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（由适用提供）：高尔基体配合物是一种中央膜细胞器，用于细胞内贩运，糖基化和膜和秘密蛋白质的排序。高尔基人的基本结构是一堆扁平的水库，但是如何组装这种结构在细胞分裂期间遗传的知识很少。在过去的几年中，我们开发了一种采用生物化学，细胞生物学，电子显微镜以及最近的蛋白质组学和糖果学的独特多学科方法，并结合了一种新型的体外重构测定法，为高尔基结构形成和功能提供了机械解释。我们发现，Grasp55和Grasp65通过形成将高尔基体机理固定在堆栈中的有丝分裂调节的反寡聚物，在高尔基体结构形成中起互补和基本作用。使用Grasp55/65作为操纵高尔基堆形成的工具，我们提供了第一个证据，表明高尔基堆积会阻碍蛋白质运输以确保准确的翻译后修饰。我们假设抓握蛋白通过低聚和与其他蛋白质相互作用在高尔基结构形成中起着至关重要的作用，随后调节蛋白质运输和糖基化。该提议是我们以前的研究的逻辑延续，以评估高尔基体结构如何组装，以及为什么它对蛋白质运输，糖基化和分类很重要。具体目的是：1）识别和表征新型的Grasp65相互作用的蛋白质，可调节高尔基体结构形成。我们的初步数据表明，相间细胞胞质中有蛋白质可以增强GRASP65低聚。我们已经确定了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。这也将有助于我们了解GRASP如何执行多个功能，如先前报道。 2）确定高尔基体在蛋白质运输，糖基化和分类中的结构功能关系。我们将使用最近开发的CRISPR/CAS9技术敲除GRASP55/65，并表达细胞中的GRASP55/65突变体来确定高尔基体破坏和对蛋白质运输，修饰和分泌的恢复的后果来操纵Golgi结构。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和阿尔茨海默氏病。高尔基缺陷可能会影响大型贩运，分类和修改蛋白质的数量并在细胞内部和细胞表面引起全局效应，从而损害各种细胞功能。在理解其在人类疾病CA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其重要细胞功能的关系。我们提出的研究将确定高尔基人破坏对蛋白质运输和加工的后果，从而提供有关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理之间关系的基本信息。