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

基本信息

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
项目类别：
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&apos 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

项目摘要

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和结构蛋白的周转。发育的这一普遍方面在很大程度上是由多泛素化介导的，该多泛素化靶向26S-蛋白酶中的蛋白质以处置。 Cullin-Ring-type E3泛素（UB） - 岩石酶（CRLS）的主要聚集蛋白连接酶，SCF（SKP1-CULLIN1-FBOX）子类已直接与许多生理（包括细胞周期）和发育过程有关。一个关键特征是它们的使用o在诸如磷酸化之类的启动事件之后，选择靶标的特异性因子。大量证据表明，CRL UB - 连接酶也受到调节。我们在模型生物体中的发现揭示了调节SCF类本身的新机制。这些机制似乎在包括许多重要人类病原体在内的生物中很普遍。新型机制涉及通过羟基化和随后通过5种糖进行连续修饰对SKP1适配器的共价修饰，以最终形成五糖。我们定义了最后一个项目期间和以前的途径的遗传学和酶学。这些研究还揭示了O2发育依赖性的显着变化，我们追溯到类似于人类中相应过程的prolyl 4-羟化酶的O2传感器功能。我们还发现，连续的糖基化步骤调节O2传感，并且途径中的最终糖基转移酶AGTA具有与酶无关的功能，这些功能也是适当发育所必需的。现在，我们的最新发现表明，这些修饰改变了SKP1的构象，从而抑制其同构化并促进SKP1与Fbox蛋白的结合，从而导致其自动泛素化和过早降解。此外，AGTA通过糖基化介导的新型自限制机制来竞争地结合未修饰的SKP1。由于这些相互作用与E3SCFUB - 连接酶的组装有关，因此我们假设SKP1修饰酶最终控制了〜50个预测的Fbox蛋白中许多许多预测的Fbox蛋白的泛素化，其中大多数在发育过程中受到了差异性调节，并且也有潜在的目标底物。该项目的目的是定义其发生方式的生化机制。该模型很重要，因为它提供了一种对E3SCFUB - 连接酶进行特定调节的新模式，对发育产生了重大影响，并且在致病性生物中的6-酶途径发生的发生是为将来开发的大型药物靶标。这些研究将在dictyostelium（实验性容易的生物）中进行，我们开发了宝贵的工具来检验该假设。 AIM 1将研究羟基化，糖基化和AGTA如何在体外影响SCF复合物及其E3 UB - 连接酶活性的组装。 AIM 2将使用免疫沉淀和显微镜研究发现与SKP1复合物组装和活性的相关性。 AIM 3将采用基因和抑制剂合成研究来解决SKP1修饰与发育调控中泛素化和降解活性的联系。