Ubiquitin and Cell Cycle Regulation of Golgi Membrane Dynamics

描述(申请人提供)：高尔基复合体是一个膜结合的细胞器，在所有真核细胞中作为处理分泌蛋白质的中央管道。高尔基体结构和功能的改变与多种人类疾病有关，包括自身免疫性疾病、亨廷顿病和阿尔茨海默病、病毒感染和癌症。在了解高尔基体在人类疾病中的作用之前，需要更好地了解正常高尔基体结构形成与其重要细胞功能之间的关系。高尔基体在细胞分裂过程中的生物发生是通过分解和重组过程来调节的。它在有丝分裂过程中分解成管泡结构，这些结构被分割成子细胞，在那里它们重新组装成一个新的高尔基体。重组是由两个ATPase(NSF和p97)介导的，它们融合在膜上。我们最近发现泛素在p97介导的高尔基体膜融合中发挥作用，这为揭示其潜在机制打开了一扇新的大门。泛素化发生在有丝分裂的高尔基体拆解过程中，是后续重组所必需的。重组需要p97/p47和Monoubiquitin之间的相互作用以及p97/p47复合体的辅因子去泛素化酶VCIP135的活性。我们假设泛素化是细胞周期中高尔基体膜动力学调节的一般机制。我们将使用生化和形态学的结合方法来阐明泛素化是如何在有丝分裂高尔基体分解过程中发生的，以及它是如何调节有丝分裂后的重组的。其具体目的是：1)鉴定泛素连接酶(E3)，并在体外通过抑制该酶来阐明其功能，在体内通过敲除该蛋白来阐明其功能。2)鉴定高尔基体上的泛素化底物(S)，并利用已有的泛素连接酶和脱泛素酶对其进行确认。3)阐明有丝分裂高尔基体膜动力学中泛素化的机制。我们将确定酶与底物(S)之间的相互作用与p97介导的膜融合的关系。我们将利用shRNA和过表达技术，通过操纵细胞中的泛素连接酶和去泛素化酶来控制泛素化，从而确定在有丝分裂结束时对高尔基膜重组的影响。这些研究将为高尔基体膜动力学调控细胞周期的分子机制提供新的见解。与公共卫生相关：高尔基体是一种主要的细胞组件，充当处理膜和分泌蛋白质的中央管道，包括抗体、神经递质、激素、生长因子和消化酶。高尔基体功能障碍与包括阿尔茨海默病和癌症在内的许多疾病有关，这些疾病涉及异常的蛋白质运输和分泌。本研究对高尔基体的生物发生、结构和功能进行了研究，以期为理解疾病的发病机制提供知识。

DESCRIPTION (provided by applicant): The Golgi complex is a membrane-bound organelle that serves as a central conduit for the processing of secretory proteins in all eukaryotic cells. Alterations in the Golgi structure and function have been associated with a variety of human diseases, including autoimmune disease, Huntington's and Alzheimer's diseases, viral infections and cancer. A better understanding of the relationship between the normal Golgi structure formation and its vital cellular function is required before its role in human disease can be understood. Golgi biogenesis during cell division is mediated by a disassembly and reassembly process. It disassembles into tubularvesicular structures during mitosis, which are partitioned into the daughter cells where they are reassembled into a new Golgi apparatus. Reassembly is mediated by two ATPases (NSF and p97) that fuse the membranes. Our recent discovery that ubiquitin plays a role in p97-mediated Golgi membrane fusion opens a door for a new approach to uncover the underlying mechanism. Ubiquitination occurs during mitotic Golgi disassembly and is required for subsequent reassembly. Reassembly requires the interaction between the p97/p47 and monoubiquitin and the activity of the deubiquitinating enzyme, VCIP135, a cofactor of the p97/p47 complex. We hypothesize that ubiquitination operates as a general mechanism in regulation of Golgi membrane dynamics during the cell cycle. We will use a combination of biochemical and morphological approaches to elucidate how ubiquitination occurs during mitotic Golgi disassembly and how it regulates postmitotic reassembly. The specific aims are: 1) To identify the ubiquitin ligase (E3) and elucidate its function in vitro by inhibition of the enzyme and in vivo by knocking down the protein. 2) To identify the ubiquitinated substrate(s) on the Golgi and confirm it using the available ubiquitin ligase and deubiquitinase. 3) To elucidate the mechanism of ubiquitination in mitotic Golgi membrane dynamics. We will determine the interactions between the enzymes and the substrate(s) in relation to p97-mediated membrane fusion. We will control ubiquitination by manipulation of both the ubiquitin ligase and the deubiquitinating enzyme in cells using shRNA and overexpression techniques, and thus determine the effects on Golgi membrane reassembly at the end of mitosis. These studies will provide new insights into the molecular mechanisms of cell cycle regulation of Golgi membrane dynamics. Public Health Relevance: The Golgi apparatus is a major cellular component that serves as a central conduit for the processing of membrane and secretory proteins, including antibodies, neurotransmitters, hormones, growth factors and digestive enzymes. Dysfunction of the Golgi apparatus has been associated with many diseases including Alzheimer's disease and cancer, in which abnormal protein trafficking and secretion are involved. This proposal studies the biogenesis, structure and function of the Golgi apparatus in order to provide knowledge for understanding the pathogenesis of diseases.