Mechanism of catalytic regulation and active site loop motions in related protein tyrosine phosphatases

相关蛋白酪氨酸磷酸酶的催化​​调控和活性位点环运动机制

• 批准号: 9188816
• 负责人: JOSEPH P LORIA
• 金额: $ 32.31万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9188816
• 关键词: Acids; Active Sites; Address; Affect; Allosteric Regulation; Amino Acids; Bacteria; Bacterial Infections; Bacterial Proteins; Binding; Binding Proteins; Biochemical; Biological; Biophysics; Catalysis; Catalytic Domain; Cell physiology; Cells; Chemicals; Chimera organism; Complex; Data; Diabetes Mellitus; Disease; Distal; Enzyme Kinetics; Enzymes; Excision; Family member; Goals; Growth; Homeostasis; Human; Hydrolysis; Infection; Inflammation; Isotopes; Kinetics; Light; MAPK3 gene; MAPK8 gene; Malignant Neoplasms; Malignant neoplasm of cervix uteri; Malignant neoplasm of prostate; Maps; Medical; Mitogen-Activated Protein Kinases; Monitor; Motion; Mutation; NMR Spectroscopy; Nature; Non-Insulin-Dependent Diabetes Mellitus; Obesity; PTPN1 gene; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Post-Translational Protein Processing; Protein Kinase; Protein Tyrosine Phosphatase; Proteins; Reaction; Regulation; Relaxation; Research; Role; Serine; Site; Speed; Stimulus; Structure; Techniques; Testing; Tissues; Translational Repression; Type III Secretion System Pathway; Tyrosine; Vaccinia; Virulent; X-Ray Crystallography; Yersinia; active control; biophysical analysis; cancer type; chemical reaction; experimental study; human disease; inhibitor/antagonist; inorganic phosphate; insight; malignant breast neoplasm; mutant; pathogenic bacteria; protein folding; protein structure; public health relevance; small molecule; small molecule inhibitor

DESCRIPTION (provided by applicant): Cells must respond to internal and external stimuli for healthy function, growth, and differentiation. One primary mechanism by which cells do this is through modulation of the post-translational phosphorylation levels of many proteins. Disruption of, or aberrant protein phosphorylation levels results in numerous diseases including cancer, Type-II diabetes, obesity, and inflammation. One class of enzymes that modulate the phosphorylation state of proteins is the protein tyrosine phosphatases. Because of their important role, the rate at which these enzymes catalyze phosphate removal is crucial and is tightly regulated. This regulation can occur by allosteric mechanisms, including small molecule binding and post-translational modification. And, as recent data in the PI's lab suggests, regulation is also inherently controlled by the closing kinetics of the acid loop in these phosphatases. The long-term goal is to understand in more detail how the activity of these phosphatase enzymes is regulated. This goal will be achieved through three aims: 1) study of the allosteric mechanism of PTP1B inhibition, a human enzyme whose misregulation leads to Type-II diabetes, obesity, and certain breast cancers~ 2) characterize how the primary sequence of the acid loop in PTP1B and YopH (a phosphatase from the virulent Yersinia bacterium) controls the kinetics of loop motion~ and 3) characterize the activation mechanism of the human VHR phosphatase that results from binding to the pseudokinase, VRK-3. For these three aims the motions of the acid loop will be monitored by solution NMR relaxation dispersion techniques with focus on any alterations in loop motions due to small molecule binding, post-translational modification, mutation, or accessory protein binding. Any functional changes in these phosphatases as a result of these perturbations will be characterized by detailed kinetic studies including pH and isotope effects. The combination of these biophysical and biochemical techniques will give insight into the connections between motions and catalysis while informing on the regulatory mechanisms of medically important enzymes.

描述（由申请人提供）：细胞必须对内部和外部刺激做出反应，以实现健康的功能、生长和分化。细胞这样做的一个主要机制是通过调节许多蛋白质的翻译后磷酸化水平。蛋白质磷酸化水平的破坏或异常导致许多疾病，包括癌症、II型糖尿病、肥胖和炎症。调节蛋白质磷酸化状态的一类酶是蛋白质酪氨酸磷酸酶。由于它们的重要作用，这些酶催化磷酸盐去除的速率至关重要，并且受到严格调控。这种调节可以通过变构机制发生，包括小分子结合和翻译后修饰。而且，正如PI实验室的最新数据所表明的那样，调节也是由这些磷酸酶中酸环的闭合动力学内在控制的。长期目标是更详细地了解这些磷酸酶的活性是如何调节的。这一目标将通过三个目标实现：1）研究PTP 1B抑制的变构机制，PTP 1B是一种人类酶，其失调导致II型糖尿病、肥胖症，和某些乳腺癌~ 2）描述了PTP 1B和YopH中酸性环的一级序列如何（一种来自毒性耶尔森氏菌的磷酸酶）控制着环运动的动力学，3）表征了由与假激酶VRK-3结合引起的人VHR磷酸酶的活化机制。对于这三个目标，酸环的运动将通过溶液NMR弛豫分散技术进行监测，重点关注由于小分子结合、翻译后修饰、突变或辅助蛋白结合引起的环运动的任何改变。这些磷酸酶的任何功能变化，作为这些扰动的结果将其特征在于详细的动力学研究，包括pH值和同位素效应。这些生物物理和生物化学技术的结合将使我们深入了解运动和催化之间的联系，同时为医学上重要的酶的调节机制提供信息。