Network organization of the human autophagy system

描述（由申请人提供）：自噬构成了一种主要途径，通过该途径，大量蛋白质响应于降低的营养水平和发育信号而降解，并且该途径中的缺陷影响许多疾病状态。芽殖酵母中的自噬涉及超过30个ATG基因，它们共同调节自噬过程中的主要步骤：产生双双层膜囊泡（自噬体），其捕获细胞内容物和细胞器，并将该自噬体递送至溶酶体进行降解。出现了四种主要的信号系统：1）参与信号传导的Atg 1 p激酶复合物，2）参与在自噬体膜表面产生PtdIns 3 P（P3）的III类PI 3激酶复合物，3）Atg 8 p/Atg 12 p泛素样（UBL）缀合系统，其负责将Atg 8 p缀合至磷脂酰乙醇胺，从而促进其并入自噬体，和4）前自噬体循环复合物，其负责在需要Atg 1 p的途径中将Atg 9 p、Atg 2 p和Atg 18 p组装到前自噬体上。尽管自噬在哺乳动物细胞中发挥着明确的作用，但对该途径的组织，所涉及的蛋白质以及途径内的模块如何相互作用知之甚少。 我们对65个已知和候选的人类自噬蛋白进行了蛋白质组学分析，揭示了一个由439个候选蛋白质之间的763个相互作用组成的自噬相互作用网络（AIN），实验验证率> 70%。该分析揭示了蛋白激酶、泛素途径组分和与囊泡运输相关的蛋白质参与的许多实例。C末端脂化的UBL蛋白ATG 8（人类中的MAP 1 LC 3和GABARAP家族成员）使用保守的表面将含有LC 3相互作用区（LIR）的自噬受体募集到自噬体。六个MAP 1 LC 3/GABARAP蛋白在体内形成了一个包含67个蛋白质之间的182个相互作用的子网络，家族成员之间存在广泛的重叠，并且经常参与体外测量的LIR对接位点（LDS）。80 AIN组分的RNAi揭示了数十个基因，其缺失减少或增加自噬体数量，为参与该途径提供了功能证据。在这项提案中，我们将使用系统的方法来进一步完善，发展和传播这第一代AIN地图，并将开始检查新发现的组件如何调节蛋白激酶，泛素化，和ATG 8子网络的活动的途径。这些研究将集中在定义AIN网络中的动态变化和伴随自噬激活的网络中蛋白质的修饰，从而提供对这一重要细胞系统的相互作用景观的一瞥。 公共卫生相关性：真核细胞如何以及为什么“吃”它们的细胞质内容物的问题已经吸引了细胞生物学家几十年，并且一直是发现一般称为“自噬”的信号转导系统的中心驱动力。这个系统与许多疾病状态，以及衰老和生物体发育有关，但这个过程的许多方面在分子水平上仍然知之甚少。这项工作旨在采用蛋白质组学和遗传学的方法来制定一个路线图，了解物理和遗传的相互作用，这是建立这个系统的重要。

DESCRIPTION (provided by applicant): Autophagy constitutes a primary pathway through which bulk proteins are degraded in response to reduced nutrient levels and developmental signals, and defects in this pathway affect many disease states. Autophagy in budding yeast involves more than 30 ATG genes that together, regulate the primary steps in the autophagy process: generation of a double bi-layer membrane vesicle (the autophagosome) that captures cellular contents and organelles and delivery of this autophagosome to the lysosome for degradation. Four major signaling systems have emerged: 1) the Atg1p kinase complex involved in signaling, 2) the Class III PI3 Kinase complex involved in generating PtdIns3P (P3) on the surface of autophagosomal membranes, 3) the Atg8p/Atg12p ubiquitin-like (UBL) conjugation system, which is responsible for conjugating Atg8p to phosphatidylethanolamine thereby facilitating its incorporation into autophagosome, and 4) the pre- autophagosome cycling complex, which is responsible for assembling Atg9p, Atg2p, and Atg18p onto the pre- autophagosome in a pathway that requires Atg1p. Despite the clear role that autophagy plays in mammalian cells, much less is known about the organization of the pathway, the proteins that are involved, and how modules within the pathway interact with each other. We have performed a proteomic analysis of 65 known and candidate human autophagy proteins, revealing an Autophagy Interaction Network (AIN) composed of 763 interactions between 439 candidate proteins, with an experimental validation rate of >70%. This analysis revealed numerous examples of involvement of protein kinases, ubiquitin pathway components, and proteins linked to vesicle trafficking. The C- terminally lipidated UBL protein ATG8 (MAP1LC3 and GABARAP family members in humans) uses a conserved surface to recruit autophagy receptors containing the LC3-interacting region (LIR) to autophagosomes. The six MAP1LC3/GABARAP proteins formed a sub-network containing 182 interactions among 67 proteins in vivo, with extensive overlap between family members and frequent involvement of the LIR-docking site (LDS) measured in vitro. RNAi of 80 AIN components revealed dozens of genes whose depletion reduces or increases autophagosome number, providing functional evidence for involvement in the pathway. In this proposal, we will use systematic approaches to further refine, develop, and disseminate this first generation AIN map and will begin to examine how newly identified components regulate the activities of protein kinase, ubiquitination, and ATG8 sub-networks within the pathway. These studies will focus on defining dynamic changes in the AIN network and modifications of proteins in the network that accompany activation of autophagy, thereby providing a glimpse into the interaction landscape of this important cellular system. PUBLIC HEALTH RELEVANCE: The question of how and why eukaryotic cells "eat" their cytoplasmic contents has fascinated cell biologists for decades and has been the central driving force in the discovery of a signal transduction system referred to generically as "autophagy". This system is linked to many diseases states, as well as to aging and organismal development, yet there are many aspects of this process that remain poorly understood at the molecular level. This work seeks to employ proteomic and genetic approaches to develop a roadmap for understanding the physical and genetic interactions that are important for establishment of this system.