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GOLGI BIOGENESIS AND FUNCTION

高尔基体的生物发生和功能

基本信息

批准号：
8963053
负责人：
Yanzhuang Wang
金额：
$ 30.15万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2019-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8963053
关键词：
Affect Alzheimer&apos s Disease Antibodies Architecture Autoimmune Diseases Binding Biochemistry Biogenesis Biological Biological Assay Cell Cycle Cell Cycle Progression Cell Nucleus Cell division Cell physiology Cell surface Cell-Matrix Junction Cells Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Cytosol Data Defect Diabetes Mellitus Disease Electron Microscopy Ensure Enzymes Functional disorder Glues Golgi Apparatus Growth Growth Factor Hormones Huntington Disease In Vitro Inherited Interphase Cell Knock-out Link Malignant Neoplasms Mammalian Cell Mediating Membrane Membrane Protein Traffic Membrane Proteins Methods Modification Molecular Neurodegenerative Disorders Neurotransmitters Organelles Pathogenesis Pharmacological Treatment Physiological Play Post-Translational Protein Processing Process Protein Glycosylation Proteins Proteomics RNA Interference Reporting Role Sorting - Cell Movement Stimulus Structure Structure-Activity Relationship Techniques Testing Virus Diseases base genome editing glycosylation human disease insight interdisciplinary approach migration mutant novel protein transport public health relevance reconstitution response restoration secretion process secretory protein tissue/cell culture tool trafficking

项目摘要

DESCRIPTION (provided by applicant): The Golgi complex is a central membrane organelle for intracellular trafficking, glycosylation and sorting of membrane and secretory proteins. The basic structure of the Golgi is a stack of flattened cisternae, but how this structure is assembled and inherited during cell division is poorly understood. In the last few years, we have developed a unique multidisciplinary approach employing biochemistry, cell biology, electron microscopy, and more recently proteomics and glycomics, combined with a novel in vitro reconstitution assay, to provide a mechanistic explanation for Golgi structure formation and function. We found that GRASP55 and GRASP65 play complementary and essential roles in Golgi structure formation by forming mitotically regulated trans- oligomers that hold the Golgi membranes into stacks. Using GRASP55/65 as tools to manipulate Golgi stack formation, we provided the first evidence that Golgi stacking impedes protein trafficking to ensure accurate post-translational modifications. We hypothesize that the GRASP proteins play essential roles in Golgi structure formation through oligomerization and interaction with other proteins, which subsequently regulates protein trafficking and glycosylation. This proposal is a logical continuation of our previous studies to assess how the Golgi structure is assembled and why it is important for protein trafficking, glycosylation and sorting. The specific aims are: 1) Identify and characterize novel GRASP65 interacting proteins that regulate Golgi structure formation. Our preliminary data showed that there are proteins in the interphase cell cytosol that enhance GRASP65 oligomerization. We have identified 20 proteins that interact with GRASP65 and we will characterize their roles in Golgi structure formation. This will also help us understand how GRASPs perform multiple functions as previously reported. 2) Determine the structure- function relationship of the Golgi in protein trafficking, glycosylation, and sorting. We will manipulate th Golgi structure by knocking out GRASP55/65, using the recently developed CRISPR/Cas9 technique, and by expressing GRASP55/65 mutants in cells to determine the consequences of Golgi destruction and restoration on protein trafficking, modifications and secretion. Significantly, alterations in Golgi structure and function have been associated with a variety of human diseases, including cancer, autoimmune disease, viral infections, and Huntington's and Alzheimer's diseases. Golgi defects may affect the trafficking, sorting and modification of a large number of proteins and cause global effects inside the cell and on the cell surface that compromise a variety of cellular functions. A better understanding of Golgi structure formation and the relationship to its vital cellular function is required before its role in human disease ca be understood. Our proposed study will determine the consequence of Golgi destruction on protein trafficking and processing, and thus provide fundamental information on the role of the Golgi under normal conditions and the relationship between Golgi defects and disease pathogenesis.

描述（由申请人提供）：高尔基复合体是一种中央膜细胞器，用于细胞内运输、糖基化和膜蛋白和分泌蛋白的分选。高尔基体的基本结构是一堆扁平的池，但这种结构是如何组装的呢？以及在细胞分裂过程中遗传的基因，我们对此知之甚少在过去的几年中，我们已经开发了一个独特的多学科的方法，采用生物化学，细胞生物学，电子显微镜，最近蛋白质组学和糖组学，结合一种新的体外重建试验，提供一个高尔基体结构的形成和功能的机制解释。我们发现GRASP 55和GRASP 65通过形成有丝分裂调节的反式低聚物将高尔基体膜堆叠起来，在高尔基体结构形成中发挥互补且重要的作用。使用GRASP 55/65作为操纵高尔基体堆叠形成的工具，我们提供了第一个证据，高尔基体堆叠阻碍蛋白质运输，以确保准确的翻译后修饰。我们推测GRASP蛋白通过寡聚化和与其他蛋白的相互作用在高尔基体结构形成中发挥重要作用，随后调节蛋白质运输和糖基化。这个建议是我们以前研究的逻辑延续，以评估高尔基体结构是如何组装的，以及为什么它对蛋白质运输，糖基化和分选很重要。具体目标是：1）识别和表征新的GRASP 65相互作用蛋白，调节高尔基体结构的形成。我们的初步数据表明，在间期细胞胞质溶胶中存在增强GRASP 65寡聚化的蛋白质。我们已经确定了20个蛋白质与GRASP 65相互作用，我们将其在高尔基体结构形成的作用。这也将帮助我们了解GRASPs如何执行先前报告的多种功能。2)确定高尔基体在蛋白质运输、糖基化和分选中的结构-功能关系。我们将使用最近开发的CRISPR/Cas9技术通过敲除GRASP 55/65来操纵高尔基体结构，并通过在细胞中表达GRASP 55/65突变体来确定高尔基体破坏和恢复对蛋白质运输，修饰和分泌的影响。值得注意的是，高尔基体结构和功能的改变与多种人类疾病相关，包括癌症、自身免疫性疾病、病毒感染以及亨廷顿病和阿尔茨海默病。高尔基体的缺陷可能会影响一个大的许多蛋白质，并导致细胞内和细胞表面的全球影响，损害各种细胞功能。在了解高尔基体在人类疾病中的作用之前，需要更好地了解高尔基体结构的形成及其与重要细胞功能的关系。我们提出的研究将确定高尔基体破坏对蛋白质运输和加工的后果，从而为正常条件下高尔基体的作用以及高尔基体缺陷与疾病发病机制之间的关系提供基本信息。