Physiology of Class III PI 3-kinase Signaling 2

III 类 PI 3 激酶信号传导的生理学 2

基本信息

批准号：
8085281
负责人：
Jonathan M. Backer
金额：
$ 34.03万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8085281
关键词：
1-Phosphatidylinositol 3-Kinase Address Aging Amino Acids Autophagocytosis Autophagosome Binding Binding Sites Biochemical Biological Models Calmodulin Cell Survival Cell physiology Cells Clinical Color Complex Cultured Cells Cytosol Data Degenerative Disorder Development Disease Down-Regulation Engineering Enzymes Fatty acid glycerol esters Fluorescence Fluorescence Recovery After Photobleaching Gatekeeping Grant Hepatocyte Immune response Individual Label Laboratories Lead Life Lipids Location Lysosomes Maintenance Mammalian Cell Mammals Measures Mediating Membrane Methods Molecular Chaperones Movement Mus Muscle Mutation Nerve Degeneration Neurodegenerative Disorders Neurologic Neurons Normal tissue morphology Nutrient Organelles Pathway interactions Perinatal Pharmacologic Substance Phenotype Phosphotransferases Physiology Play Pregnancy Process Protein Kinase Proteins Recruitment Activity Regulation Role S-nitro-N-acetylpenicillamine Serum Signal Transduction Spectrum Analysis Starvation Stress Structure Structure of beta Cell of islet Syndrome Time Translations Yeasts Zebrafish analog base cellular imaging chemical genetics design detection of nutrient feeding fly human disease in vivo inhibitor/antagonist insight mutant novel pathogen protein complex reconstitution research study response small molecule stoichiometry sugar trafficking wasting

项目摘要

DESCRIPTION (provided by applicant): Autophagy is a cellular response to nutrient stress, in which the formation of a double-walled membrane structure sequesters cytosolic components and organelles and delivers them to the lysosome for degradation. This liberates nutrients for use in new biosynthetic activity. Autophagy is critical for perinatal survival in mice, plays a role in normal tissue maintenance in neurons, hepatocytes, and pancreatic beta cells, and contributes to innate immune responses to pathogens. Its activation under pathological conditions leads to disease states such as muscle wasting, and decreases in autophagic activity may contribute to neurological decline in aging and in neurodegenerative disease. Thus, pharmacological modulation of autophagy may have significant clinical benefit. One approach to modulating autophagy would be through the Class III PI 3-kinase, hVps34, which is required for autophagy in yeast, flies and mammalian cells. hVps34 exists in multi-protein complexes containing components specific for autophagy (Atg14L) or vesicular trafficking (UVRAG), as well as components that function in both these pathways (hVps15, beclin-1). The mechanisms that regulate hVps34 activity, its recruitment to these complexes, and their subcellular localization, are not well understood. This proposal addresses these questions through a focused analysis of hVps34 regulation in mammalian cells and in zebrafish. Aim 1 uses a chemical genetic approach to examine the regulation of hVps34 by the hVps15 protein kinase. It is based on our exciting data showing that the binding of hVps15 to hVps34, previously thought to require hVps15 activity, is in fact kinase independent and only requires hVps15 binding to ATP. Using hVps15 mutants that are able to utilize ATP analogues, we will define kinase dependent and independent signaling by hVps15, and identify hVps15 substrates. Aim 2 examines the dynamics of hVps34 complex formation, both at the whole cell level and, using fluorescence recovery after photobleaching (FRAP), on the autophagosomal membrane. We will determine whether nutrient starvation regulates subunit exchange between hVps34 complexes, and ask whether hVps34-associated proteins are recruited in tandem, or individually, to autophagosomal membranes. Aim 3 uses fluorescence fluctuation spectroscopy, a method able to define the stoichiometry of cytosolic hVps34 complexes in living cells, to measure the regulation of complex formation by nutrients. Finally, Aim 4 uses novel mutants that selectively disrupt hVps34 binding to calmodulin and hVps15 binding to Rab5 and Rab7, block hVps34 degradation, and abolish hVps34 lipid kinase activity while preserving its protein kinase activity. These mutants will be used in a knockdown/rescue approach in cultured cells and in zebrafish, to define mechanisms that regulate hVps34 signaling in vivo. Taken together, these studies will provide important new information on how hVps34 is regulated by nutrient stress, and how it is targeted to autophagosomal membranes. Given that hVps34 is one of the key kinases involved in autophagy, a better understanding of its regulation will lead to new insights into this critical cellular process. PUBLIC HEALTH RELEVANCE: Normal tissues respond to changes in nutrient availability by activating a process known as autophagy, in which cellular contents are degraded to small molecules (amino acids, sugars and fats) that can be used to support continued cell survival. A growing body of data suggests that abnormal regulation of autophagy can lead to human disease, ranging from muscle wasting to neurodegeneration and aging. This proposal studies the regulation hVps34, a key enzyme that is required for autophagy, with the hope that a better understanding of the mechanisms to drive autophagy can be exploited for the development of new pharmaceuticals for the treatment of degenerative diseases.

描述（申请人提供）：自噬是细胞对营养应激的一种反应，其中双壁膜结构的形成隔离细胞溶质成分和细胞器，并将它们输送到溶酶体进行降解。这会释放出营养物质用于新的生物合成活动。自噬对于小鼠围产期生存至关重要，在神经元、肝细胞和胰腺β细胞的正常组织维持中发挥作用，并有助于对病原体的先天免疫反应。它在病理条件下的激活会导致肌肉萎缩等疾病状态，而自噬活性的降低可能会导致衰老和神经退行性疾病中的神经功能衰退。因此，自噬的药理学调节可能具有显着的临床益处。调节自噬的一种方法是通过 III 类 PI 3 激酶 hVps34，它是酵母、果蝇和哺乳动物细胞自噬所必需的。 hVps34 存在于多蛋白复合物中，其中含有自噬 (Atg14L) 或囊泡运输 (UVRAG) 特异性成分，以及在这两种途径中发挥作用的成分 (hVps15、beclin-1)。调节 hVps34 活性、其向这些复合物的募集及其亚细胞定位的机制尚不清楚。该提案通过重点分析哺乳动物细胞和斑马鱼中的 hVps34 调控来解决这些问题。目标 1 使用化学遗传学方法来检查 hVps15 蛋白激酶对 hVps34 的调节。它基于我们令人兴奋的数据，显示 hVps15 与 hVps34 的结合（以前认为需要 hVps15 活性）实际上是独立于激酶的，只需要 hVps15 与 ATP 结合。使用能够利用 ATP 类似物的 hVps15 突变体，我们将定义 hVps15 的激酶依赖性和独立信号传导，并鉴定 hVps15 底物。目标 2 检查 hVps34 复合物形成的动态，包括在全细胞水平以及在自噬体膜上使用光漂白后荧光恢复 (FRAP)。我们将确定营养饥饿是否调节 hVps34 复合物之间的亚基交换，并询问 hVps34 相关蛋白是否串联或单独募集至自噬体膜。目标 3 使用荧光波动光谱法（一种能够定义活细胞中胞质 hVps34 复合物的化学计量的方法）来测量营养素对复合物形成的调节。最后，Aim 4 使用新型突变体选择性破坏 hVps34 与钙调蛋白的结合以及 hVps15 与 Rab5 和 Rab7 的结合，阻止 hVps34 降解，并消除 hVps34 脂质激酶活性，同时保留其蛋白激酶活性。这些突变体将用于培养细胞和斑马鱼的击倒/拯救方法，以确定体内调节 hVps34 信号传导的机制。总而言之，这些研究将提供有关 hVps34 如何受营养应激调节以及它如何靶向自噬体膜的重要新信息。鉴于 hVps34 是参与自噬的关键激酶之一，更好地了解其调节将带来对这一关键细胞过程的新见解。公共健康相关性：正常组织通过激活一种称为自噬的过程来应对营养物质可用性的变化，在该过程中，细胞内容物被降解为可用于支持细胞持续生存的小分子（氨基酸、糖和脂肪）。越来越多的数据表明，自噬的异常调节可能导致人类疾病，从肌肉萎缩到神经退行性变和衰老。该提案研究了自噬所需的关键酶 hVps34 的调节，希望能够更好地了解驱动自噬的机制，以开发治疗退行性疾病的新药物。