Investigating the relationship between allostery and substrate specificity in protein tyrosine phosphatases

研究蛋白酪氨酸磷酸酶变构与底物特异性之间的关系

• 批准号: 10531934
• 负责人: JOSEPH P LORIA
• 金额: $ 38.72万
• 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 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10531934
• 关键词: Active Sites; Allosteric Regulation; Allosteric Site; Amino Acids; Architecture; Aspartic Acid; Binding; Binding Proteins; Binding Sites; Biochemical; Biochemistry; Biological; Biological Assay; Biological Process; Biophysics; Catalysis; Cells; Cellular biology; Chemicals; Computing Methodologies; Disease; Distal; Distant; Drug Targeting; ERBB2 gene; Enzymes; Epidermal Growth Factor Receptor; Excision; Exhibits; Family member; Funding; Goals; H1-related protein-tyrosine phosphatase; Health; Human; Immune System Diseases; In Vitro; Inflammation; Insulin Receptor; JAK2 gene; Kinetics; Ligand Binding; Ligands; Link; MAPK1 gene; MAPK3 gene; MAPK8 gene; Malignant Neoplasms; Medical; Methods; Molecular; Monitor; Motion; Mutate; Mutation; NMR Spectroscopy; Non-Insulin-Dependent Diabetes Mellitus; Nuclear Magnetic Resonance; Obesity; Paint; Pathway Analysis; Peptides; Pharmaceutical Preparations; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Productivity; Protein Dephosphorylation; Protein Family; Protein Tyrosine Phosphatase; Proteins; Research; Role; Signaling Protein; Site; Specificity; Stat5 protein; Structure; Substrate Specificity; Sumoylation Pathway; Testing; Therapeutic; Thermodynamics; Tyrosine; Vaccinia; Validation; amino group; biophysical analysis; computer studies; experimental study; extracellular; human disease; improved; in vivo; inhibitor; inorganic phosphate; insight; molecular dynamics; novel; protein function; protein structure; protein tyrosine phosphatase 1B; screening; small molecule; success

Protein tyrosine phosphatases (PTPs) are enzymes that regulate an enormous number of biological processes through the modulation of their target proteins (substrates). PTPs do this by catalyzing the removal of a phosphate group from the amino acid tyrosine in their targets, which alters the activity of the target enzyme. The misregulation of PTPs are linked to a number of diseases including type II diabetes, obesity, cancer, and inflammation and therefore PTPs are viewed as potential drug targets. A primary obstacle to drugging PTPs is that the sites of their enzymatic action (active sites) are nearly identical, thus targeting a specific PTP enzyme by a drug focused on the active site has not been a productive endeavor. A solution to the problem is to target the drug to sites on PTPs that are distant from the active site, the so-called allosteric sites, which are not identical across the family of PTPs. A difficulty with this approach is the challenge of identification and characterization of these allosteric sites to obtain a better understanding of how they interact, from long molecular distances, with the active site. We hypothesize that different substrates alter the protein structure in a distinct manner and cause different allosteric sites to be exposed. We plan to test this hypothesis and characterize these allosteric sites through a powerful combination of solution nuclear magnetic resonance (NMR) spectroscopy, biochemical, and computational approaches in two medically important human enzymes, protein tyrosine phosphatase 1B (PTP1B) and Vaccinia H1-related (VHR) phosphatase. Our aims are: To identify and characterize substrate dependent allosteric sites in PTP1B and VHR using different substrate peptides from the natural in vivo targets of these PTPs. We will identify these sites by monitoring changes in NMR chemical shift and dynamics parameters. In complementary experiments we will use novel computational methods developed by our research team to further understand the mechanism of allostery in these enzymes. Subsequently we will validate our newly identified allosteric sites by mutation followed by functional assays and computational methods to better understand the impact of the allosteric sites on the substrate specific enzymatic activity in PTP1B and VHR.

蛋白酪氨酸磷酸酶（PTPs）是调节大量生物过程的酶 通过调节其靶蛋白（底物）。PTP通过催化去除一种 它们可以从靶点的氨基酸酪氨酸中去除磷酸基团，从而改变靶酶的活性。 PTPs的失调与许多疾病有关，包括II型糖尿病、肥胖症、癌症和糖尿病。 炎症和因此PTP被视为潜在的药物靶标。给PTPs下药的主要障碍是 它们的酶促作用位点（活性位点）几乎相同，因此靶向特定的PTP酶 通过药物集中在活性部位一直不是一个富有成效的奋进。解决这个问题的一个办法是瞄准 药物作用于远离活性位点的PTP上的位点，即所谓的变构位点， 在PTPs家族中是相同的。这种方法的一个困难是识别的挑战， 表征这些变构位点，以更好地了解它们如何相互作用，从长 分子距离，与活性位点。我们假设不同的底物改变了蛋白质的结构， 不同的方式，并导致不同的变构位点被暴露。我们计划测试这个假设， 通过溶液核磁共振的强大组合来表征这些变构位点， (NMR)光谱学，生物化学和计算方法在两个医学上重要的人类酶， 蛋白酪氨酸磷酸酶1B（PTP1B）和牛痘H1相关（VHR）磷酸酶。我们的目标是： 使用不同底物鉴定和表征PTP 1B和VHR中的底物依赖性变构位点 来自这些PTP的天然体内靶标的肽。我们将通过监测以下方面的变化来确定这些地点： NMR化学位移和动力学参数。在补充实验中，我们将使用新型计算 我们的研究团队开发的方法，以进一步了解这些酶的变构机制。 随后，我们将通过突变验证我们新鉴定的变构位点，然后进行功能测定， 计算方法，以更好地了解变构位点对底物特异性的影响， PTP1B和VHR的酶活性。