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Supplement: GOLGI BIOGENESIS AND FUNCTION

补充：高尔基体的生物发生和功能

基本信息

批准号：
9894924
负责人：
Yanzhuang Wang
金额：
$ 11.34万
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 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9894924
关键词：
Affect Alzheimer&apos s Disease Antibodies Autoimmune Diseases Biochemistry Biogenesis Biological Assay CRISPR/Cas technology Cell Cycle Cell division Cell physiology Cell surface Cells Cellular biology Cytosol Data Defect Diabetes Mellitus Disease Electron Microscopy Ensure Enzymes Functional disorder GORASP2 gene GRASP gene Golgi Apparatus Growth Factor Hormones Huntington Disease In Vitro Inherited Interphase Cell Knock-out Malignant Neoplasms Membrane Membrane Proteins Mitotic Modification Neurotransmitters Organelles Pathogenesis Play Post-Translational Protein Processing Protein Glycosylation Proteins Proteomics Reporting Role Sorting - Cell Movement Structure Structure-Activity Relationship Techniques Virus Diseases glycosylation human disease interdisciplinary approach mutant novel protein transport reconstitution restoration secretory protein tool trafficking

项目摘要

PROJECT SUMMARY 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 the 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 can 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.

项目摘要高尔基复合体是一种中央膜细胞器，用于细胞内运输，糖基化和分选。膜和分泌蛋白。高尔基体的基本结构是一堆扁平的池，这种结构是在细胞分裂期间组装和遗传的，人们对此知之甚少。在过去的几年里，我们开发了一种独特的多学科方法，采用生物化学，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测定，高尔基体结构形成和功能的机制解释。我们发现GRASP 55和GRASP 65 在高尔基体结构形成中起着补充和重要的作用，通过形成有丝分裂调节的反式- 将高尔基体膜堆叠起来的低聚物。以GRASP 55/65为工具操纵高尔基体形成，我们提供了第一个证据，高尔基体堆积阻碍蛋白质运输，以确保准确的翻译后修饰。我们推测GRASP蛋白在高尔基体中起重要作用通过寡聚化和与其他蛋白质相互作用形成结构，调节蛋白质运输和糖基化。这一建议是我们先前建议的合理延续。研究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很重要，糖基化和分选。具体目的是：1）鉴定和表征新的GRASP 65相互作用调节高尔基体结构形成的蛋白质。我们的初步数据显示，增强GRASP 65寡聚化间期细胞胞质溶胶。我们已经鉴定出20种蛋白质与GRASP 65，我们将其功能的高尔基体结构的形成。这也将帮助我们了解GRASP如何执行先前报告的多种功能。2)确定结构- 高尔基体在蛋白质运输、糖基化和分选中的功能关系。我们将操纵高尔基体通过敲除GRASP 55/65，使用最近开发的CRISPR/Cas9技术，在细胞中表达GRASP 55/65突变体以确定高尔基体破坏和恢复的后果蛋白质运输、修饰和分泌。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、病毒感染和亨廷顿氏病和阿尔茨海默氏病。高尔基体的缺陷可能会影响细胞的运输、分选和修饰，大量的蛋白质，并导致细胞内和细胞表面的全球影响，各种细胞功能。更好地理解高尔基体结构的形成及其与其生命的关系在了解细胞在人类疾病中的作用之前，需要了解细胞功能。我们建议的研究将确定高尔基体破坏对蛋白质运输和加工的后果，从而提供关于正常条件下高尔基体作用的基本信息以及高尔基体之间的关系缺陷和疾病的发病机制。