Functional Dissection of Autophagosome Biogenesis

 描述（由申请人提供）：自噬是通过自我消化维持细胞稳态的基本机制。异常的自噬与许多人类疾病如癌症、衰老和神经变性密切相关。深入了解自噬的机制对于开发针对这些疾病的治疗策略至关重要。自噬体的生物发生与大分子的捕获和破坏密切相关，大分子的周转在自噬过程中进行。它需要蛋白质-膜相互作用的时空编排，这还没有完全理解。Ge博士建立了一种基于自噬体生物发生的无细胞重建的功能测定法，通过该方法，他通过鉴定ER-高尔基体中间隔室作为自噬体的关键膜来源，向理解自噬体生物发生迈出了进一步的一步。在这项研究中，Ge博士试图扩展这种功能测定，以破译产生自噬体的潜在分子作用。在目标1中，Ge博士将建立一套新的无细胞测定法，结合细胞成像和遗传学，通过关注自噬信号诱导的膜动员事件产生自噬膜前体来研究自噬体生物合成的初始步骤。在目标2中，Ge博士将开发一种系统的蛋白质分级方法，以识别自噬体生物发生中的新型蛋白质因子，并定义每个因子在自噬的蛋白质-膜网络中的功能作用。葛博士的长期目标是了解自噬在生理和病理环境中的分子本质，以达到自噬调节治疗的目的。指导阶段的培训将使Ge博士能够领导一个独立的研究团队，使用生化重建，细胞成像，遗传操作和质谱来解决自噬的基本问题，以及了解自噬相关疾病的发病机制。该奖项下的培训将包括：学习新技术，如超分辨率成像，电子显微镜和定量质谱，获得更多的生化重建和分馏经验，扩大自噬的知识和专业知识，以及教学和写作。研究和培训的完成将极大地促进葛博士作为独立研究者的过渡和成功。

 DESCRIPTION (provided by applicant): Autophagy is a fundamental mechanism for maintenance of cellular homeostasis through self-digestion. Abnormal autophagy is closely related to many human disorders such as cancer, aging, and neurodegeneration. A deep mechanistic understanding of autophagy is crucial for the development of therapeutic strategies against these diseases. Autophagosome biogenesis is intimately associated with the capture and destruction of macromolecules whose turnover is executed in the autophagic process. It entails a spatiotemporal orchestration of protein-membrane interactions which is not fully understood. Dr. Ge has established a functional assay based on cell-free reconstitution of autophagosome biogenesis, through which he has made a further step towards the understanding of autophagosome biogenesis by identifying the ER-Golgi intermediate compartment as a key membrane source of the autophagosome. In this study, Dr. Ge seeks to extend this functional assay to decipher the underlying molecular actions generating the autophagosome. In Aim 1, Dr. Ge will establish a set of new cell-free assays together with cell-imaging and genetics to investigate the initial step of autophagosome biogenesis by focusing on an autophagic signal-induced membrane mobilization event generating the autophagic membrane precursor. In Aim 2, Dr. Ge will develop a systematic protein fractionation approach to identify novel protein factors in autophagosome biogenesis, as well as to define the functional role of each factor in the protein-membrane network of autophagy. Dr. Ge's long term goal is to understand the molecular nature of autophagy in physiological and pathological settings for the purpose of autophagy-modulating therapy. Training in the mentored phase will prepare Dr. Ge to lead an independent research team using biochemical reconstitution, cell imaging, genetic manipulation, and mass spectrometry to address the fundamental questions of autophagy, as well as to understand the pathogenesis of autophagy-related diseases. Training under this award will include: learning new techniques, such as super-resolution imaging, electron microscopy and quantitative mass spectrometry, acquiring more experience in biochemical reconstitution and fractionation, and expanding knowledge and expertise in autophagy, as well as teaching and writing. Completion of the research and training will greatly facilitate Dr. Ge's transition and success as an independent investigator.