GOLGI BIOGENESIS AND FUNCTION

项目总结 高尔基体是一个中央细胞膜细胞器，处理各种各样的蛋白质。至 为了更好地发挥其复杂的功能，高尔基体薄膜需要形成独特的堆叠结构。值得注意的是， 异常高尔基体碎裂在越来越多的疾病中被描述，这些疾病影响着数百万人 美国人和全世界更多的人，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如何形成这种堆积结构，以及它如何在 疾病在很大程度上仍不为人所知。在过去的几年里，我们发展了一种多学科的方法 利用生物化学、细胞生物学、蛋白质组学和糖组学，结合一种新的体外 重建分析，以解决这些根本问题。我们发现高尔基体堆积蛋白 GRASP55和GRASP65都形成反式寡聚体，将高尔基体池粘合在一起。使用抓手 作为操纵高尔基堆积形成的工具，我们提供了高尔基堆积阻碍蛋白质的第一个证据 贩运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 拆解和重组过程，这是由控制脑池堆积的磷酸化调节的 通过GRAP和单素化调节p97/P47介导的有丝分裂后高尔基体膜 核聚变。我们鉴定了HACE1、Synaxin 5和VCIP135为泛素连接酶、底物和脱泛素酶。 分别在后一种过程中。在阿尔茨海默病(AD)中，我们发现β-淀粉样蛋白(Aβ)积聚 激活CDK5，使GRASP65磷酸化，导致高尔基体碎裂。值得注意的是，拯救 通过表达GRASP65的磷酸化缺陷突变体的高尔基体结构减少β的分泌 上调淀粉样前体蛋白(APP)的非淀粉样变性切割，暗示高尔基体是一种 阿尔茨海默病的潜在治疗靶点。我们的总体假设是高尔基体基质蛋白， 包括抓取，将高尔基膜组织成堆叠结构，确保蛋白质的保真度 修改、处理和排序。这项Mira提案整合了两个中心项目的资助研究 细胞生物学中关于高尔基体结构和功能的问题：1)堆积型高尔基体结构是如何形成的， 2)高尔基体堆积的形成对其作用的重要性。我们将探索高尔基结构的机制 GRAPs、高尔基体基质和膜融合蛋白的形成及其调控 细胞周期。我们将确定高尔基体在有丝分裂中的结构与功能的关系 是完全分解的，在抓握耗尽的细胞中，高尔基池没有堆叠，在 高尔基体支离破碎时的压力或疾病状态。在接下来的5-10年，我们希望建立一个可测试的 形成和维持高尔基体结构的多分子模型，同时容纳各种 生理和病理条件下的贩运事件。我们的长期目标是开发分子 阻断阿尔茨海默病患者高尔基体缺陷并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.