Glycoregulation of Skp1 in the cytoplasm and nucleus

描述(由申请人提供)：感知和响应环境中氧气的变化对好氧生物至关重要。后生动物通过在氧气充足的情况下破坏一种名为缺氧诱导因子(HIF)的转录因子的稳定来实现这一点。这是通过依赖O2的脯氨基4-羟基酶(PHD)来实现的，它修饰HIF-1�亚单位中的脯氨酸残基，使其成为E3多泛素连接酶的靶标，最终将其分派到26S-蛋白酶体进行降解。PHD是一种非血红素双加氧酶，它使用O2和酮戊二酸来羟化一个脯氨酸残基，形成琥珀酸和二氧化碳。原生动物有博士学位，但缺乏HIF，因此感知氧气的方式不同。在社会阿米巴中，我们发现它的PHD修饰了Skp1中的一个脯氨酸，Skp1是SCF(Skp1/Cullin1/F-box Protein/Rbx1)多泛素连接酶复合体的一个组成部分。Skp1的Pro羟基化不影响其稳定性，但允许其被一系列糖基转移酶修饰。反过来，糖基化稳定了Skp1与F-box蛋白的结合，F-box蛋白是SCF多泛素连接酶组装所必需的。基因组分析和生化分析表明，这种Skp1修饰途径在原虫寄生虫弓形虫中保守，而在其人类宿主中不保守。此外，弓形虫PhD的缺失或两种糖基转移酶中的任何一种都会导致寄生虫在低氧但生理水平下的复制缺陷，并导致其Skp1的变化 互动组。由于其医学重要性，我们将重点研究弓形虫并追求三个特定的目标：i)Skp1是弓形虫PhD的关键底物吗？新的糖基化对PhyA的激活有什么贡献？2)Pro羟化对Skp1和多泛素连接酶组装有何影响？Iii)寄生虫PHD是如何被调节的，这些数据如何被用来开发针对寄生虫的药理PHD抑制剂？为了最大限度地提高这一更新申请的进展，我们汇集了3名独立研究人员的专业知识：i)一名专门研究原生动物Skp1和多泛素连接酶的糖生物学家，ii)一名在低氧研究方面经验丰富的弓形虫专家，以及iii)一名专门研究非血红素�乙戊二酸依赖的双加氧酶及其抑制的有机化学家。

DESCRIPTION (provided by applicant): Sensing and responding to changes in environmental O2 is critical for aerobic organisms. Metazoans accomplish this by destabilizing a transcription factor called hypoxia inducible factor (HIF) in O2 sufficiency. This is achieved by a O2-dependent prolyl 4-hydroxylase (PHD) that modifies proline residues in the HIF-1�ubunit making it the target of an E3 polyubiquitin ligase and ultimately dispatching it to the 26S-proteasome for degradation. PHDs are non-heme dioxygenases that use O2 and ketoglutarate to hydroxylate a proline residue and form succinate and CO2. Protozoans have PHDs but lack HIF and thus sense oxygen differently. In the social amoeba Dictyostelium, we discovered that its PHD modifies a proline in Skp1, which is a component of the SCF (Skp1/Cullin1/F-box protein/Rbx1) polyubiquitin ligase complex. Skp1 prolylhydroxylation does not affect its stability, but allows it to be modified by a series of glycosyltransferases. In turn glycosylation stabilizes the binding of Skp1 to F-box proteins essential for assembly of the SCF polyubiquitin ligases. Genome analysis and biochemical assays demonstrated that this Skp1 modification pathway is conserved in the protozoan parasite Toxoplasma gondii, but not in its human host. Moreover, loss of Toxoplasma PHD, or either of two glycosyltransferases, leads to a defect in parasite replication at low but physiological oxygen levels, and to changes in its Skp1 interactome. Because of its medical importance, we will focus on Toxoplasma and pursue three specific aims: i) Is Skp1 the key substrate for the Toxoplasma PHD and what is the contribution of novel glycosylation to PhyA activation? ii) How is Skp1 and polyubiquitin ligase assembly affected by prolyl hydroxylation? iii) How is the parasite PHD regulated and how can these data be used to develop parasite-specific pharmacological PHD inhibitors? To maximize progress in this renewal application, we have pooled the expertise of 3 independent investigators: i) a glycobiologist specialized in protozoan Skp1 and polyubiquitin ligases, ii) a Toxoplasma specialist experienced in hypoxia research, and iii) an organic chemist specializing in non-heme �etoglutarate-dependent dioxygenases and their inhibition.