Regulation of TOR Signaling and Autophagy in Drosophila

描述(申请人提供)：这个项目的长期目标是了解自噬的调节和生理作用，自噬是蛋白质、细胞器和大量细胞质被隔离在自噬小泡中并被运送到溶酶体进行降解的过程。这个过程在细胞中扮演着几个不同的重要角色，作用是回收老化或受损的成分，提供营养来源以应对饥饿，在某些情况下还会启动细胞死亡。这些细胞功能是自噬对广泛的人类疾病产生影响的基础。关于自噬的基本问题仍有待解决，包括自噬如何受到营养和其他信号的调控，底物如何选择性地被降解，以及自噬如何在不同的环境中促进细胞存活或死亡。这项拟议的研究将在果蝇系统中使用基于遗传、生化和成像的方法来帮助确定自噬调节的机制及其在完整有机体中的功能。最近对果蝇和哺乳动物细胞的研究已经描绘出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。这一营养感知通路的中心成分，雷帕霉素的丝氨酸/苏氨酸蛋白激酶靶标(TOR)，被证明控制着一个包含自噬相关激酶Atg1和磷蛋白Atg13的多组分复合体的活性。本项目的具体目标是：1)测试Atg1/13复杂调控的潜在模型，包括磷酸化、多聚化以及与新的抑制辅助因子的关联。PKA在这一调节中的潜在作用以及Atg1相关的激酶Ulk3的功能将被表征。2)研究ATG1抑制TOR信号的新的自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。作为对Jun激酶(JNK)信号转导作用的初步暗示，我们将探讨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)通过基因和蛋白质组筛选鉴定新的自噬调节子和底物的功能。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高度影响。这一建议利用了最近开发的试剂，包括几个自噬相关(ATG)基因的敲除，体内自噬活性的标记，以及新的遗传操作方法。从果蝇自噬研究中获得的信息将为以哺乳动物细胞培养为基础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.