Physiology of Class III PI 3-kinase Signaling 2

III 类 PI 3 激酶信号传导的生理学 2

• 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
• 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

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（由申请人提供）：自噬是对养分应激的细胞反应，其中双壁膜结构的形成序列隔离了胞质成分和细胞器，并将其传递到溶酶体中以降解。这将释放用于新生物合成活性的营养。自噬对于小鼠的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起作用，并有助于对病原体的先天免疫反应。它在病理状况下的激活导致疾病状态，例如肌肉浪费，自噬活动的降低可能导致衰老和神经退行性疾病的神经系统下降。因此，自噬的药理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级PI 3-激酶HVPS34，这是酵母，苍蝇和哺乳动物细胞中自噬所必需的。 HVPS34存在于包含针对自噬（ATG14L）或囊泡运输（UVRAG）的多组分的多蛋白络合物中，以及在这两种途径中起作用的组件（HVPS15，Beclin-1）。调节HVPS34活性，其对这些复合物的募集及其亚细胞定位的机制尚不清楚。该提案通过对哺乳动物细胞和斑马鱼中HVPS34调节的重点分析来解决这些问题。 AIM 1使用一种化学遗传方法来检查HVPS15蛋白激酶对HVPS34的调节。它基于我们令人兴奋的数据，表明HVPS15与以前认为需要HVPS15活性的HVPS34的结合实际上是独立的，并且仅需要HVPS15与ATP结合。使用能够利用ATP类似物的HVPS15突变体，我们将通过HVPS15定义激酶依赖性和独立的信号，并识别HVPS15底物。 AIM 2在自噬体膜上检查了整个细胞水平的HVPS34复合物形成的动力学，并使用光漂白（FRAP）后的荧光回收率进行了动力学。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相关的蛋白是串联募集还是单独募集到自噬体膜。 AIM 3使用荧光波动光谱，该方法能够定义活细胞中胞质HVPS34复合物的化学计量法，以测量营养物质对复合物形成的调节。最后，AIM 4使用新型突变体，这些突变体有选择地破坏HVPS34与钙调蛋白结合，HVPS15与RAB5和RAB7结合，阻断HVPS34降解以及废除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中的敲低/救援方法，以定义调节体内HVPS34信号传导的机制。综上所述，这些研究将提供有关HVPS34如何受到营养应激以及如何针对自噬体膜的重要新信息。鉴于HVPS34是自噬涉及的关键激酶之一，因此对其调节的更好理解将导致对这种关键细胞过程的新见解。 公共卫生相关性：正常组织通过激活一种称为自噬的过程来应对养分的可用性变化，其中细胞含量被降解为可用于支持持续细胞存活的小分子（氨基酸，糖和脂肪）。越来越多的数据表明，自噬的异常调节会导致人类疾病，从肌肉浪费到神经变性和衰老。该建议研究了自噬所需的关键酶HVPS34调节，希望可以更好地理解驱动自噬的机制，以开发用于治疗退行性疾病的新药物。