Structural basis of PP2A phosphatase diseases

项目总结 蛋白磷酸酶2A(PP2A)是一种主要的丝氨酸/苏氨酸磷酸酶，具有复杂的调节和组成。 放松对PP2A全酶的管制会导致毁灭性的人类疾病，包括多种类型的癌症和 神经紊乱。近年来，全外显子组/基因组测序用于分子诊断鉴定 癌症和神经疾病中PP2A亚单位和底物的广泛疾病突变。诊断 发育障碍中新发现的PP2A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。PP2A基因B56家族几个成员的新基因突变 调节亚基，如B56δ和B56γ，会引起神经疾病，称为乔丹综合症。这个 在癌症患者中也发现了同样的体细胞突变。建立在我们初步的低温EM分子动力学基础上 模拟，生化，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 破译癌症和智力障碍中B56疾病突变的机制。我们将确定 WT的冷冻-EM结构和PP2A-B56δ全酶的疾病变体(特定目标1)，调查如何 智力残疾(ID)突变改变了全酶动力学、激活磷酸化和与 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)(特定目标2)，并检验我们的假设 B56δ和B56γID突变干扰cAMP阴性的区分和合并机制 分别通过MAPK和CREB信号传递反馈信号(具体目标3)。这些研究将揭示 关于一种涉及折叠长长的无序区域的高动态调节机制 全酶核心和含有大部分残基的超长动态界面在约旦突变 综合征、多个激活磷酸化位点和抑制磷酸酶的调节元件 活性中心和底物结合槽。

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.