GOLGI MATRIX ASSEMBLY AND DISASSEMBLY IN THE CELL CYCLE

描述（由申请人提供）：高尔基复合体是一种膜结合细胞器，是所有真核细胞（包括激素、生长因子、抗体和消化酶）中运输、糖基化、分类和处理膜和分泌蛋白的中心单元。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、亨廷顿氏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观察到高尔基体碎片化，异常糖基化是癌症的一个标志。在了解正常高尔基结构在人类疾病中的作用之前，需要详细的动态模型及其与重要细胞功能的关系。独特的高尔基结构（扁平的池池排列成堆叠）被认为依赖于与高尔基体相关的蛋白质网络，即“高尔基矩阵”。目前关于高尔基矩阵的组成和函数的资料非常有限。基于观察到在细胞分裂的每个周期中高尔基体的拆卸和重组，我们假设在有丝分裂期间产生和维持间期高尔基结构的高尔基基质必须被拆卸，这一过程受到广泛的有丝分裂磷酸化的调节，该磷酸化破坏了蛋白质-蛋白质的相互作用。这次高尔基体研究人员（王博士）和生物质谱专家（安德鲁斯博士）的合作应用了一种系统方法，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测定法，在有丝分裂期间重建高尔基体的拆卸，并在有丝分裂后重新组装。这使我们能够制备高质量和高纯度的间期和有丝分裂高尔基膜，用于蛋白质组学定量分析，并进行针对性的介入研究。我们将使用我们新的蛋白质组学方案来量化蛋白质-蛋白质相互作用和蛋白质磷酸化事件。相关分析将使我们能够将细胞周期中特定的磷酸化事件与高尔基基质中的蛋白质相互作用联系起来，这可以在我们的体外实验中得到验证和表征。在本研究中，我们将：1)利用定量蛋白质组学分析间期和有丝分裂高尔基基质的组成；2)通过交联和蛋白质组学分析确定间期和有丝分裂过程中高尔基基质和膜中的蛋白质-蛋白质相互作用；3)确定磷酸化在体内体外高尔基基质组装和拆卸以及蛋白质-蛋白质和蛋白质-膜相互作用中的作用。体外发现将在完整细胞中使用我们的新交联剂以及细胞生物学和生化技术进行验证。这些研究将为了解正常细胞中的高尔基体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.