Supplement: GOLGI BIOGENESIS AND FUNCTION

项目概要 高尔基体是处理多种蛋白质的中央细胞膜细胞器。至 为了最好地发挥其复杂功能，高尔基膜需要形成独特的堆叠结构。值得注意的是， 越来越多的疾病描述了异常的高尔基体断裂，这些疾病影响了数百万人。 美国人和世界各地无数的人，包括癌症和神经退行性疾病。尽管如此，如何 高尔基体在生理条件下形成这种堆叠结构以及它如何在疾病中变得有缺陷 仍然很大程度上不为人知。在过去的几年里，我们开发了一种多学科方法 生物化学、细胞生物学、蛋白质组学和糖组学，结合新颖的体外重构测定， 解决这些基本问题。我们发现高尔基体堆积蛋白 GRASP55 和 GRASP65 都 形成反式寡聚物将高尔基体“粘合”成堆叠。使用 GRASP 作为操作高尔基体堆栈的工具 形成，我们提供了第一个证据表明高尔基体堆积阻碍蛋白质运输，以确保准确 糖基化和分选。在细胞分裂期间，高尔基体经历拆卸和重组过程， 它受到磷酸化的调节，磷酸化通过 GRASP 控制脑池堆积，并通过 单泛素化调节 p97/p47 介导的有丝分裂后高尔基体膜融合。我们确定了 HACE1， 在后一个过程中，突触融合蛋白 5 和 VCIP135 分别作为泛素连接酶、底物和去泛素酶。 在阿尔茨海默病 (AD) 中，我们发现 β-淀粉样蛋白 (Aβ) 积累会激活 Cdk5，从而 磷酸化 GRASP65 并导致高尔基体断裂。值得注意的是，通过表达来拯救高尔基体结构 GRASP65 的磷酸化缺陷突变体通过提高非淀粉样蛋白生成裂解来减少 Aβ 分泌 淀粉样前体蛋白 (APP) 的存在，表明高尔基体是 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 堆叠结构确保蛋白质修饰、加工和分选的保真度。这个米拉 该提案整合了细胞生物学中两个核心问题的资助研究，即高尔基体结构和 功能：1) 堆叠式高尔基体结构是如何形成的，以及 2) 为什么高尔基体堆叠结构对其重要 功能。我们将重点探讨GRASP、高尔基体基质和高尔基体结构形成的机制。 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 在 GRASP 耗尽的细胞中，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 高尔基体池未堆叠的地方，以及当高尔基体处于压力或疾病条件下的细胞中 支离破碎。在接下来的5-10年里，我们希望建立一个可测试的多分子模型，这些分子可以形成和 维持高尔基体的结构，同时适应生理和环境下的各种贩运事件 病理状况。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。

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.