Integrated Theoretical and NMR Studies of PTEN-lipid Interactions

描述(申请人提供)：PTEN首次作为一种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。目前，PTEN已被证明具有基于胞浆和细胞核的关键作用，其中一些作用是不依赖于磷酸酶的。那么PTEN如何对其抗癌功能进行分类，使PI(3，4，5)P3去磷酸化，使其在细胞内的其他功能发挥作用？活细胞中PTEN的单分子TIRFM研究表明，PTEN膜与膜驻留时间之间存在动态联系，膜停留时间约200ms。矛盾的是，表面等离子体共振研究表明，PTEN在阴离子双层膜上停留了约700 S，表现出很高的非特异性静电亲和力。最近由Nesbitt等人开发的计算模型。将这些差异与PTEN可能建立的两种膜相互作用方向的预测相一致，即1)非特异性静电驱动的方向，可导致PI(4，5)P2在假定的PI(4，5)P2结合结构域(PBD)处聚集；2)底物驱动的方向最小化PTEN活性部位与膜表面之间的距离，但非特异性静电亲和力显著降低。这种后一种取向将增加PTEN在催化后解离的可能性，因为3‘-磷酸的特定结合自由能将丢失。为了开始检验这个模型提出的假设，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的共振，并通过化学位移张量分析确定头基的取向。这些仅针对脂质的初步研究对于分析13C-15N标记的PTEN与含有PI(4，5)P2的囊泡结合的31P化学位移相关性是必要的。下一步，将优化PTEN在大肠杆菌中的表达，以获得足够数量的标记蛋白用于核磁共振分析。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-端二级结构。PTEN的N-末端在晶体结构中未被分解，并且参与了PI(4，5)P2的特异结合部位，该结合部位对PTEN在PM中的募集是必不可少的因此，PTEN-PI(4，5)P2的相互作用将被SS核磁共振所询问，以确定结合PI(4，5)P2的PTEN残基。上述每个发现都将被整合到一个新的PTEN计算模型中，该模型对接在阴离子双层上，以改进以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取向假设所需的初步基础工作。 与公共健康相关：磷脂酰肌醇是一种专门的脂类，其运作类似于交通信号：它们指导蛋白质类细胞机械，驱动生命基本的细胞过程。大约30种磷脂酰肌醇修饰蛋白，包括肿瘤抑制基因PTEN，维持着磷脂酰肌醇的适当平衡。这些蛋白质未能控制磷脂酰肌醇水平，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、不孕不育、糖尿病和唐氏综合症。在此概述的建议试图观察和定义赋予其有效信号能力的磷脂酰肌醇的原子学特征，并定义PTEN与对于PTEN的抗癌特性重要的磷脂酰肌醇的原子学相互作用。

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.