Supplement: Gogli Biogenesis and Function

补充：Gogli 生物发生和功能

基本信息

批准号：
10808229
负责人：
Yanzhuang Wang
金额：
$ 1.09万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10808229
关键词：
Address Affect Alzheimer&apos s Disease Alzheimer&apos s disease patient American Amyloid beta-Protein Amyloid beta-Protein Precursor Biochemistry Biogenesis Biological Assay Cell Cycle Cell division Cells Cellular Membrane Cellular biology Chimeric Proteins Complex Defect Development Disease Ensure Event Functional disorder Funding GORASP2 gene Glues Goals Golgi Apparatus In Vitro Malignant Neoplasms Mediating Membrane Membrane Fusion Membrane Proteins Mitosis Modeling Molecular Monoubiquitination Neurodegenerative Disorders Organelles Pathologic Phosphorylation Physiological Post-Translational Protein Processing Process Proteins Proteomics Regulation Research Sorting Stress Structure Structure-Activity Relationship Techniques abeta accumulation glycosylation interdisciplinary approach mutant novel postmitotic protein transport reconstitution secretory protein syntaxin 5 therapeutic target tool trafficking ubiquitin isopeptidase ubiquitin ligase

项目摘要

Project Summary The Golgi apparatus is a central cellular membrane organelle that processes a wide variety of proteins. To best perform its complex functions, Golgi membranes need to form a unique stacked structure. Notably, abnormal Golgi fragmentation has been described in an increasing number of diseases that affect millions of Americans and countless more worldwide, including cancer and neurodegenerative diseases. Despite this, how the Golgi forms this stacked structure under physiological conditions and how it becomes defective in diseases remain largely unknown. Over the last few years, we have developed a multidisciplinary approach employing biochemistry, cell biology, proteomics and glycomics, in combination with a novel in vitro reconstitution assay, to address these fundamental questions. We found that the Golgi stacking proteins GRASP55 and GRASP65 both form trans-oligomers to “glue” the Golgi cisternae into stacks. Using GRASPs as tools to manipulate Golgi stack formation, we provided the first evidence that Golgi stacking impedes protein trafficking to ensure accurate glycosylation and sorting. During cell division, the Golgi undergoes a disassembly and reassembly process, which is regulated by phosphorylation that controls cisternal stacking through GRASPs and by monoubiquitination that regulates p97/p47-mediated post-mitotic Golgi membrane fusion. We identified HACE1, syntaxin 5, and VCIP135 as the ubiquitin ligase, substrate, and deubiquitinase, respectively, in the latter process. In Alzheimer’s disease (AD), we found that beta-amyloid (Aβ) accumulation activates Cdk5, which phosphorylates GRASP65 and causes Golgi fragmentation. Significantly, rescue of Golgi structure by expressing a phosphorylation deficient mutant of GRASP65 reduces Aβ secretion by elevating non-amyloidogenic cleavage of the amyloid precursor protein (APP), implicating the Golgi as a potential therapeutic target for AD treatment. Our overall hypothesis is that Golgi matrix proteins, including GRASPs, organize Golgi membranes into a stacked structure to ensure the fidelity of protein modification, processing, and sorting. This MIRA proposal consolidates funded research on two central questions in cell biology concerning Golgi structure and function: 1) how the stacked Golgi structure is formed, and 2) why Golgi stack formation is important for its function. We will explore the mechanism of Golgi structure formation by focusing on GRASPs, Golgi matrix and membrane fusion proteins, as well as their regulation in the cell cycle. We will determine the structure-function relationship of the Golgi in mitosis when the Golgi stack is completely disassembled, in GRASP-depleted cells where Golgi cisternae are unstacked, and in cells under stress or disease conditions when the Golgi is fragmented. In the next 5-10 years, we hope to build a testable model of multiple molecules that form and maintain the structure of the Golgi while accommodating a variety of trafficking events under physiological and pathological conditions. Our long-term goal is to develop molecular tools to block Golgi defects in AD patients and to delay the development of the disease.

项目摘要高尔基体是一种中央细胞膜细胞器，可处理多种蛋白质。到最佳执行其复杂功能，高尔基体机制需要形成独特的堆叠结构。尤其，越来越多的疾病已经描述了异常的高尔基体碎片化美国人和无数全球，包括癌症和神经退行性疾病。尽管如此，高尔基如何在生理条件下形成这种堆叠结构以及如何在疾病在很大程度上仍然未知。在过去的几年中，我们开发了一种多学科的方法采用生物化学，细胞生物学，蛋白质组学和糖菌，结合新型体外重组测定法，以解决这些基本问题。我们发现高尔基堆积蛋白 Grasp55和Grasp65均形成反植物，将高尔基蓄积器“粘合”到堆栈中。使用grasps 作为操纵高尔基堆积地层的工具，我们提供了第一个证据，表明高尔基堆积会阻碍蛋白质贩运以确保准确的糖基化和分类。在细胞分裂期间，高尔基体经历了拆卸和重新组装过程，该过程受磷酸化的调节，控制文化堆叠通过grasps和通过调节p97/p47介导的有丝分裂后高尔基膜的单泛素化融合。我们确定HACE1，语法5和VCIP135是泛素连接酶，底物和去泛素酶，在阿尔茨海默氏病（AD）中，我们发现β-淀粉样蛋白（Aβ）积累激活CDK5，该CDK5磷酸化grasp65并导致高尔基体碎片化。值得注意的是，营救高尔基体结构通过表达磷酸化缺陷的GRASP65突变体可降低Aβ的分泌升高淀粉样前体蛋白（APP）的非淀粉样生成裂解，将高尔基人隐含为A AD治疗的潜在治疗靶点。我们的总体假设是高尔基基质蛋白，包括grasps，将高尔基膜组织成堆叠结构，以确保蛋白质的保真度修改，处理和分类。该MIRA提案合并了对两个中央的资助研究有关高尔基体结构和功能的细胞生物学问题：1）如何形成堆叠的高尔基体结构， 2）为什么高尔基堆积形成对其功能很重要。我们将探索高尔基体结构的机制通过关注Grasps，Golgi基质和膜融合蛋白的格式，及其调节细胞周期。我们将确定高尔基体堆栈中高尔基体在有丝分裂中的结构功能关系完全拆解，在golgi硫氏菌的抓地细胞中，未藏在细胞中高尔基分裂时的压力或疾病条件。在接下来的5 - 10年中，我们希望建立可测试的多种分子的模型，这些分子形成和维护高尔基体的结构，同时进行多种在身体和病理状况下的贩运事件。我们的长期目标是发展分子阻止AD患者的高尔基体缺陷并延迟疾病发展的工具。