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囊泡内并被递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，用于回收老化或受损的成分，提供营养来源以应对饥饿，并在某些情况下启动细胞死亡。这些细胞功能是自噬对广泛的人类疾病的影响的基础。有关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，如何选择性地靶向降解底物，以及自噬如何在不同情况下促进细胞存活或细胞死亡。 拟议的研究将在果蝇系统中使用遗传，生物化学和基于成像的方法，以帮助定义自噬调节机制及其在完整生物体中的功能。最近在果蝇和哺乳动物细胞中的研究已经描绘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子。这种营养传感途径的中心成分，雷帕霉素的Ser/Thr蛋白激酶靶标（TOR），被证明可以控制含有自噬相关激酶Atg 1和磷蛋白Atg 13的多组分复合物的活性。该项目的具体目标是：1）测试Atg 1/13复合物调节的潜在模型，包括磷酸化，多聚化和与新型抑制性辅因子的关联的影响。PKA在这种调节中的潜在作用以及Atg 1相关激酶Ulk 3的功能将被表征。 2)研究Atg 1抑制TOR信号传导的一种新的自我增强反馈信号的机制和后果。 3)研究Atg 1过表达导致自噬依赖性细胞死亡的机制。作为Jun激酶（Jnk）信号传导作用的初步指示，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剂和底物的功能。选择这些目标的实验是因为它们可能对自噬领域的关键问题产生很大影响。该建议利用最近开发的试剂，包括敲除几个自噬相关（Atg）基因，自噬活性的体内标记，和新的遗传操作方法。从果蝇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整体动物补充。 公共卫生相关性：自噬的缺陷导致对人类健康的几个领域的不利影响，包括癌症、神经变性、炎性疾病、肌病和缺血性损伤。涉及抑制或增强自噬的潜在治疗方法将受益于更好地理解自噬的调节因子和效应因子如何相互作用以控制这一过程。

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. PUBLIC HEALTH RELEVANCE: Defects in autophagy lead to adverse effects on several areas of human health, including cancer, neurodegeneration, inflammatory disease, myopathies, and ischemic injury. Potential therapeutic approaches that involve inhibiting or potentiating autophagy will benefit from greater understanding of how regulators and effectors of autophagy interact to control this process.