Structural and Biochemical Insights into PP2A Holoenzyme Biogenesis

PP2A 全酶生物发生的结构和生化见解

基本信息

批准号：
8598906
负责人：
Yongna Xing
金额：
$ 29.39万
依托单位：
UNIVERSITY OF WISCONSIN-MADISON
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-15 至 2016-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8598906
关键词：
Active Sites Address Affect Alzheimer&apos s Disease Binding Binding Proteins Biochemical Biochemistry Biogenesis Biophysics Cell Cycle Cell Death Cell Survival Cell physiology Cells Cellular biology Client Complex Coupling Crystallization Crystallography Cytoskeleton DNA Damage Defect Engineering Ensure Enzymes Health Holoenzymes Human In Vitro Knock-out Knowledge Mediating Metals Methylation Methyltransferase Modeling Modification Molecular Chaperones Molecular Conformation Mutation Nature Nuclear Receptors PPP2R4 gene Pathway interactions Phosphoric Monoester Hydrolases Phosphotransferases Process Protein phosphatase Proteins Publications Regulation Research Resolution Role Signaling Molecule Signaling Protein Site Structure Substrate Specificity Testing Ubiquitination base cancer therapy cancer type drug sensitivity enzyme activity genetic regulatory protein human disease insight mutant novel preference protein activation protein folding response yeast genetics

项目摘要

DESCRIPTION (provided by applicant): Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase that regulates diverse pathways and cellular processes. Deregulation of PP2A is associated with many types of cancers and Alzheimer's Disease. PP2A is highly regulated at two major levels: trimeric holoenzyme controls substrate specificity; modulation of active site conformation regulates the level of enzyme activity. Our recent advance in understanding PP2A-specific methyltransferase LCMT-1 shows that PP2A methylation, a modification that enhances holoenzyme assembly, is stimulated by PP2A's phosphatase activity. Our studies further suggest compelling mechanisms for PP2A inhibitory protein a4 and PP2A phosphatase activator (PTPA), and point to hierarchy controls and a linear pathway of holoenzyme biogenesis: partially-folded PP2A is stabilized by a4 in an inactive form, and converted to an active form by PTPA; activated PP2A is then selectively methylated and enhanced to form substrate-specific holoenzymes. We recently made key breakthrough in crystallization of PP2A bound to a4 and PTPA, the highly dynamic complexes with highly regulated interactions. The research proposed here will combine x-ray crystallography with biochemistry, biophysics, yeast genetics, and cell biology to determine how a4, PTPA and PP2A methylation control PP2A structure and function to precisely drive holoenzyme biogenesis. We will determine the high-resolution structure of the PP2A-a4 complex, and address how their interaction controls PP2A stability and affects cell survival (Aim 1). We will determine the structural basis for the chaperone function of PTPA to the PP2A active site to gain insight into PP2A activation, controls of catalytic metal loading and substrate preferences, and elucidate mechanisms of PTPA in PP2A activation and cell survival (Aim 2). We will determine how defects in a4 and PTPA affect subsequent steps of holoenzyme biogenesis, and decipher the role of methylation in controlling holoenzyme structure, conformation and stability (Aim 3). These studies will reveal compelling mechanisms and hierarchy controls of holoenzyme biogenesis that restrict ambiguous phosphatase activity and ensure formation of active holoenzymes, which has a fundamental impact on cell cycle, survival, and drug sensitivity.

描述（由申请人提供）：蛋白磷酸酶 2A (PP2A) 是一种主要的 Ser/Thr 磷酸酶，可调节多种途径和细胞过程。 PP2A 的失调与多种癌症和阿尔茨海默病有关。 PP2A 在两个主要水平上受到高度调控：三聚体全酶控制底物特异性；活性位点构象的调节调节酶活性的水平。我们最近在了解 PP2A 特异性甲基转移酶 LCMT-1 方面取得的进展表明，PP2A 甲基化（一种增强全酶组装的修饰）受到 PP2A 磷酸酶活性的刺激。我们的研究进一步表明了 PP2A 抑制蛋白 a4 和 PP2A 磷酸酶激活剂 (PTPA) 的令人信服的机制，并指出了全酶生物发生的层次控制和线性途径：部分折叠的 PP2A 由非活性形式的 a4 稳定，并转化为活性形式由 PTPA 形成；然后，活化的 PP2A 被选择性甲基化并增强，形成底物特异性全酶。我们最近在 PP2A 与 a4 和 PTPA 结合的结晶方面取得了关键突破，这是一种高度动态的复合物，相互作用受到高度调控。这里提出的研究将X射线晶体学与生物化学、生物物理学、酵母遗传学和细胞生物学相结合，以确定a4、PTPA和PP2A甲基化如何控制PP2A结构和功能，从而精确驱动全酶生物发生。我们将确定 PP2A-a4 复合物的高分辨率结构，并探讨它们的相互作用如何控制 PP2A 稳定性并影响细胞存活（目标 1）。我们将确定 PTPA 对 PP2A 活性位点的伴侣功能的结构基础，以深入了解 PP2A 激活、催化金属负载和底物偏好的控制，并阐明 PTPA 在 PP2A 激活和细胞存活中的机制（目标 2）。我们将确定a4和PTPA的缺陷如何影响全酶生物发生的后续步骤，并破译甲基化在控制全酶结构、构象和稳定性中的作用（目标3）。这些研究将揭示全酶生物合成的引人注目的机制和层次控制，限制模糊的磷酸酶活性并确保活性全酶的形成，这对细胞周期、存活和药物敏感性具有根本性影响。