Integrated Theoretical and NMR Studies of PTEN-lipid Interactions

描述（由申请人提供）：PTEN首次作为肿瘤抑制因子首次出现，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。现在PTEN已被证明具有关键的细胞质和细胞核的作用，其中一些是磷酸酶独立的。那么PTEN是如何将它的抗癌功能与它在细胞其他部位的功能区分开来，使PI(3,4,5)P3去磷酸化的呢？活细胞中PTEN的单分子TIRFM研究揭示了PTEN与膜的动态关联，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 s，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调和了这些差异，并预测PTEN可能建立两种膜相互作用取向(1)非特异性静电驱动取向，可导致PI(4,5)P2在假定的PI（4,5)P2结合域（PBD）积累，2）底物驱动取向使PTEN活性位点与膜表面之间的距离最小化，但显着降低了非特异性静电亲和力。后一种取向应该增加催化后PTEN解离的可能性，因为3'-磷酸的特定结合自由能将会丢失。为了开始验证该模型提出的假设，将获得PI(4,5)P2和PI(3,4,5)P3的31P光谱，以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取向。这些初步的脂质研究对于分析31P与13c - 15n标记的PTEN结合到含有PI(4,5)P2的囊泡的化学位移相关性是必要的。接下来，PTEN在大肠杆菌中的表达将被优化，以获得足够数量的标记蛋白用于核磁共振分析。利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，通过与TALOS数据库比较分配的共振来确定n端二级结构。PTEN的n端在晶体结构中未解析，并参与PI(4,5)P2特异性结合位点，该位点对于PTEN募集到PM至关重要。因此，SSNMR将询问PTEN-PI(4,5)P2相互作用，以识别结合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的计算模型中，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。通过执行拟定的Research Training Plan，我将获得专业知识，并完成正式检验两个取向假设所需的初步基础工作。

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. PUBLIC HEALTH RELEVANCE: Phosphoinositides are specialized lipids that operate analogously to traffic signals: they direct the proteinaceous cellular machinery that drives the cellular processes fundamental to life. Approximately 30 phosphoinositide-modifying proteins, including the tumor suppressor PTEN, maintain the proper balance of phosphoinositides. Failure of these proteins to keep phosphoinositide levels in check is the basis for numerous human diseases and dysfunctions including cancer, bipolar disorder, infertility, diabetes and Down syndrome. The proposal outlined herein seeks to observe and define the atomistic characteristics of phosphoinositides that confer their potent signaling abilities and to define the atomistic interactions of PTEN with phosphoinositides important for PTEN's anti-cancer properties.