBP1特异性直接结合调控糖脂代谢平衡，进而调节肝细胞癌发生、发展的分子机制，并为肝细胞癌的治疗提供潜在的药物和靶点。

在本项目中，我们采用高通量的化学蛋白质组学方法首次建立了正常肝脏中代谢中间产物香叶基香叶基焦磷酸（GGPP）的直接结合蛋白作用网络，并筛选和验证了多个参与肝脏糖脂代谢调控的GGPP结合蛋白。我们发现，GGPP直接结合在糖异生限速酶果糖-1,6-二磷酸酶（FBP1）同源四聚体的中央空腔，并驱动其构象由非活性的T态向活性的R态转变；而FBP1蛋白的R50位点对于GGPP的结合和激活作用至关重要。本项目从分子水平、细胞水平和动物模型等多个层面，系统的阐明了GGPP通过与其靶点蛋白FBP1特异性直接结合并变构调节其活性，协调肝脏葡萄糖代谢和胆固醇合成的平衡，进而调控肝细胞癌发生、发展的分子机制，并为肝细胞癌的治疗提供新的潜在靶点、药物和治疗方法。