蛋白激酶PINK1S调控蛋白质翻译的分子机制研究

Proteasomal insufficiency leads to a general shutdown of protein synthesis by inhibiting initiation complex formation. Emerging evidence demonstrates that polypeptide elongation can also be regulated during proteasomal inhibition. However, the molecular mechanism between polypeptide elongation and proteasomal stress remains unclear. We have found that decrease in proteasome activities triggers accumulation of PINK1S. Through phosphorylating P62, PINK1S helps reroute proteasome-targeted polyubiquitinated proteins to aggresome and degraded by autophagy. Furthermore, our preliminary data suggest that cytosolic accumulation of PINK1S also reduces global translation. Immunoprecipitation experiments show that PINK1S can directly bind to elongation factor eEF1A1. In this project, we propose to study the functional connections between PINK1S-mediated phosphorylation of eEF1A1 and protein translation. First, we will analyze the characteristics of translation in pink1 knockout cells by ribosome profiling and RNA-seq. Then, we propose to identify the phosphorylation sites in eEF1A1 by mass-spec analysis. Finally, we will confirm that PINK1S-induced translational inhibition whether protect neurons from proteasome inhibitor-induced stress. This study will further our understanding of the proteasomal stress response pathways and provide a theory for the development of new therapeutic approaches for protein degradation-related disorders, such as neurodegeneration.

蛋白酶体活力不足会重塑细胞内蛋白质合成途径，该调控过程主要发生在翻译的起始阶段。最近研究发现，翻译的延伸在蛋白酶活力降低引起的应激反应中也受到严格调控，但是其中的分子机制还不清楚。在前期的研究中，我们发现蛋白激酶PINK1S作为蛋白酶体活力的“感应开关”，通过磷酸化P62加速泛素化蛋白以自噬方式清除。另外，PINK1S在细胞质中积累不仅会抑制蛋白质的合成；而且还能结合并磷酸化翻译延伸因子eEF1A1。为了阐明PINK1S调控翻译的分子机制，本项目拟通过核糖体图谱展示和RNA-seq技术，分析pink1敲除细胞内mRNA翻译的特点；随后应用质谱技术，鉴定eEF1A1中的磷酸化位点；最后在动物水平上证实PINK1S介导的翻译抑制对神经元的保护作用。本项目的开展，不仅协助我们了解蛋白酶体活力不足引起的细胞应激机制，也将为蛋白质降解紊乱所引发的疾病，如神经退行性疾病的治疗提供新的切入点。

蛋白质翻译重塑是细胞应对胁迫的重要调控方式之一。当蛋白酶体活力不足时，细胞通过多种途径抑制细胞内大部分蛋白质的翻译过程，特异性激活胁迫抵抗基因的表达。关于其分子机制的研究，主要集中在翻译起始阶段，至于翻译的延伸是否也参与胁迫应答还不清楚。细胞内蛋白酶体被抑制时，作为蛋白酶体活力的感应开关，PINK1S迅速在细胞质内积累。我们的研究发现细胞质内积累的PINK1S被迅速招募到核糖体上，暗示PINK1可能直接参与蛋白质的翻译调控过程；过表达细胞质定位的PINK1S能够抑制蛋白质合成；敲除pink1 扰乱MG132诱导的蛋白质翻译重塑过程；分子水平上，细胞质PINK1S通过结合并磷酸化翻译延伸因子eEF1A1来调节细胞内蛋白质的合成。本研究揭示了翻译的延伸过程也直接参与胁迫应答过程，而PINK1是其中重要的调控因子。