Vamp8磷酸化调控自噬体-溶酶体融合的分子机制

Autophagy is a highly conserved cellular biological phenomenon in eukaryotic cells and plays an important role in many human diseases such as cancer, neurodegenerative diseases and infectious diseases. Since autophagy has been implicated in major human diseases, the studies of autophagy have gradually increased in recent years. However, the studies mainly focused on the autophagosome formation stage and few studies have been performed on autophagosome-lysosome fusion. The applicant's preliminary data showed that the SNARE protein Vamp8 involved in autophagosome-lysosome fusion can be phosphorylated and this phosphorylation negatively regulates autophagosome-lysosome fusion. However, the detailed molecular mechanism on how Vamp8 regulates autophagosome-lysosome fusion need to be further studied. The applicant intends to combine the technology of molecular biology and biophysics to identify the phosphorylation sites on Vamp8, the kinases responsible for Vamp8 phosphorylation and dissect the molecular mechanism on how the Vamp8 phosphorylation regulates autophagosome-lysosome fusion in vivo and in vitro. This study will not only provide a new mechanism for autophagy regulation, but also provide a new strategy for the treatment of autophagy-related diseases.

细胞自噬是真核细胞中高度保守的细胞生物学现象，在癌症、神经退行性疾病和感染性疾病等多种人类重大疾病中具有重要作用。正因为自噬与人类重大疾病的密切关系，近年来对于细胞自噬的研究逐渐增多。但是已有研究主要集中在自噬体生成阶段，对自噬体-溶酶体融合的研究较少。申请人的前期研究结果发现参与自噬体-溶酶体融合的SNARE蛋白Vamp8可以被磷酸化，而且此磷酸化负向调控自噬体-溶酶体融合，但其分子机制还不清楚。申请人拟结合分子细胞生物学、生物物理学的技术和手段，鉴定Vamp8上的磷酸化位点，确定其上游激酶并通过体内外实验解析Vamp8磷酸化调控自噬体-溶酶体融合的分子机制。此研究课题将不仅为细胞自噬调控提供新的机制，而且也为自噬相关疾病的治疗提供新靶标。

在此研究中，我们证明mTORC1可以通过磷酸化VAMP8来抑制STX17-SNAP29-VAMP8 SNARE复合物的形成，从而抑制自噬体与溶酶体的融合，且在体外膜融合实验证明磷酸化的VAMP8无法促进自噬体与溶酶体的融合。此外，我们发现了一个参与自噬体与溶酶体融合的Sec1/Munc18-like(SM)蛋白：SCFD1。SCFD1由去磷酸化的VAMP8招募至自噬溶酶体上，并且是SNARE复合物合成和自噬体与溶酶体融合的关键调控因子。我们发现磷酸化VAMP8或缺失SCFD1都会抑制自噬体与溶酶体融合，并且在小鼠肝脏中特异性表达磷酸化的VAMP8会导致肝脏脂滴积累。总的来说，mTORC1介导的VAMP8磷酸化可以抑制SCFD1被招募至自噬溶酶体从而抑制STX17-SNAP29-VAMP8 SNARE复合物的形成，进而抑制自噬体与溶酶体的融合。