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所选调节和结构蛋白的更新。发育的这一普遍方面在很大程度上是由多聚泛素化介导的，多聚泛素化的目标是在 26S 蛋白酶体中处理的蛋白质。多聚泛素连接酶的一个主要类别，即 cullin-RING 型 E3 泛素 (Ub) 连接酶 (CRL) 的 SCF (Skp1-cullin1-Fbox) 子类，直接涉及许多生理（包括细胞周期）和发育过程。一个关键特征是它们在磷酸化等启动事件后使用特异性因子来选择靶标。大量证据表明 CRL Ub 连接酶也受到监管。我们在模式生物盘基网柄菌中的发现揭示了调节 SCF 类别本身的新机制。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。新机制涉及通过脯氨酰羟基化对 Skp1 衔接子进行共价修饰，并随后通过 5 个糖进行连续修饰，最终形成五糖。我们在项目的最后和早期阶段定义了该途径的遗传学和酶学。这些研究还揭示了发育过程中 O2 依赖性的显着变化，我们将其追溯到脯氨酰 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，连续的糖基化步骤会调节 O2 感应，并且该途径中的最终糖基转移酶 AgtA 具有酶独立的功能，这也是正确发育所必需的。我们的最新发现表明，这些修饰改变了 Skp1 的构象，从而抑制其同二聚化并促进 Skp1 与 Fbox 蛋白的结合，导致其自动多泛素化和过早降解。此外，AgtA 通过糖基化介导的新型自我限制机制竞争性结合未修饰的 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 修饰酶最终控制了约 50 种预测的 Fbox 蛋白中的许多蛋白的泛素化，其中大多数在发育过程中受到差异性调节，也可能控制它们的目标底物。该项目的目标是确定这种情况发生的生化机制。该模型很重要，因为它提供了一种对 E3SCFUb 连接酶进行特异性调节的新模式，对发育产生重大影响，并且致病性原生生物中 6 酶途径的出现为未来的开发提供了一个大的药物靶标。这些研究将在盘基网柄菌属中进行，这是一种实验简便的生物体，我们开发了宝贵的工具来检验这一假设。目标 1 将研究羟基化、糖基化和 AgtA 如何影响 SCF 复合物的组装及其体外 E3 Ub 连接酶活性。目标 2 将使用免疫沉淀和显微镜检查这些发现与细胞中 Skp1 复合物组装和活性的相关性。目标 3 将采用基因和抑制剂合成研究来解决 Skp1 修饰与发育调节中泛素化和降解活动的联系。