Transfer of 5R01GM037539 - 22 CYTOSOLIC PROLINE HYDROXYLATION AND GLYCOSYLATION

描述（由申请人提供）：细胞分化基本上取决于选定的调节蛋白和结构蛋白的周转。这种普遍的发育方面在很大程度上是由多聚泛素化介导的，其靶向蛋白质以在26 S-蛋白酶体中进行处理。SCF（Skp 1-cullin 1-Fbox）是cullin-RING-type E3 ubiquitin（Ub）-ligases（CRL）的一个亚类，是一组主要的多聚泛素连接酶，它直接参与许多生理（包括细胞周期）和发育过程。一个关键的特征是它们使用特异性因子来选择靶点，随后是引发事件，如磷酸化。相当多的证据表明，CRL Ub连接酶也受到调控。我们在模式生物Dictyosteoblasts中的发现揭示了调节SCF类本身的新的和新颖的机制。这些机制似乎在原生生物中广泛存在，包括许多重要的人类病原体。新的机制包括通过脯氨酰羟基化共价修饰Skp 1接头，随后通过5种糖进行连续修饰，最终形成五糖。我们在最后和早期的项目期间定义了该途径的遗传学和酶学。这些研究还揭示了显着的变化，在O2依赖的发展，我们追溯到O2传感器功能的脯氨酰4-羟化酶类似于在人类中的相应过程。我们还发现，连续的糖基化步骤调节O2传感，并且该途径中的最终糖基转移酶AgtA具有酶独立的功能，这些功能也是适当发育所必需的。我们的最新研究结果表明，这些修饰改变了Skp 1的构象，从而抑制了其同源二聚化并促进Skp 1与Fbox蛋白的结合，导致其自身多聚泛素化和过早降解。此外，AgtA竞争性结合未修饰的Skp 1，通过一种新的自限性机制介导的糖基化。由于这些相互作用与E3 SCFUb-连接酶的组装有关，我们假设Skp 1修饰酶最终控制约50种预测的Fbox蛋白中的许多蛋白的泛素化，其中大部分在发育过程中受到差异调节，并且可能也控制其靶底物。该项目的目标是确定这种情况如何发生的生化机制。该模型是重要的，因为它提供了对发育具有重大影响的E3 SCFUb-连接酶的特异性调节的新模式，并且致病原生生物中6-酶途径的出现为未来开发提供了大的药物靶标。这些研究将在网骨藻中进行，网骨藻是一种易于实验的生物体，我们在其中开发了宝贵的工具来验证这一假设。目的1研究羟基化、糖基化和AgtA对SCF复合物组装及其体外E3连接酶活性的影响。目的2将调查的相关性的研究结果Skp 1复合物组装和活动的细胞使用免疫沉淀和显微镜。目的3将利用基因和抑制剂合成研究来解决Skp 1修饰与发育调控中的泛素化和降解活性的联系。

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.