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类型的一个具体例子，似乎是从真核细胞的分泌途径，糖基化，但实际上可能首先在细菌细胞的细胞质中进化。我们建议，复杂的细胞质糖基化发挥独特的糖调节功能，在真核生物中，并受到不同的控制相对于“传统”的蛋白质糖基化的分泌途径。分析的初始生物体是社会性阿米巴Dictyosteoba，这里研究的途径的目标是Skp 1，E3泛素连接酶的SCF类的适配器，其目标经常被磷酸化激活，并且可能需要一种独立的共价调控模式。最值得注意的是，这种修饰涉及六个酶促步骤，导致连接到脯氨酸的高度保守残基的五糖的组装。这种修饰具有与肽相媲美的结构丰富性，被假设为仅针对Skp 1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遗传操作控制发展的O2需求，这表明该酶在O2调节中的正常作用。最近对破坏Skp 1顺序羟脯氨酸依赖性糖基化所需的酶基因的影响进行的分析提供了证据，证明O2依赖性发育存在额外水平的分层调节，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），这使我们最终能够在遗传学和生物化学上解决这些想法。首先，我们将在目标1中定义两个1-连接的半乳糖糖的连接，其加成似乎是由AgtA催化的，这将使聚糖的化学合成能够用于后面的目标。目的2将检查的基础上，在添加两种糖，以及Skp 1和催化和2-螺旋桨样结构域的AgtA相互调节彼此的活动，假设是与质量控制明显的AgtA持续合成能力。目的3将采用反向遗传和上位性分析的糖基化基因，以测试是否分层调控是线性或涉及平行的信号通路。此外，将开发新的抗体来监测细胞中Skp 1糖基化的渐进变化，这是发育的信号。最后，为了确定功能上最重要的功能的修饰途径，目的4将进行测试的进化保守性Skp 1糖调节的apicomplexan弓形虫，人类弓形虫病的代理。所获得的知识有望产生新的想法，蛋白质组是如何在选择原生生物响应外部信号，如O2和内部信号，如糖代谢产物的调节。公共卫生相关性该研究利用NIH模式生物盘基网柄阿米巴，一种与人类寄生虫内阿米巴在基因组上同源的社会性阿米巴。网骨藻由于其自由生活、无壁生活方式和单倍性基因组，为分子生物学、生化和细胞生物学研究提供了独特的优势，已被证明是筛选其他类型原生生物蛋白质糖基化途径的有用模型。一个例子是在本研究中检查的细胞质糖基化途径。生物信息学和早期的生物化学研究表明，该途径的主要部分存在于植物病原体的大疫霉属（Phytophthora）、人类弓形虫病的病原体弓形虫（Toxoplasma gondii）和其他原生生物中。自从T.弓形虫是一种细胞内病原体，网囊线虫为弓形虫Skp 1修饰途径的候选基因的生物化学和反向遗传分析提供了一个有吸引力的替代宿主。这为进一步研究T细胞O2-调节途径的功能奠定了基础。弓形虫在人群中潜伏广泛传播，如果重新激活，药物治疗极为有限。

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.