Cytosolic Proline Hydroxylation and Glycosylation

描述(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。这种普遍的发育方面在很大程度上是由多泛素化介导的，它以26S蛋白酶体中的蛋白质为处理对象。SCF(Skp1-cullin1-Fbox)是一类主要的多泛素连接酶，是cullin环型E3泛素(Ub)连接酶(CRL)的亚类，直接参与多种生理(包括细胞周期)和发育过程。一个关键的特征是，在启动事件(如磷酸化)之后，它们使用一种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Dictyostelius中的发现揭示了调节SCF类本身的新的和新的机制。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。新的机制包括通过丙酰羟化对Skp1接头进行共价修饰，然后用5种糖进行一系列修饰，最终形成五糖。我们在项目的最后和早期定义了该途径的遗传学和酶学。这些研究还揭示了发育对氧的依赖性的显着变化，我们将其追溯到Pro-4-羟基酶的氧传感器功能，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感觉，并且该途径中的最终糖基转移酶AgTA具有酶不依赖的功能，这也是正常发育所必需的。我们的最新发现表明，这些修饰改变了Skp1的构象，从而抑制了Skp1的同源二聚化，促进了Skp1与Fbox蛋白的结合，导致其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限机制竞争性地结合未修饰的Skp1。因为这些相互作用与E3SCFUb连接酶的组装有关，我们假设Skp1修饰酶最终控制着许多~50个预测的Fbox蛋白的泛素化，其中大多数在发育过程中受到差异调控，并可能控制它们的靶底物。这个项目的目标是定义这种情况发生的生化机制。这个模型之所以重要，是因为它提供了一种对E3SCFUb-连接酶具有重要影响的特异性调控的新模式，而致病原生生物中6-酶途径的出现为未来的开发提供了一个巨大的药物靶点。这些研究将在Dictyostelials中进行，这是一种实验上容易操作的有机体，我们在那里开发了无价的工具来检验这一假设。目的1体外研究羟化、糖基化和AgTA对SCF复合体组装及其E3Ub连接酶活性的影响。目的2将利用免疫沉淀和显微镜技术研究这些发现与Skp1复合体组装和细胞内活性的相关性。目的3将利用基因和抑制剂的合成研究来解决Skp1修饰与泛素化和发育调节中的降解活性之间的联系。

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.