Integrated Theoretical and NMR Studies of PTEN-lipid Interactions

PTEN-脂质相互作用的综合理论和核磁共振研究

• 批准号: 8113391
• 负责人: Anna Elizabeth Nesbitt
• 金额: $ 2.42万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-16 至 2012-01-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8113391
• 关键词: Abate; Accounting; Active Sites; Adopted; Affinity; Amino Acids; Attention; Binding; Binding Sites; Biocompatible Materials; Bipolar Disorder; Catalysis; Cell Nucleus; Cell membrane; Cell physiology; Cells; Chad; Characteristics; Charge; Chemicals; Chemistry; Complex; Computer Simulation; Cytosol; Databases; Diabetes Mellitus; Dissociation; Divalent Cations; Docking; Down Syndrome; Electrostatics; Equilibrium; Escherichia coli; Failure; Free Energy; Functional disorder; Future; Goals; Half-Life; Human; In Vitro; Infertility; Inositol; Investigation; Label; Lateral; Lead; Learning; Life; Lipid Bilayers; Lipids; Macromolecular Complexes; Malignant Neoplasms; Measures; Membrane; Membrane Proteins; Methods; Modeling; Molecular; Molecular Conformation; N-terminal; National Research Service Awards; PTEN gene; Pathway interactions; Pattern; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Preparation; Probability; Property; Proteins; Research Training; Resolution; Role; Sampling; Screening procedure; Signal Transduction; Specificity; Structure; Students; Surface; Techniques; Testing; Therapeutic; Time; Triage; Tumor Suppressor Proteins; United States National Institutes of Health; Universities; Vesicle; Work; base; career; density; design; driving force; human disease; improved; in vivo; inorganic phosphate; instrumentation; interfacial; membrane model; novel; phosphodiester; phosphoinositide-3,4,5-triphosphate; protein expression; protein purification; single molecule; skills; solid state nuclear magnetic resonance; trafficking

DESCRIPTION (provided by applicant): PTEN first debuted as a tumor suppressor essential to abrogate the PKB/Akt pathway by controlling Phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) levels at the plasma membrane (PM). Now PTEN has been shown to possess critical cytosol and nucleus based roles, some of which are phosphatase independent. How then does PTEN triage its cancer-abating function to dephosphorylate PI(3,4,5)P3 with its duties elsewhere in the cell? Single molecule TIRFM studies of PTEN in living cells have revealed a dynamic PTEN- membrane association with membrane dwell times of ~200 ms. Paradoxically, SPR studies indicate that PTEN lingers on anionic bilayers for ~700 s, demonstrating a very high nonspecific electrostatic affinity. A recent computational model developed by Nesbitt et al. reconciles these differences with the prediction that PTEN may establish two membrane interaction orientations (1) a nonspecific electrostatics driven orientation that can lead to accumulation of PI(4,5)P2 at the putative PI(4,5)P2 binding domain (PBD), 2) a substrate driven orientation minimizing the distance between the PTEN active site and the membrane surface, but with a significantly decreased nonspecific electrostatic affinity. This latter orientation should increase the probability of PTEN dissociation post-catalysis, since the specific binding free energy of the 3'-phosphate will be lost. To begin testing the hypotheses set forth by this model, 31P spectra of PI(4,5)P2 and PI(3,4,5)P3 will be acquired to characterize the phosphodiester and monoester resonances and to determine the headgroup orientations by chemical shift tensor analyses. These preliminary lipid-only studies are necessary for analyzing 31P chemical shift correlations with of 13C-15N-labeled PTEN bound to vesicles containing PI(4,5)P2. Next, PTEN expression in E. coli will be optimized to obtain sufficient quantities of labeled protein for NMR analyses. Using a specialized labeling pattern to reduce undesired signals, 2D and 3D correlation spectra will be collected to determine the N-terminal secondary structure by comparing assigned resonances to the TALOS database. The PTEN N-terminus was unresolved in a crystal structure and participates in a PI(4,5)P2 specific binding site found to be essential for PTEN recruitment to the PM. Thus, the PTEN-PI(4,5)P2 interaction will be interrogated by SSNMR to identify the PTEN residues that bind PI(4,5)P2. Each of the above findings will be integrated into a new computational model of PTEN docked at anionic bilayers to improve the previous theoretical characterization of electrostatics driven association. Through the execution of the proposed Research Training Plan, I will gain the expertise and complete the preliminary groundwork required for formally testing the two orientations hypothesis.

描述（由申请人提供）：PTEN 首次作为肿瘤抑制因子首次亮相，通过控制质膜 (PM) 上的磷脂酰肌醇 (3,4,5)-三磷酸 (PI(3,4,5)P3) 水平来消除 PKB/Akt 通路所必需的。现在，PTEN 已被证明具有关键的细胞质和细胞核作用，其中一些作用是不依赖于磷酸酶的。那么，PTEN 如何分类其抗癌功能，使 PI(3,4,5)P3 去磷酸化及其在细胞其他部位的作用呢？对活细胞中 PTEN 的单分子 TIRFM 研究揭示了动态 PTEN 膜关联，膜停留时间约为 200 ms。矛盾的是，SPR 研究表明 PTEN 在阴离子双层上停留约 700 秒，表现出非常高的非特异性静电亲和力。 Nesbitt 等人最近开发的计算模型。将这些差异与 PTEN 可能建立两种膜相互作用方向的预测相一致：(1) 非特异性静电驱动方向，可导致 PI(4,5)P2 在假定的 PI(4,5)P2 结合域 (PBD) 处积累，2) 底物驱动方向，最大限度地减少 PTEN 活性位点与膜表面之间的距离，但显着降低 非特异性静电亲和力。后一种方向应该会增加催化后 PTEN 解离的可能性，因为 3'-磷酸的特异性结合自由能将会丢失。为了开始测试该模型提出的假设，将获取 PI(4,5)P2 和 PI(3,4,5)P3 的 31P 光谱来表征磷酸二酯和单酯共振，并通过化学位移张量分析确定头基方向。这些初步的仅脂质研究对于分析 31P 化学位移与 13C-15N 标记的 PTEN 与含有 PI(4,5)P2 的囊泡结合的相关性是必要的。接下来，将优化大肠杆菌中的 PTEN 表达，以获得足够数量的标记蛋白用于 NMR 分析。使用专门的标记模式来减少不需要的信号，将收集 2D 和 3D 相关光谱，通过将分配的共振与 TALOS 数据库进行比较来确定 N 末端二级结构。 PTEN N 末端在晶体结构中未解析，并参与 PI(4,5)P2 特异性结合位点，该位点被发现对于 PTEN 募集至 PM 至关重要。因此，将通过SSNMR询问PTEN-PI(4,5)P2相互作用以鉴定结合PI(4,5)P2的PTEN残基。上述每一项发现都将被整合到一个新的 PTEN 计算模型中，该模型停靠在阴离子双层上，以改进先前静电驱动缔合的理论表征。通过执行拟议的研究培训计划，我将获得专业知识并完成正式测试两个方向假设所需的初步基础工作。