Role of the ULK1 Complex in Autophagy

ULK1 复合物在自噬中的作用

• 批准号: 8800781
• 负责人: Xuejun Jiang
• 金额: $ 38.31万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8800781
• 关键词: Affect; Amino Acids; Autophagocytosis; Autophagosome; Biochemical; Biological; Biological Assay; Cell physiology; Cells; Chemicals; Communicable Diseases; Complex; Consensus Sequence; Coupled; Development; Disease; Eukaryotic Cell; Event; Gene Proteins; Genes; Goals; Heart Diseases; Homeostasis; In Vitro; Intracellular Membranes; Knock-out; Lead; Life; Lysosomes; Malignant Neoplasms; Mammalian Cell; Mammals; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Membrane Fusion; Membrane Protein Traffic; Methods; Molecular; Neurodegenerative Disorders; Nutrient; Organelles; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Physiology; Play; Property; Protein Family; Protein Kinase; Proteins; Proteomics; Regulation; Reporting; Research; Research Proposals; Role; Series; Signal Transduction; Staging; Starvation; System; Techniques; Yeasts; adenine analog; base; cell growth; cellular imaging; chemical genetics; detection of nutrient; genetic regulatory protein; human FRAP1 protein; human disease; inhibitor/antagonist; insight; member; mutant; novel therapeutic intervention; protein complex; public health relevance; reconstitution; research study; success; tool

DESCRIPTION (provided by applicant): The goal of this proposed research is to understand the mechanisms by which the ULK1 complex, an essential upstream component of the autophagy pathway, senses the nutrient signal and relays the signal to the downstream autophagy machinery. Autophagy is a catabolic cellular process mediated by lysosomal activity and intracellular membrane trafficking and reorganization. It functions to degrade long-lived proteins and bulky organelles in order to maintain cellular homeostasis and to promote survival under stressful conditions. Autophagy is conserved in all eukaryotic cells and crucial for normal development and cell growth. Deregulation of autophagy is involved in human diseases such as cancer, neurodegenerative disorders, infectious diseases and cardiac diseases. Although many autophagy genes (ATG) have been identified, in mammals, how autophagy is induced and regulated, and how it modulates various biological events are not fully understood. We and others previously identified the ULK1-ATG13-FIP200 protein kinase complex (abbreviated as the ULK1 complex) as the direct mediator of the nutrient-sensing kinase mTOR in the autophagy pathway. The mTOR complex-1 (mTORC1) inhibits autophagy by phosphorylating both the protein kinase ULK1 and its regulatory protein ATG13, but mechanistically how mTOR-driven phosphorylation suppresses the autophagic activity of the ULK1 complex is not known. In addition, although the ULK1 complex is considered to be the most upstream component of the autophagy pathway, new evidence suggests that it also plays a role at the later autophagic membrane fusion stages. Further, because the protein kinase activity of ULK1 is essential for its autophagic activity, identification of cellular protein substrates of ULK1 is required for understanding how the ULK1 complex communicates with downstream ATG proteins. Recently, we have obtained a series of preliminary results that have provided insights into these questions. Built upon these preliminary results, in this proposal we will determine the molecular basis underlying the autophagic function of the ULK1 complex by (1) defining the role of nutrient-modulated ATG13 phosphorylation in regulating the autophagic activity of the ULK1 complex; (2) identifying cellular substrates of the protein kinase ULK1 and investigating their potential autophagy function; and (3) determining whether the ULK1 complex regulates downstream autophagic membrane fusion, and if so, the underpinning mechanism. To achieve these aims, we will employ a combination of approaches including both conventional cell biological/biochemical methods and more advanced techniques such as chemical genetics, live-cell imaging, and SILAC-based proteomics. Success of this study will elucidate the molecular basis of mammalian autophagy, a critical cellular process involved in normal physiology and various diseases.

描述（由申请人提供）：这项拟议的研究的目的是了解ULK1复合物是自噬途径的重要上游组成部分的机制，感应营养信号并将信号传递到下游自噬机械。自噬是一种由溶酶体活性和细胞内膜运输和重组介导的分解代谢细胞过程。它的功能可以降解长寿命的蛋白质和笨重的细胞器，以维持细胞稳态并在压力条件下促进生存。自噬在所有真核细胞中都是保守的，对于正常发育和细胞生长至关重要。自噬的放松调节参与了癌症，神经退行性疾病，传染病和心脏疾病等人类疾病。尽管在哺乳动物中已经确定了许多自噬基因（ATG），但如何诱导和调节自噬，以及如何调节各种生物事件。我们和其他人先前以前将ULK1-ATG13-FIP200蛋白激酶复合物（缩写为ULK1复合物）鉴定为自噬途径中营养感应激酶MTOR的直接介体。 MTOR复合物1（MTORC1）通过磷酸化蛋白激酶ULK1及其调节蛋白ATG13抑制自噬，但尚不清楚MTOR驱动的磷酸化如何抑制MTOR驱动的磷酸化。此外，尽管ULK1复合物被认为是自噬途径中最上游的组成部分，但新的证据表明，它在后来的自噬膜融合阶段也起着作用。此外，由于ULK1的蛋白激酶活性对于其自噬活性至关重要，因此需要鉴定ULK1的细胞蛋白底物来理解ULK1复合物如何与下游ATG蛋白通信。最近，我们获得了一系列初步结果，这些结果为这些问题提供了见解。基于这些初步结果，在此提案中，我们将通过（1）定义养分调节的ATG13磷酸化在调节ULK1复合物的自噬活性中的作用来确定ULK1复合物的自噬功能的分子基础； （2）鉴定蛋白激酶ULK1的细胞底物并研究其潜在的自噬功能； （3）确定ULK1复合物是否调节下游自噬膜融合，如果是的，则是基础机制。为了实现这些目标，我们将采用多种方法组合，包括常规细胞生物学/生化方法以及更先进的技术，例如化学遗传学，活细胞成像和基于SILAC的蛋白质组学。这项研究的成功将阐明哺乳动物自噬的分子基础，哺乳动物自噬是正常生理和各种疾病的关键细胞过程。