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 高尔基体是一个中央细胞膜细胞器，处理各种蛋白质。到 为了最好地执行其复杂的功能，高尔基体膜需要形成独特的堆叠结构。值得注意的是， 异常的高尔基体断裂已经在越来越多的疾病中描述，这些疾病影响数百万人， 包括癌症和神经退行性疾病。尽管如此， 高尔基体在生理条件下形成这种堆叠结构， 但基本上仍不为人所知。在过去的几年里，我们开发了一种多学科的方法， 生物化学、细胞生物学、蛋白质组学和糖组学，结合新的体外重建试验， 解决这些基本问题。我们发现高尔基体堆积蛋白GRASP55和GRASP65都是一种蛋白质， 形成反式寡聚体以将高尔基体池"粘合"成堆叠。使用GRASP作为工具操纵Golgi堆栈 形成，我们提供了第一个证据，高尔基体堆积阻碍蛋白质运输，以确保准确的 糖基化和分选。在细胞分裂过程中，高尔基体经历了一个分解和重组的过程， 其受磷酸化调节，磷酸化通过GRASP控制脑池堆积， 调节p97/p47介导的有丝分裂后高尔基体膜融合的单泛素化。我们确定了HACE 1， syntaxin 5和VCIP 135分别作为泛素连接酶、底物和去泛素化酶。 在阿尔茨海默病（AD）中，我们发现β-淀粉样蛋白（A β）的积累激活了Cdk5， 磷酸化GRASP65并导致高尔基体断裂。值得注意的是，通过表达 GRASP65磷酸化缺陷突变体通过增加非淀粉样蛋白裂解减少A β分泌 的淀粉样前体蛋白（APP），暗示高尔基体作为一个潜在的治疗AD的治疗靶点。 我们的总体假设是高尔基体基质蛋白，包括GRASP，将高尔基体膜组织成 堆叠结构，以确保蛋白质修饰、加工和分选的保真度。这个MIRA 该提案巩固了对细胞生物学中两个核心问题的资助研究， 功能：1）堆叠的高尔基体结构是如何形成的，以及2）为什么高尔基体堆叠的形成对于其 功能我们将通过对GRASPs、高尔基体基质和高尔基体结构的研究来探讨高尔基体结构形成的机制。 膜融合蛋白，以及它们在细胞周期中的调节。我们将确定结构-功能 当高尔基体堆叠完全分解时，在GRASP缺失的细胞中，有丝分裂中高尔基体的关系 其中高尔基体池未堆叠，以及在压力或疾病条件下的细胞中，当高尔基体 支离破碎在未来的5 - 10年里，我们希望建立一个可测试的多分子模型， 维持高尔基体的结构，同时适应生理和 病理条件。我们的长期目标是开发分子工具来阻断AD患者的高尔基体缺陷 并延缓疾病的发展。