Glycoregulation of Skp1 in the cytoplasm and nucleus

描述（由申请人提供）：进化已经招募了大量的翻译后修饰，以提供细胞蛋白质机制的时间，空间和功能调节。这个项目的重点是一个特定的例子，这种类型似乎是从真核细胞的分泌途径中借来的，糖基化，但实际上可能首先在细菌细胞的细胞质中进化。我们认为，复杂的细胞质糖基化在真核生物中具有独特的糖调节功能，并且相对于分泌途径中的“传统”蛋白质糖基化受到不同的控制。首先分析的生物是群居变形虫Dictyostelium，这里研究的途径的靶点是Skp1，它是SCF类E3泛素连接酶的适配器，其靶点经常被磷酸化激活，可能需要一个独立的共价调节模式。最值得注意的是，这种修饰涉及六个酶促步骤，导致五糖连接到高度保守的脯氨酸残基的组装。这种结构丰富度与肽相媲美的修饰被假设仅针对Skp1并调节其对关键发育转变（顶点）的调节。脯氨酰羟化酶表达的基因操纵控制了发育对o2的需求，表明该酶在o2调节中的正常作用。最近对Skp1的顺序羟脯氨酸依赖糖基化所需的破坏酶基因的影响的分析，为o2依赖发育的额外水平的分层调节提供了证据，这将在本项目中进行表征。我们最近发现了构建五糖所需的最后一种酶（AgtA），这使我们最终能够从遗传学和生物化学角度解决这些问题。首先，我们将在目标1中定义两个1链半乳糖的键，它们的添加似乎是由AgtA催化的，这将使糖的化学合成成为可能。Aim 2将研究添加两种糖时明显AgtA进程的基础，以及Skp1和AgtA的催化和2-螺旋桨样结构域如何相互调节彼此的活性，假设与质量控制有关。目的3将采用糖基化基因的反向遗传和上位分析来测试分层调节是线性的还是涉及平行信号通路。此外，将开发新的抗体来监测细胞中Skp1糖基化的进展变化，这是发育的信号。最后，为了确定该修饰途径在功能上最重要的特征，aim 4将对人类弓形虫的病原体——弓形虫顶复合体中Skp1糖调节的进化保守性进行测试。所获得的知识有望产生新的想法，即蛋白质组如何在选择的原生生物中响应外部信号（如O2）和内部信号（如糖代谢物）进行调节。该研究利用了美国国立卫生研究院（NIH）的模式生物盘状盘齿龙（Dictyostelium disideum），这是一种与人类寄生虫内阿米巴（Entamoeba）基因组相关的社会性阿米巴原虫。Dictyostelium由于其自由生活、无壁的生活方式和单倍体基因组，在分子、生化和细胞生物学研究中具有特殊的优势，已被证明是各种其他类型原生生物蛋白质糖基化途径选择的有用模型。一个例子是细胞质糖基化途径在本研究中检查。生物信息学和早期生化研究表明，该途径的主要部分存在于植物病原菌疫霉（Phytophthora group）、人类弓形虫病（toxoplasmosis）的病原体弓形虫（Toxoplasma gondii）等原生生物中。由于弓形虫是一种细胞内病原体，Dictyostelium为弓形虫Skp1修饰途径候选基因的生化和反向遗传分析提供了一个有吸引力的替代宿主。这将为未来直接研究弓形虫的o2调控途径的功能奠定基础，弓形虫在人群中广泛潜伏传播，如果它被重新激活，药物治疗将非常有限。

DESCRIPTION (provided by applicant): 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 1-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 2-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. PUBLIC HEALTH RELEVANCE The study utilizes the NIH model organism Dictyostelium discoideum, a social amoeba allied genomically with the human parasite Entamoeba. Dictyostelium, which offers special advantages for molecular, biochemical and cell biological studies owing to its free-living, wall-less lifestyle and haploid genome, has proven to be a useful model for select pathways of protein glycosylation of various other types of protists. One example is the cytoplasmic glycosylation pathway examined in this investigation. Bioinformatics and early biochemical studies indicate that the main part of the pathway is present in the large Phytophthora group of plant pathogens, the agent for human toxoplasmosis Toxoplasma gondii, and other protists. Since T. gondii is an intracellular pathogen, Dictyostelium offers an attractive surrogate host for the biochemical and reverse genetic analysis of the Toxoplasma genes that are candidates for the Skp1 modification pathway in this organism. This will serve as a prelude for future direct studies on the function of the pathway for O2-regulation of T. gondii, which is widely disseminated latently in the human population and for which, if it is re-activated, pharmacological therapies are extremely limited.