GOLGI MATRIX ASSEMBLY AND DISASSEMBLY IN THE CELL CYCLE

描述（由申请人提供）：高尔基体复合体是一种膜结合细胞器，在所有真核细胞中作为转运、糖基化、分选和加工膜和分泌蛋白的中心单元，包括激素、生长因子、抗体和消化酶。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、亨廷顿病和阿尔茨海默病以及病毒感染。在许多肿瘤细胞系和组织中已经观察到高尔基体断裂，并且异常糖基化是癌症的标志。在了解高尔基体在人类疾病中的作用之前，需要一个详细的正常高尔基体结构形成的动态模型及其与重要细胞功能的关系。独特的高尔基体结构（扁平的池排列成堆叠）被认为依赖于与高尔基体相关的蛋白质网络，即“高尔基体基质”。目前关于高尔基体基质的组成和功能的信息非常有限。基于高尔基体在细胞分裂的每个周期中分解和重新组装的观察，我们假设在间期产生和维持高尔基体结构的高尔基体基质在有丝分裂期间必须被分解，并且这个过程受到广泛的有丝分裂磷酸化的调节，从而破坏蛋白质-蛋白质相互作用。高尔基体研究人员（王博士）和生物质谱专家（安德鲁斯博士）之间的这种合作采用了系统方法，通过绘制其在细胞周期中的组成和组装以及磷酸化和蛋白质-蛋白质相互作用之间的关系来研究高尔基体基质的性质。我们已经开发了一种体外试验，重建有丝分裂过程中高尔基体的拆卸和有丝分裂后的重新组装。这使我们能够制备高数量和高纯度的间期和有丝分裂高尔基体膜，用于蛋白质组学定量分析和进行靶向干预研究。我们将使用我们的新的蛋白质组学协议来量化蛋白质-蛋白质相互作用和蛋白质磷酸化事件。相关性分析将使我们能够在细胞周期中将特定的磷酸化事件与高尔基体基质中的蛋白质相互作用联系起来，这可以在我们的体外试验中进行验证和表征。在这项研究中，我们将：1）利用定量蛋白质组学分析间期和有丝分裂期高尔基体中高尔基体基质的组成; 2）通过交联和蛋白质组学分析鉴定间期和有丝分裂期高尔基体基质和膜中蛋白质-蛋白质相互作用; 3）在体外和体内确定磷酸化对高尔基体基质组装和拆卸以及蛋白质-蛋白质和蛋白质-膜相互作用的作用。将使用我们的新交联剂以及细胞生物学和生物化学技术在完整细胞中验证体外发现。这些研究将为正常细胞中高尔基体的结构和功能及其在疾病状态下的功能障碍提供新的见解。

DESCRIPTION (provided by applicant): The Golgi complex is a membrane-bound organelle that serves as a central unit for trafficking, glycosylation, sorting and processing of membrane and secretory proteins in all eukaryotic cells, including hormones, growth factors, antibodies and digestive enzymes. Alterations in Golgi structure and function have been associated with a variety of human diseases, including cancer, autoimmune disease, Huntington's and Alzheimer's diseases, and viral infections. Golgi fragmentation has been observed in many tumor cell lines and tissues, and aberrant glycosylation is a hallmark of cancer. A detailed dynamic model of normal Golgi structure formation and the relationship to its vital cellular function is required before its role in human disease can be understood. The unique Golgi architecture (flattened cisternae arranged into stacks) is believed to rely on the protein network associated with the Golgi, the "Golgi matrix". Very limited information is currently available on the composition and functions of the Golgi matrix. Based on the observation that the Golgi disassembles and reassembles during each cycle of cell division, we hypothesize that the Golgi matrix that generates and maintains the Golgi structure in interphase must be disassembled during mitosis and this process is regulated by extensive mitotic phosphorylation that disrupts protein-protein interactions. This collaboration between a Golgi researcher (Dr. Wang) and a biological mass spectrometry expert (Dr. Andrews) applies a systems approach to investigate the nature of the Golgi matrix by mapping its composition and assembly in the cell cycle and the relationship between phosphorylation and protein-protein interactions. We have developed an in vitro assay that reconstitutes the disassembly of Golgi during mitosis and its reassembly after mitosis. This allows us to prepare interphase and mitotic Golgi membranes of high quantity and high purity for proteomic quantitative analysis and to perform targeted interventional studies. We will use our novel proteomic protocols to quantify protein-protein interaction and protein phosphorylation events. Correlation analysis will allow us to link specific phosphorylation events with protein interactions in the Golgi matrix during the cell cycle, which can be validated and characterized in our in vitro assay. In this study, we will: 1) Use quantitative proteomics to analyze the components of the Golgi matrix in interphase and mitotic Golgi; 2) Identify protein-protein interactions in the Golgi matrix and membranes in interphase and mitosis by crosslinking and proteomic analysis; 3) Determine the role of phosphorylation on Golgi matrix assembly and disassembly as well as in protein-protein and protein-membrane interactions in vitro and in vivo. In vitro discoveries will be validated in intact cells using our new crosslinker as well as cell biology and biochemical techniques. These studies will provide new insights into the Golgi structure and function in normal cells and its dysfunction in disease states.