Regulation of TOR Signaling and Autophagy in Drosophila

描述（由申请人提供）：该项目的长期目标是了解自噬的调控和生理作用，自噬是一种蛋白质、细胞器和大量细胞质被隔离在自噬囊泡内并被运送到溶酶体降解的过程。这个过程在细胞中起着几个不同的重要作用，回收老化或受损的成分，在饥饿时提供营养来源，在某些情况下启动细胞死亡。这些细胞功能是自噬对多种人类疾病影响的基础。关于自噬的基本问题仍有待解决，包括自噬如何受到营养物质和其他信号的调节，底物如何选择性地靶向降解，以及自噬如何在不同情况下促进细胞存活或细胞死亡。本研究将在果蝇系统中使用遗传、生化和基于成像的方法来帮助定义自噬调节机制及其在完整生物体中的功能。最近对果蝇和哺乳动物细胞的研究已经发现了在有利的营养条件下抑制自噬的保守信号通路中的关键因素。这个营养感应通路的核心成分，雷帕霉素丝氨酸/苏氨酸蛋白激酶靶点（TOR），被证明可以控制包含自噬相关激酶Atg1和磷酸化蛋白Atg13的多组分复合物的活性。该项目的具体目标是：1)测试Atg1/13复合物调控的潜在模型，包括磷酸化、多聚化的影响，以及与新型抑制辅因子的关联。PKA在这种调节中的潜在作用以及atg1相关激酶Ulk3的功能将被表征。2)研究Atg1抑制TOR信号的一种新的自我强化反馈信号的机制和后果。3)研究Atg1过表达导致自噬依赖性细胞死亡的机制。作为Jun kinase （Jnk）信号通路作用的初步提示，我们将探索Jnk诱导自噬的机制以及自噬在内源性Jnk介导的死亡中的作用。4)通过遗传和蛋白质组学筛选鉴定和表征新型自噬调节因子和底物的功能。选择这些目标的实验是因为它们可能对自噬领域重要的关键问题产生重大影响。该建议利用了最近开发的试剂，包括敲除几个自噬相关（Atg）基因、自噬活性的体内标记物和新的遗传操作方法。从果蝇的自噬研究中获得的信息将为基于哺乳动物细胞培养的研究提供重要的全动物补充。

DESCRIPTION (provided by applicant): The long term goal of this project is to understand the regulation and physiological roles of autophagy, a process by which proteins, organelles and bulk cytoplasm are sequestered within autophagic vesicles and delivered to the lysosome for degradation. This process plays several distinct, vital roles in the cell, acting to recycle aged or damaged components, to provide a source of nutrients in response to starvation, and in some cases to initiate cell death. These cellular functions underlie the impact of autophagy on a broad range of human illnesses. Fundamental questions regarding autophagy remain to be addressed, including how autophagy is regulated by nutrients and other signals, how substrates are selectively targeted for degradation, and how autophagy can promote either cell survival or cell death in different contexts. The proposed studies will use genetic, biochemical and imaging-based approaches in the Drosophila system to help define mechanisms of autophagy regulation and its functions in an intact organism. Recent studies in Drosophila and mammalian cells have delineated key factors in a conserved signaling pathway that inhibits autophagy under favorable nutrient conditions. The central component of this nutrient-sensing pathway, the Ser/Thr protein kinase Target of Rapamycin (TOR), was shown to control the activity of a multicomponent complex containing the autophagy related kinase Atg1 and the phosphoprotein Atg13. Specific aims of this project are to: 1) test potential models of Atg1/13 complex regulation, including the effects of phosphorylation, multimerization, and association with novel inhibitory cofactors. Potential roles of PKA in this regulation as well as the function of the Atg1-related kinase Ulk3 will be characterized. 2) investigate mechanisms and consequences of a novel self-reinforcing feedback signal by which Atg1 inhibits TOR signaling. 3) investigate mechanisms by which Atg1 overexpression leads to autophagy-dependent cell death. As preliminary indicate of a role for Jun kinase (Jnk) signaling, the mechanisms of autophagy induction by Jnk and the roles of autophagy in endogenous Jnk-mediated death will be explored. 4) identify and characterize the functions of novel autophagy regulators and substrates through genetic and proteomic screens. Experiments in these aims were selected for their likelihood of having a high impact on key questions important to the field of autophagy. This proposal makes use of recently developed reagents including knockouts of several autophagy-related (Atg) genes, in vivo markers of autophagic activity, and novel methods of genetic manipulation. Information gained from studies of autophagy in Drosophila will provide an important whole- animal complement to mammalian cell culture-based studies.