Cytosolic Proline Hydroxylation and Glycosylation

Skp1，主要的SCF类泛素(Ub)连接酶的一个重要亚基，对细胞的周转至关重要 主要调节蛋白，在社会阿米巴的细胞质中受到复杂的糖基化作用 网柄网柄菌属。一项确定导致这种不寻常变化的途径的项目表明，它 依赖于一种对O2敏感的细胞质Pro-4-羟基酶(P4H1)。P4H1是哺乳动物的同源基因 酶被认为是氧传感器，通过转录调节细胞的缺氧反应 因子亚单位缺氧诱导因子-α。DictyostelialP4H1的破坏抑制了氧依赖 末端分化(达到顶峰)提示P4H1是网柄苔藓中一个关键的氧感受器 这影响了E3(SCF)Ub连接酶的活性，而E3(SCF)Ub连接酶控制着顶峰。P4H1-空单元显然也失败了 诱导调控顶端发育的柄前细胞顶端组织者细胞。使用Dictyostelius作为模型系统 对于动物和致病原生生物，包括弓形虫，这是信息学和生化研究 建议分享这条调控途径，我们将探索以下有关低氧调控的关键预测 模型：1)P4H1在柄前细胞尖端组织细胞中表达并活跃，在那里它介导O2- Skp1的依赖性修饰和Skp1的核积累。2)高活性P4H1突变体 低氧芽胞形成者抑制子筛选中的定向进化将揭示O2-结合在 活着。3a)Skp1羟化位点的突变将重现P4H1-破坏的表型。3b)SCF 在体外，复杂的亚基会影响Skp1的羟化/糖基化。3C)跳过1 在体内，羟基化/糖基化会影响SCF复合体的形成。4)羟基化的GlcNAc封端 Skp1是P4H1功能所必需的。这些结果将为研究O2-的作用机制提供新的信息。 P4H1的刺激有望适用于理解哺乳动物的低氧调节 这对癌症、炎症和伤口修复都很重要。预计这些发现将定义关键的新 一种新的低氧调节机制的步骤将适用于原生动物寄生虫 也包括无脊椎动物和/或哺乳类动物。根据这项新的研究，药物发现倡议 动物P4H酶可能会在控制原生动物传染病方面发现意想不到的应用，因为 抑制剂可能会损害病原体对低氧条件或压力的适应！

Skp1, an essential subunit of the major SCF-class of ubiquitin (Ub)-ligases important for the turnover of major regulatory proteins, is subject to complex glycosylation in the cytoplasm of the social amoeba Dictyostelium. A project to define the pathway responsible for this unusual modification showed that it depends on an O2-sensitive cytoplasmic prolyl 4-hydroxylase (P4H1). P4H1 is an ortholog of mammalian enzymes proposed to serve as O2-sensors that regulate hypoxic responses of cells via the transcriptional factor subunit hypoxia inducible factor-alpha. Disruption of Dictyostelium P4H1 inhibits an O2-dependent step in terminal differentiation (culmination) suggesting that P4H1 is a critical oxygen-sensor in Dictyostelium that influences the activity of E3(SCF)Ub-ligases that control culmination. P4H1-null cells also apparently fail to induce prestalk cell tip organizer cells that regulate culmination. Using Dictyostelium as a model system for animals and pathogenic protists including Toxoplasma gondii, which informatics and biochemical studies suggest share this regulatory pathway, we will explore the following key predictions of the hypoxic regulation model: 1) P4H1 is expressed in and active in prestalk cell tip organizer cells, where it mediates O2- dependent modification of Skp1 and nuclear accumulation of Skp1. 2) Hyperactive P4H1 mutants isolated by directed evolution in a suppressor screen for hypoxic spore-formers will reveal a direct role of O2-binding in vivo. 3A) Mutation of the Skp1 hydroxylation site will recapitulate the phenotype of P4H1-disruption. 3B) SCF complex subunits will influence Skp1 hydroxylation/glycosylation in vitro. 3C) Skp1 hydroxylation/glycosylation will influence SCF complex formation in vivo. 4) GlcNAc-capping of hydroxylated Skp1 is essential for P4H1 function. The results will provide new infomration about the mechanism of O2- stimulation of P4H1 that is expected to be applicable to understanding of mammalian hypoxic regulation which is important for cancer, inflammation, and wound repair. The findings are expected to define key new steps of a novel mechanism for hypoxic regulation that will be applicable to protozoan parasites and possibly also invertebrate and/or mammalian animals. Based on the new research, drug discovery initiatives for animal P4H enzymes might find unanticipated applications in control of protozoan infectious disease, as inhibitors might compromise pathogen adaptation to hypoxic conditions or stress!