Structural basis of PP2A phosphatase diseases

PP2A磷酸酶疾病的结构基础

• 批准号: 10736835
• 负责人: Yongna Xing
• 金额: $ 36.2万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10736835
• 关键词: Active Sites; Adenine; Affect; Amino Acid Motifs; Binding; Biochemical; Brain; CREB1 gene; Cancer Patient; Catalytic Domain; Cell physiology; Cellular biology; Common Core; Complex; Cryoelectron Microscopy; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Diagnosis; Disease; Dissection; Event; Family; Feedback; Forskolin; Heart Diseases; Holoenzymes; Human; Intellectual functioning disability; Jordan; Knowledge; Link; MAP Kinase Gene; Malignant Neoplasms; Missense Mutation; Molecular Conformation; Molecular Diagnosis; Mutate; Mutation; Parkinsonian Disorders; Patients; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Play; Protein Array; Protein Dephosphorylation; Protein Deregulation; Protein Family; Protein phosphatase; Proteins; Proteome; Regulation; Regulatory Element; Research; Resolution; Role; Sampling; Signal Transduction; Site; Somatic Cell; Somatic Mutation; Structure; Syndrome; Testing; Variant; arm; cancer type; de novo mutation; developmental disease; disease-causing mutation; early onset; exome; genetic regulatory protein; genome sequencing; human disease; inhibitor; insight; member; molecular dynamics; multidisciplinary; mutant; nervous system disorder; neurodevelopment; phosphoric diester hydrolase; protein phosphatase 2A regulatory subunit 65 kDa; response; scaffold; simulation; single molecule; single-molecule FRET; whole genome

PROJECT SUMMARY Protein phosphatase 2A (PP2A) is a major Ser/Thr phosphatase with complex regulation and composition. Deregulation of PP2A holoenzymes leads to devastating human diseases, including multiple types of cancer and neurological disorders. In recent years, whole exome/genome sequencing for the molecular diagnosis identified broad disease mutations in PP2A subunits and substrates in cancer and neurological disorders. The diagnosis of de novo PP2A mutations in developmental disorders enthused unprecedented multidisciplinary phosphatase research and interactions with patient families. De novo mutations in several members of B56 family of PP2A regulatory subunits, such as B56δ and B56γ, cause neurological disorders, known as Jordan Syndrome. The same somatic mutations were found in cancer patients. Built on our preliminary cryo-EM, molecular dynamic simulations, biochemical, single molecule, reverse phase protein array, and cell biology studies, here we aim to decipher the mechanisms of B56 disease mutations in cancer and intellectual disabilities. We will determine the cryo-EM structures of WT and disease variants of the PP2A-B56δ holoenzyme (Specific Aim 1), investigate how intellectual disability (ID) mutations alter holoenzyme dynamics, activation phosphorylation, and interactions with regulatory proteins, combining biochemical dissections, advanced molecular dynamic simulation approaches, and single-molecule fluorescence resonance energy transfer (smFRET) (Specific Aim 2), and test our hypothesis on the discriminating and merging mechanisms of B56δ and B56γ ID mutations in perturbing the cAMP negative feedback signaling via MAPK and CREB signaling, respectively (Specific Aim 3). These studies will shed light on a highly dynamic regulation machinery involving the folding of long disordered regions against the holoenzyme core and a super-long dynamic interface harboring the majority of residues mutated in Jordan Syndrome, multiple activation phophorylation sites, and regulatory elements that suppress both the phosphatase active site and the substrate-binding groove.

项目概要 蛋白磷酸酶 2A (PP2A) 是一种主要的 Ser/Thr 磷酸酶，具有复杂的调控和组成。 PP2A 全酶的失调会导致毁灭性的人类疾病，包括多种类型的癌症和 神经系统疾病。近年来，用于分子诊断的全外显子组/基因组测序已确定 癌症和神经系统疾病中 PP2A 亚基和底物的广泛疾病突变。诊断 发育障碍中的从头 PP2A 突变引起了前所未有的多学科磷酸酶的热情 研究以及与患者家属的互动。 PP2A B56 家族几个成员的从头突变 调节亚基，例如 B56δ 和 B56γ，会导致神经系统疾病，称为乔丹综合症。这 在癌症患者中也发现了相同的体细胞突变。基于我们初步的冷冻电镜、分子动力学 模拟、生化、单分子、反相蛋白阵列和细胞生物学研究，我们的目标是 破译 B56 疾病突变在癌症和智力障碍中的机制。我们将确定 WT 的冷冻电镜结构和 PP2A-B56δ 全酶的疾病变体（具体目标 1），研究如何 智力障碍（ID）突变改变全酶动力学、激活磷酸化以及与 调节蛋白，结合生化解剖、先进的分子动力学模拟方法， 和单分子荧光共振能量转移 (smFRET)（具体目标 2），并检验我们的假设 关于 B56δ 和 B56γ ID 突变干扰 cAMP 阴性的区分和合并机制 分别通过 MAPK 和 CREB ​​信号传导反馈信号（具体目标 3）。这些研究将揭示 关于高度动态的调节机制，涉及长无序区域的折叠 全酶核心和超长动态界面包含约旦大部分突变残基 综合征、多重激活磷酸化位点和抑制磷酸酶的调节元件 活性位点和底物结合槽。