Mechanisms of Endosome Trafficking in Neurons

神经元内体运输的机制

• 批准号: 6606262
• 负责人: Victor Faundez
• 金额: $ 28.88万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6606262
• 关键词: PC12 cells; enzyme substrate; guanosinetriphosphatases; mass spectrometry; membrane fusion; membrane transport proteins; molecular cloning; neurons; phosphorylation; protein kinase; protein protein interaction; protein structure function; protein transport; radionuclides; synaptic vesicles; synaptogenesis; transfection

DESCRIPTION (provided by applicant): A central question to the biology of any cell, is how membrane proteins are specifically sorted by vesicle carriers. This problem is particularly pertinent to the synapse, a neuronal domain specialized in the trafficking of synaptic vesicles (SV). However, the mechansisms by which synaptic vesicle proteins make their sorting decisions are not well understood. To study this fundamental problem I have developed an adaptor-dependent neuronal vesiculation model. This mechanism is capable to sort from endosomes SV proteins using the adaptor complex AP-3 and two enzymes, a GTPase and an AP-3-associated kinase that phosphorylates the adaptor AP-3. Although phosphorylation has been recognized as an important cell regulatory mechanism, its role in adaptor function is mostly unexplored. The central hypothesis guiding this proposal is that an AP-3-associated kinase selectively regulates AP-3-dependent vesiculation and sorting functions. Our goal is to use unique cell-free reconstitution assays plus the analysis of synaptic vesicle protein trafficking in intact neurons to understand the basic mechanisms that govern SV protein sorting and to test the role of phosphorylation in controlling adaptor-dependent vesicle formation. These studies will reveal the first detailed model for the role of phosphorylation in an adaptor-dependent vesiculation process. In this proposal, we will convey the wealth of molecules, mechanisms and drugs that we have characterized in PC12 cells to dissect a novel endosome SV biogenetic pathway in neurons. In mice ablation of the AP-3 pathway results in pigment dilution, epilepsy, behavioral phenotypes and defective SV protein sorting whereas in humans generates the Hermansky-Pudlak II syndrome. The understanding of the AP-3 mechanisms and regulation will open avenues for the development of pathway-specific pharmacological agents that will impact the treatment of epilepsy, behavioral disorders and the Hermansky-Pudlak II syndrome.

描述(由申请人提供)： 对于任何细胞的生物学来说，一个中心问题是膜蛋白是如何被囊泡载体特异性地分类的。这个问题尤其与突触有关，突触是一个专门负责突触小泡(SV)运输的神经元域。然而，突触小泡蛋白做出分类决定的机制还不是很清楚。为了研究这个基本问题，我开发了一个适配器依赖的神经元囊泡模型。这一机制能够使用接头复合体AP-3和两种酶，一种GTP酶和一种使接头AP-3磷酸化的AP-3相关蛋白，从内体SV蛋白中分离出来。虽然磷酸化已被认为是一种重要的细胞调节机制，但其在接头功能中的作用大多未被研究。指导这一提议的中心假设是，AP-3相关的激酶选择性地调节AP-3依赖的囊泡形成和分类功能。我们的目标是使用独特的无细胞重构分析加上对完整神经元中突触小泡蛋白运输的分析，以了解支配SV蛋白分选的基本机制，并测试磷酸化在控制适配器依赖的小泡形成中的作用。这些研究将揭示第一个详细的模型，即磷酸化在适配器依赖的囊泡形成过程中所起的作用。在这个提案中，我们将传达丰富的分子、机制和药物，我们已经在PC12细胞中描述了这一特征，以剖析神经元中新的内体SV生物发生途径。在小鼠中，AP-3通路的消融会导致色素稀释、癫痫、行为表型和SV蛋白分离缺陷，而在人类中会产生Hermansky-Pudlak II综合征。对AP-3机制和调控的了解将为开发影响癫痫、行为障碍和Hermansky-Pudlak II综合征治疗的通路特异性药物开辟道路。