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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

基本信息

批准号：
8450844
负责人：
Yanzhuang Wang
金额：
$ 29.17万
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：
2009
资助国家：
美国
起止时间：
2009-04-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8450844
关键词：
ATP phosphohydrolase Adaptor Signaling Protein Alzheimer&apos 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

项目摘要

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细胞器，作为处理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之相关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氏病、病毒性疾病感染和癌症。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，需要了解其重要的细胞功能。高尔基体生物发生细胞分裂期间是由分解和重组过程介导的。它分解成管状囊泡有丝分裂过程中的结构，被分成子细胞并在那里重新组装进入新的高尔基体。重组是由两种 ATP 酶（NSF 和 p97）介导的，它们融合了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，这开启了一个新的研究领域。揭示潜在机制的新方法之门。泛素化发生在高尔基体有丝分裂期间拆卸并需要随后重新组装。重新组装需要之间的相互作用 p97/p47 和单泛素以及去泛素化酶 VCIP135（p97/p47 的辅助因子）的活性复杂的。我们假设泛素化作为高尔基体调节的一般机制起作用细胞周期中的膜动力学。我们将结合使用生化和形态学阐明有丝分裂高尔基体解体过程中泛素化如何发生及其如何调节有丝分裂后的方法重新组装。具体目标是：1）鉴定泛素连接酶（E3）并阐明其在体外通过抑制酶，体内通过敲低蛋白质。 2) 鉴定泛素化高尔基体上的底物，并使用可用的泛素连接酶和去泛素酶进行确认。 3）阐明有丝分裂高尔基体膜动力学中的泛素化机制。我们将确定相互作用酶和底物之间与 p97 介导的膜融合有关。我们将控制通过使用细胞中的泛素连接酶和去泛素化酶进行泛素化 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。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

HACE1 (HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1).

HACE1（包含 E3 泛素蛋白连接酶 1 的 HECT 结构域和锚蛋白重复序列）。

DOI：
10.4267/2042/49701
发表时间：
2012
期刊：
Atlas of genetics and cytogenetics in oncology and haematology
影响因子：
0
作者：
Cui,Feng;Wang,Yanzhuang
通讯作者：
Wang,Yanzhuang

The ubiquitin ligase HACE1 regulates Golgi membrane dynamics during the cell cycle.

DOI：
10.1038/ncomms1509
发表时间：
2011-10-11
期刊：
Nature communications
影响因子：
16.6
作者：
通讯作者：

Direct selection of monoclonal phosphospecific antibodies without prior phosphoamino acid mapping.

直接选择单克隆磷酸化特异性抗体，无需事先进行磷酸氨基酸图谱分析。

DOI：
10.1074/jbc.m109.008730
发表时间：
2009
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Vielemeyer,Ole;Yuan,Hebao;Moutel,Sandrine;Saint-Fort,Rénette;Tang,Danming;Nizak,Clément;Goud,Bruno;Wang,Yanzhuang;Perez,Franck
通讯作者：
Perez,Franck

Monoubiquitination of Syntaxin 5 Regulates Golgi Membrane Dynamics during the Cell Cycle.

语法5的单泛素化调节细胞周期中的高尔基膜动力学。