Uncovering the mechanism of OGT substrate recognition and membrane localization

揭示OGT底物识别和膜定位的机制

• 批准号: 8252500
• 负责人: Rodrigo Fermin Ortiz-Meoz
• 金额: $ 4.92万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8252500
• 关键词: Acetylglucosamine; Affect; Affinity; Alzheimer' s Disease; Binding; Binding Proteins; Biochemical; Biological Assay; Biological Models; Breast Cancer Cell; Catalysis; Cell membrane; Cell model; Cell physiology; Cells; Cellular Membrane; Centrifugation; Chromatin; Detection; Diabetes Mellitus; Disease; Dissection; Environment; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Foundations; Future; Glean; Glucose; Goals; Growth; Human; Human Cell Line; Incubated; Individual; Insulin Signaling Pathway; Kinetics; Knowledge; Lead; Learning; Length; Link; Malignant Neoplasms; Maps; Measurement; Membrane; Methodology; Methods; Microscopic; Modeling; Modification; Monitor; Mutate; Mutation; Non-Insulin-Dependent Diabetes Mellitus; O-GlcNAc transferase; Pathway interactions; Peptides; Peripheral; Point Mutation; Process; Protein Glycosylation; Protein Isoforms; Protein Microchips; Protein Profiling Microarrays; Protein Region; Proteins; Protocols documentation; Public Health; Recruitment Activity; Reporting; Research; Research Proposals; Role; Scheme; Screening procedure; Series; Serine; Site; Stimulus; Structure; Substrate Interaction; Techniques; Testing; Thermodynamics; Threonine; Transfection; Walkers; Work; base; biochemical model; cancer cell; cellular targeting; enzyme activity; enzyme structure; extracellular; glycosylation; human disease; in vitro Assay; in vivo; inhibitor/antagonist; insight; insulin signaling; member; mutant; novel; prevent; research study; sugar; tool; tumor growth

When glucose (sugar) is present in high levels in cells, it is re-directed into a pathway that leads to the stimulation of an enzyme which is responsible for the addition of 2-N-acetylglucosamine (O-GlcNAc) to over 1,000 proteins in the cell. The correct activity of this enzyme (O-GlcNAc transferase or OGT) is critical for controlling the ability of a cell to respond to its environment and prevent aberrant growth and proliferation. The function of this enzyme has been linked to proper insulin signaling and diabetes, cancer and Alzheimer's disease. Recently it has been shown that the activity of OGT is increased in breast cancer cells. Reduction of enzyme levels and its activity by either biochemical methods or an OGT-specific inhibitor lead to an inhibition of tumor growth and decreased cancer cell invasion. The long term goal of the proposed is to characterize the mechanism of OGT function; details of which are critical for the treatment of the cellular processes and diseases it has been linked to. The objectives of this proposal are to 1) develop a biochemical model of OGT function 2) search for OGT substrates using protein microarrays and 3) examine the role of OGT as a peripheral membrane binding protein. In order to develop a biochemical model of OGT function, the recently solved crystal structure of the enzyme will be utilized to mutate regions of the protein that affect its ability to properly recognize its cellular targets. With this information, much can be learned about the mechanism that OGT utilizes to differentially glycosylate proteins in the cell. To expand the knowledge of the cellular targets (and the processes those targets are responsible for) of OGT, a microscopic array that contains over 9000 human proteins will be incubated with OGT and its mutants. After detection of the proteins that have been glycosylated, a comprehensive map of OGT targets can be built. This map can be used, in the future, to selectively inhibit glycosylation of a subset of OGT targets. During insulin signaling, OGT is effectively recruited to the inner membrane of cells where it acts on members of the insulin signaling pathway. The mechanism of OGT's recruitment / binding to the cell membrane remains poorly understood. A series of biochemical and cell-based methods will be employed to determine the particular membrane components that OGT binds to and which region of the enzyme is responsible for this action. Taken together, the objectives in this proposal and the methods used to achieve them will greatly enhance our knowledge of OGT's glycosylation mechanism.

当细胞中葡萄糖（糖）含量较高时，它会被重新引导至一条途径，刺激一种酶，该酶负责将 2-N-乙酰氨基葡萄糖 (O-GlcNAc) 添加到细胞中的 1,000 多种蛋白质中。这种酶（O-GlcNAc 转移酶或 OGT）的正确活性对于控制细胞对其环境做出反应并防止异常生长和增殖的能力至关重要。这种酶的功能与适当的胰岛素信号传导以及糖尿病、癌症和阿尔茨海默病有关。最近有研究表明，乳腺癌细胞中 OGT 的活性增加。通过生化方法或 OGT 特异性抑制剂降低酶水平及其活性，从而抑制肿瘤生长并减少癌细胞侵袭。该提案的长期目标是表征 OGT 功能的机制；其中的细节对于治疗与其相关的细胞过程和疾病至关重要。 该提案的目标是 1) 开发 OGT 功能的生化模型 2) 使用蛋白质微阵列寻找 OGT 底物 3) 检查 OGT 作为外周膜结合蛋白的作用。为了开发 OGT 功能的生化模型，最近解决的酶晶体结构将用于突变影响其正确识别细胞靶标能力的蛋白质区域。有了这些信息，就可以了解 OGT 用于差异糖基化细胞中蛋白质的机制。为了扩展对 OGT 细胞靶标（以及这些靶标负责的过程）的了解，包含 9000 多种人类蛋白质的显微阵列将与 OGT 及其突变体一起孵育。检测到已糖基化的蛋白质后，可以构建 OGT 靶标的综合图谱。该图谱将来可用于选择性抑制 OGT 靶标子集的糖基化。在胰岛素信号传导过程中，OGT 被有效地募集到细胞内膜，并作用于胰岛素信号传导通路的成员。 OGT 募集/与细胞膜结合的机制仍知之甚少。将采用一系列生化和基于细胞的方法来确定 OGT 结合的特定膜成分以及酶的哪个区域负责这种作用。总而言之，该提案的目标以及实现这些目标的方法将极大地增强我们对 OGT 糖基化机制的了解。