Network organization of the human autophagy system

描述（由申请人提供）：自噬是机体蛋白质在营养水平和发育信号降低时降解的主要途径，该途径中的缺陷影响许多疾病状态。出芽酵母的自噬涉及30多个ATG基因，它们共同调节自噬过程的主要步骤：产生双双层膜囊泡（自噬体），捕获细胞内容物和细胞器，并将自噬体运送到溶酶体进行降解。出现了四种主要的信号系统：1)参与信号传导的Atg1p激酶复合体，2)参与在自噬体膜表面生成PtdIns3P （P3）的III类PI3激酶复合体，3)Atg8p/Atg12p泛素样（UBL）偶联系统，负责将Atg8p偶联到磷脂酰乙醇胺，从而促进其并入自噬体，4)自噬体前循环复合体，负责组装Atg9p， Atg2p，和Atg18p在需要Atg1p的途径中进入自噬前体。尽管自噬在哺乳动物细胞中起着明确的作用，但对该途径的组织、所涉及的蛋白质以及该途径内的模块如何相互作用知之甚少。我们对65种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种候选蛋白之间763种相互作用组成的自噬相互作用网络（autophagy Interaction Network， AIN），实验验证率为70%。该分析揭示了许多涉及蛋白激酶、泛素途径成分和与囊泡运输相关的蛋白质的例子。C末端脂化的UBL蛋白ATG8（人类的MAP1LC3和GABARAP家族成员）使用一个保守的表面将含有lc3相互作用区（LIR）的自噬受体招募到自噬体中。6个MAP1LC3/GABARAP蛋白在体内形成了一个包含67个蛋白之间182个相互作用的子网络，家族成员之间有广泛的重叠，并且经常参与体外测量的lir对接位点（LDS）。80个AIN组分的RNAi揭示了数十个基因的缺失减少或增加了自噬体的数量，为参与该途径提供了功能证据。在本提案中，我们将使用系统的方法进一步完善、发展和传播这第一代AIN图谱，并将开始研究新发现的成分如何调节通路内的蛋白激酶、泛素化和ATG8子网络的活性。这些研究将侧重于定义AIN网络的动态变化以及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相互作用景观的一瞥。

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.