Ubiquitin and Cell Cycle Regulation of Golgi Membrane Dynamics

泛素和高尔基膜动力学的细胞周期调节

• 批准号: 8242103
• 负责人: Yanzhuang Wang
• 金额: $ 30.01万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8242103
• 关键词: ATP phosphohydrolase; Adaptor Signaling Protein; Alzheimer' s Disease; Animals; Antibodies; Autoimmune Diseases; Binding; Biochemical; Biogenesis; Biological Assay; Capsid Proteins; Cell Cycle; Cell Cycle Regulation; Cell division; Cell physiology; Cells; Coat Protein Complex I; Complex; Coupled; Cytosol; Data; Deubiquitinating Enzyme; Disease; Enzyme Inhibition; Enzymes; Eukaryotic Cell; Functional disorder; Goals; Golgi Apparatus; Growth Factor; Hormones; Huntington Disease; In Vitro; Incubated; Knowledge; Malignant Neoplasms; Mediating; Membrane; Membrane Fusion; Membrane Proteins; Mitosis; Mitotic; Molecular; Neurotransmitters; Organelles; Pathogenesis; Phosphotransferases; Play; Process; Protein Secretion; Proteins; Regulation; Role; SNAP receptor; Structure; Techniques; Tubular formation; UBA Domain; Ubiquitin; Ubiquitin Like Proteins; Ubiquitination; Vesicle; Viral Cancer; Virus Diseases; Work; base; cofactor; daughter cell; enzyme substrate; human disease; in vivo; insight; knock-down; multicatalytic endopeptidase complex; mutant; novel strategies; overexpression; protein degradation; protein transport; reconstitution; secretory protein; small hairpin RNA; ubiquitin ligase; ubiquitin-protein ligase; uncontrolled cell growth

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.

高尔基复合体是一种膜结合的细胞器，作为处理细胞内蛋白质的中心管道。 所有真核细胞中的分泌蛋白。高尔基体结构和功能的改变与 与多种人类疾病，包括自身免疫性疾病、亨廷顿氏病和阿尔茨海默氏病，病毒性 感染和癌症。更好地理解正常高尔基体结构形成之间的关系 在了解其在人类疾病中的作用之前，需要其重要的细胞功能。高尔基体生物发生 在细胞分裂过程中是由分解和重组过程介导的。它分解成管状囊泡状 在有丝分裂过程中，这些结构被划分成子细胞，在子细胞中它们被重新组装 变成一个新的高尔基体重组由两种ATP酶（NSF和p97）介导， 我们最近发现泛素在p97介导的高尔基体膜融合中起作用，这一发现开启了 一个新的方法来揭示潜在的机制的大门。泛素化发生在高尔基体有丝分裂期间 拆卸和后续重新组装所需。重组需要 p97/p47和monoubiquitin以及去泛素化酶VCIP 135的活性，VCIP 135是p97/p47的辅因子 复杂.我们推测，泛素化作为一种普遍的机制，在调节高尔基体 细胞周期中的膜动力学。我们将结合生物化学和形态学 方法来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 重组具体目的是：1）鉴定泛素连接酶（E3），并阐明其功能。 体外通过抑制酶，体内通过敲低蛋白质。2)为了识别泛素化的 在高尔基体上的底物，并使用可用的泛素连接酶和去泛素化酶确认它。3)阐明 有丝分裂高尔基体膜动力学中的泛素化机制。我们将确定 酶和底物之间的相互作用与p97介导的膜融合有关。我们将控制 通过操纵细胞中的遍在蛋白连接酶和去遍在蛋白化酶来进行遍在蛋白化， shRNA和过表达技术，从而确定对高尔基体膜重组的影响， 有丝分裂的结束这些研究将为细胞周期调控的分子机制提供新的见解 高尔基体膜动力学