GOLGI BIOGENESIS AND FUNCTION

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， 高尔基体如何在生理条件下形成这种堆叠结构，以及它如何在 疾病在很大程度上仍然未知。在过去的几年里，我们开发了一种多学科的方法， 采用生物化学、细胞生物学、蛋白质组学和糖组学，结合新的体外 重组分析，以解决这些基本问题。我们发现高尔基体堆积蛋白 GRASP 55和GRASP 65都形成反式寡聚体以将高尔基体池“粘合”成堆叠。使用GRASPs 作为操纵高尔基体堆积形成的工具，我们首次提供了高尔基体堆积阻碍蛋白质形成的证据。 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 拆卸和重新组装过程，这是由磷酸化调控，控制脑池堆积 通过GRASPs和通过调节p97/p47介导的有丝分裂后高尔基体膜的单泛素化 核聚变我们鉴定了HACE 1、突触融合蛋白5和VCIP 135作为泛素连接酶、底物和去泛素化酶， 在后一个过程中。在阿尔茨海默病（AD）中，我们发现β-淀粉样蛋白（Aβ）的积累 激活Cdk 5，使GRASP 65磷酸化并导致高尔基体片段化。值得注意的是， 高尔基体结构通过表达GRASP 65的磷酸化缺陷突变体减少Aβ分泌， 提高淀粉样前体蛋白（APP）的非淀粉样蛋白裂解，暗示高尔基体是一个 AD治疗的潜在治疗靶点。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，以确保蛋白质的保真度 修改、加工和分类。这项MIRA提案巩固了两个中心的资助研究 细胞生物学中关于高尔基体结构和功能的问题：1）堆叠的高尔基体结构如何形成， 2）为什么高尔基体的形成对其功能很重要。我们将探讨高尔基体结构的机制 通过关注GRASPs，高尔基体基质和膜融合蛋白的形成，以及它们在 细胞周期我们将确定高尔基体在有丝分裂时的结构-功能关系， 在高尔基体池未堆积的GRASP缺失细胞中， 压力或疾病条件下，当高尔基体是破碎的。在未来的5-10年内，我们希望建立一个可测试的 一种多分子模型，形成并维持高尔基体的结构，同时容纳各种 生理和病理条件下的贩运事件。我们的长期目标是发展分子生物学 阻断AD患者高尔基体缺陷和延缓疾病发展的工具。

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.