Integrated Theoretical and NMR Studies of PTEN-lipid Interactions

描述（由申请人提供）：PTEN首次作为通过控制质膜（PM）处的磷脂酰肌醇（3，4，5）-三磷酸（PI（3，4，5）P3）水平来消除PKB/Akt途径所必需的肿瘤抑制因子而首次亮相。现在已经显示出PTEN具有关键的基于细胞质和细胞核的作用，其中一些是磷酸酶非依赖性的。那么，PTEN如何将其癌症减轻功能分类以使PI（3，4，5）P3与其在细胞中其他地方的职责去磷酸化？活细胞中PTEN的单分子TIRFM研究揭示了动态PTEN-膜缔合，膜停留时间约为200 ms。特别地，SPR研究表明，PTEN在阴离子双层上停留约700 s，证明了非常高的非特异性静电亲和力。最近由Nesperson等开发的计算模型使这些差异与下述预测相一致：PTEN可以建立两种膜相互作用取向（1）非特异性静电驱动取向，其可以导致PI（4，5）P2在推定的PI（4，5）P2结合结构域（PBD）处的积累，2）底物驱动取向，其使PTEN活性位点和膜表面之间的距离最小化，但非特异性静电亲和力显著降低。后一种取向应增加催化后PTEN解离的可能性，因为3 '-磷酸的特异性结合自由能将丢失。为了开始测试该模型提出的假设，将采集PI（4，5）P2和PI（3，4，5）P3的31 P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。这些初步的仅脂质的研究对于分析31 P化学位移与结合到含有PI（4，5）P2的囊泡的13 C-15 N-标记的PTEN的相关性是必要的。下一步，在E.大肠杆菌将进行优化，以获得足够量的标记蛋白质用于核磁共振分析。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，通过将指定的共振与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被解析，并且参与PI（4，5）P2特异性结合位点，该位点被发现对于将PTEN募集至PM是必需的。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。上述研究结果将被整合到一个新的计算模型的PTEN停靠在阴离子双层，以改善以前的理论表征的静电驱动的协会。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。 公共卫生相关性：磷酸肌醇是一种特殊的脂质，其作用类似于交通信号：它们指导蛋白质细胞机制，驱动生命的基本细胞过程。大约30种磷酸肌醇修饰蛋白，包括肿瘤抑制因子PTEN，维持磷酸肌醇的适当平衡。这些蛋白质不能保持磷酸肌醇水平在检查中是许多人类疾病和功能障碍的基础，包括癌症，双相情感障碍，不育症，糖尿病和唐氏综合症。本文概述的建议试图观察和定义赋予其有效信号传导能力的磷酸肌醇的原子特征，并定义PTEN与对PTEN的抗癌性质重要的磷酸肌醇的原子相互作用。

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.