GTP结合蛋白Rab32参与肝脏脂质自噬清除的分子机制研究

The basic pathological change of non-alcoholic fatty liver disease (NAFLD) is the accumulation of lipid in liver. A recent study showed that lipid droplets can be degraded in hepatocytes by a specific autophagy-related process. This finding shed light on a novel treatment strategy of NAFLD. The complex of autophagy-related protein 9(Atg9) interacted with Atg2 -WIPI may have a role in providing the growing phagophore with donor membranes by allowing Atg9 containing vesicles to cycle between the site of expansion and the donor. However, little was known about the source of donor membranes and the mechanism of transportation of Atg9 containing vesicles. There are many questions remained to uncover. How does the Atg9 complex work? Whether there are any other proteins involved? Our previous studies have found that GTP binding protein- Rab32 was interacted with Atg2 and WIPI. Thus we hypothesized that Rab32 might participate in lipophagy to regulate the accumulation of lipid according to our previous work and the report that silencing of Atg2 caused a block of autophagic flux and accumulation of lipid. The purposes of this study are to explore the role of Rab32 in hepatic lipid accumulation, to reveal the effect way of Rab32 and to clarify the molecular mechanism of Rab32 in regulation of hepatic lipid accumulation by immunofluorescence assay, co-immunoprecipitation assay, mass spectrometry technology, protein-protein interaction assay and so on. This study can provide the theoretical foundation of the therapeutic strategy in NAFLD.

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）最基本的病理变化是肝细胞脂质的沉积。研究发现脂质可以通过自噬方式清除，这为NAFLD的治疗提供了新途径。自噬蛋白Atg9与Atg2-WIPI形成复合物参与了自噬体膜的转运。但目前对组成脂质自噬体膜结构的脂质来源和转运模式知之甚少，在脂质自噬过程中Atg9复合物是怎样工作的，是否还有其它分子参与，其机制还不清楚。我们前期研究发现GTP结合蛋白Rab32与Atg2及WIPI有相互作用。这结合文献报道的沉默Atg2可阻断自噬流而导致脂质的沉积，提示Rab32可能参与肝脏脂质自噬清除。本项目拟在前期工作的基础上，利用免疫荧光、免疫共沉淀质谱检测和蛋白相互作用等技术，探讨Rab32在肝脏脂质沉积中的作用，以此揭示Rab32发挥功能的效应途径，明确其调控的分子机制，为缓解和治疗NAFLD提供理论基础。

非酒精性脂肪性肝病（Non-alcoholic fatty liver disease, NAFLD）最基本的病理变化是肝细胞脂质的沉积。研究发现脂质可以通过自噬方式清除，这为NAFLD的治疗提供了新途径。自噬蛋白Atg9与Atg2-WIPI形成复合物参与了自噬体膜的转运。但目前对组成脂质自噬体膜结构的脂质来源和转运模式知之甚少，在脂质自噬过程中Atg9复合物是怎样工作的，是否还有其它分子参与，其机制还不清楚。我们前期研究发现GTP结合蛋白Rab32与Atg2及WIPI有相互作用。本项目拟在前期工作的基础上，利用免疫荧光、免疫共沉淀质谱检测和蛋白相互作用等技术，探讨Rab32在肝脏脂质沉积中的作用，以此揭示Rab32发挥功能的效应途径，明确其调控的分子机制。我们的结果显示在细胞系中Cas9敲除Rab32后出现脂滴增多，甘油三酯与胆固醇增加；过表达Rab32，脂滴减少，甘油三酯与胆固醇减少； Rab32全敲C57小鼠和肝Rab32特敲小鼠肝脏脂质含量较野生鼠增加；Rab32全敲的斑马鱼肝脏中的脂质含量也较野生斑马鱼增加。本研究通过体外细胞系和体内动物实验证实了Rab32敲除导致肝脏脂质沉积，且该现象在不同动物体系中保守存在。qPCR定量野生型C57小鼠与肝脏特异性Rab32敲除鼠（n=3）中与肝脏脂质摄取、从头合成、脂质氧化与外排相关基因。结果显示两组间基因的表达无明显差异。提取野生鼠和Rab32肝脏特异性敲除鼠的肝原代细胞，加入自噬激活剂雷帕霉素与溶酶体降解阻断剂Baf A后，western blot检测自噬蛋白LC3与溶酶体降解蛋白p62的表达。结果显示：Rab32敲除后LC3Ⅱ/LC3Ⅰ降低，自噬受抑制，影响自噬的阶段在自噬活化之后溶酶体降解之前。western blot检测了这个阶段自噬有关的蛋白的表达。结果显示Rab32敲除后Atg9、ULK1增加，Atg18减少。这说明Rab32的敲除影响了自噬启动的早期阶段。本项目为缓解和治疗NAFLD提供了理论基础。

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