Transfer of 5R01GM037539 - 22 CYTOSOLIC PROLINE HYDROXYLATION AND GLYCOSYLATION

5R01GM037539 - 22 胞质脯氨酸羟基化和糖基化的转移

• 批准号: 9071719
• 负责人: CHRISTOPHER M. WEST
• 金额: $ 43.38万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9071719
• 关键词: 26S proteasome; Acanthamoeba; Address; Affect; Animal Model; Animals; Antibodies; Back; Binding; Binding Proteins; Biochemical; Biological Assay; Boxing; Cell Cycle; Cell Differentiation process; Cell physiology; Cells; Client; Co-Immunoprecipitations; Communicable Diseases; Complex; Cullin 1; Cullin Proteins; Data; Dependence; Development; Developmental Process; Dictyostelium; Double Effect; Drug Targeting; Employee Strikes; Encephalitis; Enzymatic Biochemistry; Enzymes; Eukaryota; Event; Future; Gel Chromatography; Genes; Genetic; Goals; Gray unit of radiation dose; Health; Homodimerization; Human; Hydroxylation; Immunofluorescence Immunologic; Immunoprecipitation; Keratitis; Knock-out; Lead; Legionella pneumophila; Legionnaires' Disease; Ligase; Livestock; Mediating; Microscopy; Modeling; Modification; Molecular Conformation; Mutagenesis; Organism; Parasitic infection; Pathway interactions; Phosphorylation; Physiological; Phytophthora; Plants; Polysaccharides; Polyubiquitin; Polyubiquitination; Process; Procollagen-Proline Dioxygenase; Production; Proline; Proteasome Inhibitor; Protein Binding; Protein Isoforms; Proteins; Publishing; Reagent; Regulation; Role; Signal Transduction; Soybeans; Specificity; Structural Protein; Structure; Testing; Toxoplasma gondii; Toxoplasmosis; Ubiquitin; Ubiquitination; Western Blotting; base; dimer; enzyme pathway; genetic analysis; genetic inhibitor; genetic regulatory protein; glycosylation; glycosyltransferase; human disease; in vitro activity; induced pluripotent stem cell; inhibitor/antagonist; insight; mutant; novel; nutrition; overexpression; pathogen; premature; protein complex; protein protein interaction; sensor; sugar; tool; ubiquitin ligase; ubiquitin-protein ligase; vector

DESCRIPTION (provided by applicant): Cell differentiation depends fundamentally on the turnover of selected regulatory and structural proteins. This universal aspect of development is mediated in large part by polyubiquitination, which targets proteins for disposal in the 26S-proteasome. A major group of polyubiquitin ligases, the SCF (Skp1-cullin1-Fbox) subclass of cullin-RING-type E3 ubiquitin (Ub)-ligases (CRLs), has been directly implicated in numerous physiological (including cell cycle) and developmental processes. A critical feature is their use o a specificity factor for selecting targets, following a priming event such as phosphorylation. Considerable evidence indicates that CRL Ub-ligases are also regulated. Our findings in the model organism Dictyostelium reveal new and novel mechanisms for regulating the SCF class itself. These mechanisms appear to be widespread in protists including many important human pathogens. The novel mechanisms involve covalent modification of the Skp1 adaptor by prolylhydroxylation and subsequent serial modification by 5 sugars to ultimately form a pentasaccharide. We defined the genetics and enzymology of the pathway in the last and earlier project periods. These studies also revealed striking changes in the O2 dependence of development which we traced back to an O2 sensor function of the prolyl 4-hydroxylase analogous to a corresponding process in humans. We also discovered that successive glycosylation steps modulate O2 sensing, and that the final glycosyltransferase in the pathway, AgtA, has enzyme-independent functions that are also necessary for proper development. Our newest findings now suggest that these modifications alter the conformation of Skp1, which inhibits its homodimerization and promotes binding of Skp1 to Fbox proteins, leading to their auto-polyubiquitination and premature degradation. Furthermore, AgtA competitively binds unmodified Skp1, by a novel self-limiting mechanism mediated by glycosylation. Because these interactions pertain to the assembly of E3SCFUb-ligases, we hypothesize that the Skp1 modification enzymes ultimately control the ubiquitination of many of the ~50 predicted Fbox proteins, most of which are differentially regulated during development, and potentially their target substrates as well. The goal of this project is to define the biochemical mechanism of how this occurs. This model is important be- cause it offers a novel mode of specific regulation of E3SCFUb-ligases with major impact on development, and the occurrence of the 6-enzyme pathway in pathogenic protists presents a large drug target for future exploitation. The studies will be conducted in Dictyostelium, an experimentally facile organism where we have developed invaluable tools to test the hypothesis. Aim 1 will investigate how hydroxylation, glycosylation, and AgtA affect assembly of SCF complexes and their E3 Ub-ligase activities in vitro. Aim 2 will investigate the relevance of the findings to Skp1 complex assembly and activities in cells using immunoprecipitation and microscopy. Aim 3 will employ gene and inhibitor synthetic studies to address the linkage of Skp1 modification to ubiquitination and degradation activities in developmental regulation.

描述（由申请人提供）：细胞分化从根本上取决于选定的调节蛋白和结构蛋白的周转。这种普遍的发育在很大程度上是由多泛素化介导的，多泛素化作用的目标是在26s蛋白酶体中处理蛋白质。cullin- ring型E3泛素(Ub)-连接酶（CRLs）的SCF （Skp1-cullin1-Fbox）亚类是多泛素连接酶的一个主要群体，它直接参与了许多生理（包括细胞周期）和发育过程。一个关键特征是它们在启动事件（如磷酸化）之后使用特异性因子来选择靶标。大量证据表明，CRL ub连接酶也受到调控。我们在模式生物盘基ostelium中的发现揭示了调节SCF类本身的新机制。这些机制似乎广泛存在于原生生物中，包括许多重要的人类病原体。新的机制包括Skp1接头的共价修饰，通过脯羟化和随后的5个糖的一系列修饰，最终形成一个五糖。我们在项目后期和早期定义了该途径的遗传学和酶学。这些研究还揭示了O2依赖性的显著变化，我们将其追溯到proyll 4-羟化酶的O2传感器功能，类似于人类的相应过程。我们还发现，连续的糖基化步骤调节O2传感，并且该途径中的最后一个糖基转移酶AgtA具有酶独立的功能，这也是正常发育所必需的。我们的最新研究结果表明，这些修饰改变了Skp1的构象，从而抑制了Skp1的同二聚化，促进了Skp1与Fbox蛋白的结合，导致它们的自身多泛素化和过早降解。此外，AgtA通过糖基化介导的一种新的自我限制机制，竞争性地结合未修饰的Skp1。由于这些相互作用与e3scfu连接酶的组装有关，我们假设Skp1修饰酶最终控制了许多~50种预测的Fbox蛋白的泛素化，其中大多数在发育过程中受到差异调节，并且可能也是它们的靶底物。这个项目的目标是定义这种情况发生的生化机制。该模型很重要，因为它为e3scfu连接酶的特异性调控提供了一种新的模式，对发育有重大影响，并且6-酶途径在致病性原生生物中的出现为未来开发提供了一个巨大的药物靶点。这些研究将在盘基ostelium中进行，这是一种易于实验的生物，我们已经开发了宝贵的工具来测试这一假设。目的1将研究羟基化、糖基化和AgtA如何影响体外SCF复合物的组装及其E3 ub连接酶的活性。目的2将使用免疫沉淀和显微镜研究这些发现与细胞中Skp1复合物组装和活性的相关性。目的3将利用基因和抑制剂合成研究来解决Skp1修饰与泛素化和发育调控中降解活性的联系。