Supplement: GOLGI BIOGENESIS AND FUNCTION

补充：高尔基体的生物发生和功能

• 批准号: 10580215
• 负责人: Yanzhuang Wang
• 金额: $ 13.63万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 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/10580215
• 关键词: Address; Affect; Alzheimer' s Disease; Alzheimer' 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; Mitotic; Modeling; Molecular; Monoubiquitination; Neurodegenerative Disorders; Organelles; Pathologic; Phosphorylation; Physiological; Post-Translational Protein Processing; Process; Proteins; Proteomics; Regulation; Research; Sorting - Cell Movement; Stress; Structure; Structure-Activity Relationship; Techniques; abeta accumulation; glycosylation; interdisciplinary approach; mutant; novel; protein transport; reconstitution; secretory protein; syntaxin 5; therapeutic target; tool; trafficking; ubiquitin ligase

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，怎么样 高尔基体在生理条件下形成了这种堆叠结构及其在疾病中如何变得有缺陷 在很大程度上未知。在过去的几年中，我们开发了一种使用多学科的方法 生物化学，细胞生物学，蛋白质组学和糖基因与一种新型的体外重构测定法相结合 解决这些基本问题。我们发现Golgi堆叠蛋白质抓取55和Grasp65都 形成反植物体，将高尔基蓄积器“粘合”到堆栈中。使用grasps作为操纵高尔基堆栈的工具 形成，我们提供了第一个证据，表明高尔基堆积会阻碍蛋白质运输以确保准确 糖基化和排序。在细胞分裂期间，高尔基体经历了拆卸和重新组装的过程， 它受磷酸化的调节，该磷酸化控制了通过grasps和 调节p97/p47介导的有丝分裂后高尔基膜融合的单泛素化。我们确定了Hace1， 在以后的过程中，语法5和VCIP135分别为泛素连接酶，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，这 磷酸化抓紧65并导致高尔基体碎片化。值得注意的是，通过表达高尔基结构的营救 GRASP65的磷酸化缺陷突变体通过升高非淀粉样蛋白裂解来降低Aβ分泌 淀粉样蛋白前体蛋白（APP）隐含了高尔基体作为AD治疗的潜在治疗靶标。 我们的总体假设是高尔基基质蛋白（包括grasps）将高尔基膜组织到 堆叠结构，以确保蛋白质修饰，加工和分类的保真度。这个mira 提案合并有关高尔基结构和细胞生物学中的两个核心问题的资助研究 功能：1）如何形成堆叠的高尔基体结构，以及2）为什么高尔基堆积形成对其很重要 功能。我们将通过关注Grasps，Golgi Matrix和 膜融合蛋白及其在细胞周期中的调节。我们将确定结构功能 当高尔基体堆栈完全分解时，高尔基体在有丝分裂中的关系，在抓紧的细胞中 在高尔基硫酸水中未藏的地方，在高尔基体处于压力或疾病条件下的细胞中 分散。在接下来的5 - 10年中，我们希望建立一个可测试的多个分子的模型 在生理和 病理状况。我们的长期目标是开发分子工具以阻止AD患者的高尔基体缺陷 并延迟疾病的发展。