Structural Basis for Small Molecule-Mediated Allosteric Activation of Protein Phosphatase 2A

统称为PP2A(蛋白磷酸酶2A)的一组磷酸酶代表 重要的肿瘤抑制因子家族。作为一名研究员在导师的指导下工作 关于Thomas Look博士，候选人最近发现奋乃静(PPZ)，一种经典的 抗精神病药物及其相关小分子iHAP1选择性地推动 含有15个可能的B56ϵ(PPP2R5E)亚单位的异三聚体PP2A络合物 不同的调控B亚基(K.Morita等人牢房。2020)。界定结构机制 通过这些小分子以极高的特异性变构激活PP2A 极其重要，因为这样的知识将从根本上推动磷酸酶领域的发展 生物学，同时形成了药物化学家将这些工具化合物优化为新的基础 用于临床研究的抗癌疗法。在这份拨款提案中，候选人寻求 要了解PP2A变构激活的基本机制 从结构的角度看磷酸酶，在Eric博士的主要指导下工作 他是生物化学和结构生物学领域的新兴领导者。中环 假设PPZ/iHAP1与AC异源二聚体结合并变构改变其构象 --从高分辨率的晶体结构中可以明显看出--因此B56ϵ 亚基很容易招募，从而推动PP2A-B56ϵ的形成和激活 细胞中的磷酸酶。眼下的目标是确定三维 PPZ/iHAP反应形成的PP2A全酶复合体的蛋白质结构 用X射线确定PP2A亚基与PPZ/iHAP化合物的分子接触 结晶学。这一目标将在两个具体目标中追求：1)确定三维 在PPZ或iHAP化合物存在下的PP2A全酶复合体的结构 这些小分子化合物对特定PP2A亚单位复合体的变构激活机制 分子，以及2)决定变构能力的生化机制 小分子PP2A激活剂，推动PP2A调节B亚单位与细胞的动态交换 AC异源二聚体，从对照细胞中的Striatin家族蛋白到B56ϵ或B55α蛋白 IHAP/PPZ和SMAP。这一策略意义重大，因为结果可能揭示出新的 特定的PP2A磷酸酶复合体的变构激活机制，进而 将是解剖这个不同的磷酸酶家族的正常生理作用的关键 了解它们作为肿瘤抑制因子的作用以及它们在恶性肿瘤中是如何被颠覆的 转型。该提案旨在为应聘者提供以下方面的高级培训 结构生物学，以及手稿/赠款写作和领导素质方面的指导。 在新的初级导师Eric Fischer博士的指导下，以及正在进行的 共同导师Thomas Look博士和由以下成员组成的科学咨询委员会的贡献 完成这项工作所需的专家，这项建议应该大大增强候选人的 职业目标是成为一名独立调查员。

The group of phosphatases collectively referred to as PP2A (protein phosphatase 2A) represent an important family of tumor suppressors. As a research fellow working under the mentorship of Dr. Thomas Look, the candidate has recently discovered that perphenazine (PPZ), a classic antipsychotic drug, and its related small molecule iHAP1 selectively drive the formation of heterotrimeric PP2A complexes containing the B56ϵ (PPP2R5E) subunit out of 15 possible distinct regulatory B subunits (K. Morita et al. Cell. 2020). Defining the structural mechanisms through which these small molecules allosterically activate PP2A with extreme specificity is of paramount importance because such knowledge will fundamentally advance the field of phosphatase biology, while forming the basis for medicinal chemists to optimize these tool compounds as novel anticancer therapeutics for clinical study. In this grant proposal, the candidate seeks to understand the fundamental mechanisms responsible for this allosteric activation of PP2A phosphatase from a structural perspective, working under the primary mentorship of Dr. Eric Fischer, an emerging leader in biochemistry and the field of structural biology. The central hypothesis is that PPZ/iHAP1 binds to the AC heterodimer and allosterically alters its conformation -- in ways that will be evident from the high-resolution crystal structure -- so that the B56ϵ subunit is readily recruited, thus driving the formation and activation of the PP2A-B56ϵ phosphatase in the cell. The immediate objectives are to determine the three-dimensional protein structures of the PP2A holoenzyme complexes formed in response to PPZ/iHAP, and to define the molecular contacts of the PP2A subunits with PPZ/iHAP compounds by X-ray crystallography. This goal will be pursued in two specific aims: 1) Determine the three-dimensional structure of the PP2A holoenzyme complex in the presence of PPZ or iHAP compounds to decipher the mechanisms underlying allosteric activation of specific PP2A subunit complexes by these small molecules, and 2) Determine the biochemical mechanisms underlying the ability of allosteric small-molecule PP2A activators to drive dynamic exchange of the PP2A regulatory B subunits with the AC heterodimer, from the Striatin family proteins in control cells to the B56ϵ or B55α proteins for iHAP/PPZ and SMAP, respectively. This strategy is significant because the results could reveal new mechanisms underlying allosteric activation of specific PP2A phosphatase complexes, which in turn will be key to dissecting the normal physiologic roles of this diverse family of phosphatases and to understanding their roles as tumor suppressors and how they are subverted in malignant transformation. This proposal has been designed to provide the candidate with advanced training in structural biology, as well as mentoring in manuscript/grant writing and leadership qualities. Bolstered by the guidance of a new primary mentor, Dr. Eric Fischer, together with the ongoing contributions of co-mentor Dr. Thomas Look, and the scientific advisory committee composed of experts necessary to complete this work, this proposal should significantly enhance the candidate's career goal of becoming an independent investigator.