Glycoregulation of Skp1 in the cytoplasm and nucleus

摘要 进化已经招募了大量的翻译后修饰来提供时间、空间和 对细胞蛋白质机械的功能调节。本项目关注的是一个特定的示例 一种似乎是从真核细胞的分泌途径中借来的类型，糖基化，但 实际上可能最早是在细菌细胞的细胞质中进化的。我们认为，复杂的细胞质 糖基化在真核生物中发挥着独特的糖调节功能，并受到不同的调控。 相对于分泌途径中的“常规”蛋白质糖基化。分析的初始有机体是 该途径的靶标为Skp1，它是SCF的一个适配子 一类E3泛素连接酶，其靶标经常被磷酸化激活，对其有 可能需要一种独立的共价监管模式。最值得注意的是，这种修改 涉及六个酶步骤，导致连接到高度保守的 脯氨酸残留物。这种修饰，其结构的丰富性与多肽相媲美，被假设为 仅针对Skp1，并调节其对关键发育转变(高潮)的调节。遗传 对脯氨酸羟基酶表达的操纵控制着发育所需的O2-，这表明 这种酶在O2-调节中的正常作用。干扰酶基因作用的最新分析 Skp1的顺序羟脯氨酸依赖的糖基化所需的蛋白质提供了其他 氧依赖发育的分级调控水平，这将在本项目中描述。 我们最近发现了构建五糖所需的最后一种酶(AgTA)，这让我们 最后，从基因和生物化学的角度来解决这些想法。首先，我们将在目标1中定义 连接的两个半乳糖的连接，它们的添加似乎是由AgTA催化的，它将 使化学合成的多聚糖用于以后的目的。目标2将审查表观Agta的基础 添加这两种糖的过程，以及Skp1和AgTA的催化和螺旋桨样结构域是如何 相互规范彼此的活动，假设与质量控制有关。AIM 3将聘用 对糖基化基因进行反向遗传和上位性分析，以测试分级调控是否 线性的或涉及平行的信号通路。此外，还将开发新的抗体来监测 细胞中Skp1糖基化的进行性变化，这是发育的信号。最后，要确定 在功能上，Aim 4将对修饰途径的最重要特征进行测试 弓形虫顶端复合体Skp1糖调节的进化保守性 人类弓形虫病。所获得的知识有望产生关于蛋白质组如何的新想法 受选择的原生生物对外部信号(如O2)和内部信号(如糖)的调节 代谢物。

SUMMARY Evolution has enlisted a large variety of posttranslational modifications to provide temporal, spatial and functional regulation of the protein machinery of the cell. This project focuses on a specific example of a type that has seemingly been borrowed from the secretory pathway of eukaryotic cells, glycosylation, but might actually have first evolved in the cytoplasm of bacterial cells. We propose that complex cytoplasmic glycosylation exerts unique glycoregulatory functions in eukaryotes, and is subject to distinct controls relative to `conventional' protein glycosylation in the secretory pathway. The initial organism of analysis is the social amoeba Dictyostelium, and the target of the pathway studied here is Skp1, an adaptor of the SCF class of E3 ubiquitin ligases whose targets are frequently activated by phosphorylation and for which there may be a need for an independent mode of covalent regulation. Most remarkable is that this modification involves six enzymatic steps resulting in the assembly of a pentasaccharide attached to a highly conserved residue of proline. This modification, with a structural richness rivaling that of a peptide, is hypothesized to target only Skp1 and modulate its regulation of a critical developmental transition (culmination). Genetic manipulation of prolyl hydroxylase expression controls the O2-requirement for development suggesting a normal role for this enzyme in O2-regulation. Recent analysis of the effects of disrupting enzyme genes required for the sequential hydroxyproline-dependent glycosylation of Skp1 gives evidence for additional levels of hierarchical regulation of O2-dependent development, which is to be characterized in this project. Our recent discovery of the last enzyme (AgtA) needed to construct the pentasaccharide has positioned us finally to address these ideas genetically and biochemically. At the outset, we will in aim 1 define the linkages of the two ¿-linked galactose sugars whose additions appear to be catalyzed by AgtA, which will enable chemical synthesis of the glycan for the later aims. Aim 2 will examine the basis for apparent AgtA processivity in adding the two sugars, and how Skp1 and the catalytic and ¿-propeller-like domains of AgtA mutually regulate each other's activity, hypothesized to be associated with quality control. Aim 3 will employ reverse genetic and epistatic analysis of the glycosylation genes to test whether hierarchical regulation is linear or involves parallel signaling pathways. In addition, new antibodies will be developed to monitor progressive variations in Skp1 glycosylation in the cells which signal development. Finally, to identify the functionally most important features of the modification pathway, aim 4 will carry out tests for the evolutionary conservation of Skp1 glycoregulation in the apicomplexan Toxoplasma gondii, the agent for human toxoplasmosis. The knowledge gained is expected to generate new ideas of how the proteome is regulated in select protists in response to external signals such as O2 and internal signals such as sugar metabolites.