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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(申请人提供)：高尔基复合体是一个中央膜细胞器，用于细胞内运输、糖基化和膜蛋白和分泌蛋白的分类。高尔基体的基本结构是一堆扁平的池子，但这个结构是如何组装的并在细胞分裂过程中遗传，人们对此知之甚少。在过去的几年里，我们发展了一种独特的多学科方法，利用生物化学、细胞生物学、电子显微镜以及最近的蛋白质组学和糖组学，结合一种新的体外重建实验，为高尔基体结构的形成和功能提供机制解释。我们发现，GRASP55和GRASP65通过形成有丝分裂调节的反式寡聚体，将高尔基体膜堆叠在一起，在高尔基体结构的形成中发挥着互补和必不可少的作用。使用GRASP55/65作为工具来操纵高尔基堆叠的形成，我们提供了第一个证据，证明高尔基堆叠阻碍蛋白质运输，以确保准确的翻译后修饰。我们假设GRAP蛋白通过寡聚和与其他蛋白质的相互作用在高尔基体结构的形成中发挥重要作用，从而调节蛋白质的运输和糖基化。这一建议是我们之前研究的合乎逻辑的继续，目的是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、糖基化和排序是重要的。具体目标是：1)识别和表征调节高尔基体结构形成的新的GRASP65相互作用蛋白。我们的初步数据显示，间期细胞胞浆中存在促进GRASP65寡聚的蛋白质。我们已经鉴定了20种与GRASP65相互作用的蛋白质，我们将表征它们在高尔基体结构形成中的作用。这也将帮助我们了解抓握如何执行之前报道的多种功能。2)确定高尔基体在蛋白质转运、糖基化和分选中的结构与功能关系。我们将通过敲除GRASP55/65，使用最近开发的CRISPR/Cas9技术，并通过在细胞中表达GRASP55/65突变体来操纵高尔基体结构，以确定高尔基体破坏和修复对蛋白质运输、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有关，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。高尔基缺陷可能会影响大量的贩运、分拣和改装大量的蛋白质，并在细胞内和细胞表面引起整体影响，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。我们的研究将确定高尔基体破坏对蛋白质运输和加工的影响，从而为了解高尔基体在正常条件下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础信息。