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.

项目摘要 蛋白磷酸酶2A（PP 2A）是一种主要的Ser/Thr磷酸酶，具有复杂的调节和组成。 PP 2A全酶的失调导致毁灭性的人类疾病，包括多种类型的癌症， 神经系统疾病近年来，全外显子组/基因组测序用于分子诊断鉴定 PP 2A亚基和底物在癌症和神经系统疾病中的广泛疾病突变。诊断 在发育障碍中的从头PP 2A突变激发了前所未有的多学科磷酸酶 研究和与患者家属的互动。PP 2A基因B56家族几个成员的新突变 调节亚基，如B56δ和B56γ，导致神经系统疾病，称为乔丹综合征。的 在癌症患者中也发现了相同的体细胞突变。建立在我们初步的冷冻EM、分子动力学基础上 模拟，生物化学，单分子，反相蛋白质阵列和细胞生物学研究，在这里，我们的目标是 破译癌症和智力残疾中B56疾病突变的机制。康贝特人将以 WT和PP 2A-B56 δ全酶的疾病变体的冷冻电镜结构（具体目的1），研究如何 智力残疾（ID）突变改变全酶动力学，活化磷酸化， 调节蛋白，结合生化解剖，先进的分子动态模拟方法， 和单分子荧光共振能量转移（smFRET）（具体目标2），并测试我们的假设 B56δ和B56γ ID突变在干扰cAMP阴性的细胞中的区分和合并机制 分别通过MAPK和CREB信号传导的反馈信号传导（具体目标3）。这些研究将揭示 在一个高度动态的调节机制，涉及折叠长无序区域对 全酶核心和一个超长的动态界面，包含约旦突变的大部分残基 综合征，多个激活磷酸化位点，以及抑制磷酸酶 活性位点和底物结合沟。