Protein Phosphatase 1 Holoenzyme Formation and Subunit Exchange

蛋白磷酸酶 1 全酶形成和亚基交换

基本信息

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
项目类别：
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

项目摘要

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.

抽象的磷酸化是细胞中最普遍、可逆的翻译后修饰之一。负责控制细胞磷酸化状态的是激酶，它催化磷酸化的转移 ATP 的 γ-磷酸部分与底物和磷酸酶催化逆水解反应，从磷酸化底物中去除磷酸部分因此，磷酸酶动态逆转。因为磷酸化对于从细胞生长到细胞生长的所有生物过程都至关重要。分化到发育、激酶和磷酸酶相互作用的位置和持续时间当这种严格的检查时，必须在细胞内的时间和空间上进行精确的调节。调节被破坏，磷酸化信号传导失调，其后果通常是在这里，我们正在研究丝氨酸/苏氨酸蛋白磷酸酶 1 (PP1) 的细胞组装。调节蛋白 SDS22 和抑制剂 3 (I-3) 被认为对此过程至关重要，通过利用生物化学和结构生物学，我们证明该模型以类似伴侣的方式发挥作用。不正确，SDS22 和 I-3 可以单独发挥 PP1 抑制剂的作用，但是 SDS22 通过抑制 PP1。正如我们所展示的，SDS22 意外地扭曲了 PP1 活性位点，从而使底物发生变化。因此，只要 SDS22 与 PP1 结合，I-3 就无法结合金属，因此可以。将 PP1 活性所需的金属转运至 PP1 活性位点金属负载后，I-3 被转运。然而，尽管负载了 I-3，SDS22 仍然以 p37 依赖性方式释放。维持 PP1 处于非活性状态因此，SDS22 是细胞中 PP1 活性的关键调节因子。研究项目采用了结合 X 射线晶体学和 NMR 的强大集成方法光谱学与生化和细胞生物学实验相结合，以获得对分子的新见解这些调节器用于控制 PP1 活性的机制我们将： 1) 确定 PP1 的结构。游离形式的调节剂，2) 确定 PP1 二聚体和三聚体全酶的结构，3) 确定这些重要复合物如何指导和调节细胞中的 PP1 信号传导。这些结构在分子水平上阐明这些键的生物功能和作用模式由于 PP1 全酶在人类疾病中具有关键作用，因此拟议的工作将提供通过靶向 PP1 全酶形成选择性抑制 PP1 活性的新策略亚基交换，这对于理解不同的 PPP 如何导致疾病至关重要。