Protein Phosphatase 1 Holoenzyme Formation and Subunit Exchange

蛋白磷酸酶 1 全酶形成和亚基交换

• 批准号: 9985412
• 负责人: Wolfgang Peti
• 金额: $ 4.39万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-10 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9985412
• 关键词: ATP phosphohydrolase; Active Sites; Adaptor Signaling Protein; Allosteric Regulation; Area; Binding; Binding Proteins; Biochemical; Biochemistry; Biochemistry and Cellular Biology; Biogenesis; Biological; Biological Process; Biology; Biophysics; Cell Growth Processes; Cell physiology; Cells; Cellular biology; Complex; Coupled; Cysteine; Data; Development; Disease; Drug Targeting; Enzymes; Essential Genes; Eukaryota; Eukaryotic Cell; Excision; Goals; Health; Histidine; Holoenzymes; Hydrolysis; Individual; Knowledge; Laboratories; Location; Malignant Neoplasms; Manuscripts; Metals; Mitosis; Modeling; Molecular; Molecular Chaperones; NMR Spectroscopy; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Process; Protein Dephosphorylation; Protein Serine/Threonine Phosphatase; Protein phosphatase; Proteins; Publishing; Reaction; Regulation; Research; Research Personnel; Research Project Grants; Role; Route; Serine/Threonine Phosphorylation; Signal Transduction; Structural Protein; Structure; Substrate Specificity; Techniques; Testing; Threonine Phosphorylation Site; Time; Work; X-Ray Crystallography; Yeasts; base; dimer; experimental study; genetic regulatory protein; human disease; in vivo; inhibitor/antagonist; inorganic phosphate; insight; novel; novel strategies; protein phosphatase inhibitor-1; protein structure; reconstitution; structural biology; ubiquitin-protein ligase

ABSTRACT Phosphorylation is one of the most ubiquitous, reversible posttranslational modifications in cells. The enzymes responsible for controlling the phosphorylation state of the cell are kinases, which catalyze the transfer of the γ-phosphate moiety of ATP to substrates, and phosphatases, which catalyze the reverse hydrolysis reaction, the removal of the phosphate moiety from phosphorylated substrates. Thus, phosphatases dynamically reverse the effects of kinases. Because phosphorylation is critical for all biological processes from cell growth to differentiation to development, the location and duration of the reciprocal actions of kinases and phosphatases must be exquisitely regulated both temporally and spatially within the cell. Consequently, when this tight regulation is disrupted, dysregulation of phosphorylation signaling ensues and the consequence is most often disease. Here we are investigating the cellular assembly of the serine/threonine protein phosphatase 1 (PP1). The regulatory protein SDS22 and the inhibitor-3 (I-3) have been proposed to be critical for this process, by functioning in a chaperone-like fashion. Using biochemistry and structural biology we show that this model is incorrect. Rather SDS22 and I-3 can individually function as a PP1 inhibitor. However, SDS22 inhibits PP1 via a completely novel mechanism. As we show, SDS22 unexpectedly distorts the PP1 active site so substrates can no longer bind. Thus as long as SDS22 is bound to PP1, PP1 is inactive. I-3 binds metals and thus can transport metals, which are necessary for PP1 activity, to the PP1 active site. Upon metal loading, I-3 is released in a p37-dependent manner from PP1. However, despite I-3 metal-loading, SDS22 continues to maintain PP1 in an inactive state. Thus, SDS22 is the key regulator of PP1 activity in cells. The presented research project uses a powerful integrated approach that combines X-ray crystallography and NMR spectroscopy with biochemical and cell biology experiments to obtain novel insights into the molecular mechanisms used by these regulators to control PP1 activity. We will: 1) determine the structures of the regulators in their free forms, 2) determine the structures of the PP1 dimeric and trimeric holoenzymes and 3) determine how these essential complexes direct and regulate PP1 signaling in cells. We will then leverage these structures to elucidate, at a molecular level, the biological functions and modes of action of these key PP1 holoenzymes. Because PP1 holoenzymes have critical roles in human diseases, the proposed work will provide novel strategies for selectively inhibiting PP1 activity by targeting the PP1 holoenzyme formation and subunit exchange, which is essential for understanding how distinct PPPs contribute to disease.

摘要 磷酸化是细胞中最普遍的、可逆的翻译后修饰之一。的酶 负责控制细胞磷酸化状态的是激酶，其催化磷酸化酶的转移。 ATP的γ-磷酸部分转化为底物和磷酸酶，催化逆水解反应， 从磷酸化底物中除去磷酸部分。因此，磷酸酶动态逆转 激酶的作用。因为磷酸化对于从细胞生长到 分化到发育，激酶和磷酸酶相互作用的位置和持续时间 必须在细胞内的时间和空间上进行精细的调节。因此，当这种紧 调节被破坏，磷酸化信号传导的失调加剧，其结果是最常见的 疾病在这里，我们正在研究丝氨酸/苏氨酸蛋白磷酸酶1（PP 1）的细胞组装。 调节蛋白SDS 22和抑制剂-3（I-3）已被认为是这一过程的关键， 像监护人一样工作使用生物化学和结构生物学，我们表明，这个模型是 不正确.相反，SDS 22和I-3可以单独作为PP 1抑制剂发挥作用。然而，SDS 22通过以下途径抑制PP 1： 一种全新的机制正如我们所展示的，SDS 22出乎意料地扭曲了PP 1活性位点， 不能再束缚了。因此，只要SDS 22与PP 1结合，PP 1就无活性。I-3结合金属，因此可以 将PP 1活性所必需的金属转运到PP 1活性位点。在金属负载时，I-3是 以p37依赖的方式从PP 1释放。然而，尽管I-3金属负载，SDS 22继续 保持PP 1处于非活动状态。因此，SDS 22是细胞中PP 1活性的关键调节因子。所呈现的 一个研究项目使用了一种强大的综合方法，结合了X射线晶体学和NMR 光谱与生物化学和细胞生物学实验，以获得新的见解， 这些调节器用于控制PP 1活性的机制。我们将：1）确定 自由形式的调节剂，2）确定PP 1二聚体和三聚体全酶的结构和3） 确定这些基本复合物如何指导和调节细胞中的PP 1信号传导。然后我们会利用 在分子水平上阐明这些关键结构的生物学功能和作用模式， PP 1全酶。由于PP 1全酶在人类疾病中具有关键作用，因此拟议的工作将 提供了通过靶向PP 1全酶形成来选择性抑制PP 1活性的新策略， 亚基交换，这对于理解不同的PPP如何促进疾病至关重要。