Ubiquitin and Cell Cycle Regulation of Golgi Membrane Dynamics

描述（由申请人提供）：高尔基复合体是一种膜结合细胞器，在所有真核细胞中充当分泌蛋白加工的中心管道。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。更好地了解正常高尔基体结构的形成和其重要的细胞功能之间的关系，才能了解其在人类疾病中的作用。高尔基体在细胞分裂过程中的生物合成是由一个拆卸和重组过程介导的。它在有丝分裂过程中分解成管状泡状结构，这些结构被划分成子细胞，在子细胞中它们重新组装成新的高尔基体。重组是由两种ATP酶（NSF和p97）介导的，它们使膜融合。 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，为揭示潜在机制的新方法打开了一扇门。泛素化发生在有丝分裂高尔基体解体，并需要随后的重组。重组需要p97/p47和monoubiquitin之间的相互作用和去泛素化酶VCIP 135的活性，VCIP 135是p97/p47复合物的辅因子。我们推测，泛素化作为一种一般机制，在细胞周期中的高尔基体膜的动态调节。我们将使用生物化学和形态学的方法相结合，以阐明泛素化如何发生在有丝分裂高尔基体拆卸和它如何调节有丝分裂后的重组。具体目标是：1）鉴定泛素连接酶（E3），并通过体外抑制酶和体内敲除蛋白来阐明其功能。2)鉴定高尔基体上的泛素化底物，并使用可用的泛素连接酶和去泛素化酶进行确认。3)阐明泛素化在有丝分裂高尔基体膜动力学中的作用机制。我们将确定酶和底物之间的相互作用与p97介导的膜融合。我们将通过使用shRNA和过表达技术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体膜重组的影响。这些研究将提供新的见解细胞周期调控的高尔基体膜动力学的分子机制。 公共卫生相关性：高尔基体是一个主要的细胞组成部分，作为一个中央管道，用于处理膜和分泌蛋白，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许多疾病有关，包括阿尔茨海默病和癌症，其中涉及异常蛋白质运输和分泌。该建议研究高尔基体的生物起源、结构和功能，以便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.