Uncovering the mechanism of OGT substrate recognition and membrane localization

当葡萄糖(糖)在细胞中处于高水平时，它被重新定向到一条导致刺激酶的途径，该酶负责将2-N-乙酰氨基葡萄糖(O-GlcNAc)添加到细胞中的1000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环境的反应能力和防止异常生长和增殖至关重要。这种酶的功能与适当的胰岛素信号和糖尿病、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和活性，可以抑制肿瘤生长，减少癌细胞侵袭。拟议的长期目标是描述OGT功能的机制；这些细节对于治疗与其相关的细胞过程和疾病至关重要。 该方案的目的是：1)建立OGT功能的生化模型；2)利用蛋白质芯片寻找OGT底物；3)研究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，将利用最近解决的酶的晶体结构来突变蛋白质的区域，这些区域影响其正确识别其细胞靶标的能力。有了这些信息，就可以更多地了解OGT在细胞中差异糖化蛋白质的机制。为了扩大对OGT细胞靶点(以及这些靶点所负责的过程)的了解，一个包含9000多个人类蛋白质的显微阵列将与OGT及其突变体孵育。在检测到糖基化的蛋白质后，可以建立OGT靶标的全面图谱。这个图谱将来可以用来选择性地抑制OGT靶标的子集的糖基化。在胰岛素信号转导过程中，OGT被有效地募集到细胞内膜上，作用于胰岛素信号转导通路的成员。OGT与细胞膜的募集/结合机制尚不清楚。将使用一系列生化和基于细胞的方法来确定OGT结合到的特定膜成分以及酶的哪个区域负责这一作用。综上所述，这项建议中的目标和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。

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.