Glycoregulation of Skp1 in the cytoplasm and nucleus

描述（由申请人提供）：感知和响应环境中O2的变化对有氧生物至关重要。后生动物通过破坏一种称为缺氧诱导因子（HIF）的转录因子在氧气充分性中的稳定来实现这一点。这是通过o2依赖性脯氨酸4-羟化酶（PHD）实现的，该酶修饰HIF-1α亚基中的脯氨酸残基，使其成为E3多泛素连接酶的目标，并最终将其发送到26s蛋白酶体进行降解。phd是非血红素双加氧酶，它利用O2和酮戊二酸将脯氨酸残基羟基化，形成琥珀酸盐和二氧化碳。原生动物有博士学位，但缺乏HIF，因此对氧气的感觉不同。在社会性变形虫Dictyostelium中，我们发现它的PHD修饰Skp1中的脯氨酸，Skp1是SCF （Skp1/Cullin1/F-box蛋白/Rbx1）多泛素连接酶复合物的一个组成部分。Skp1脯羟化不影响其稳定性，但允许它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，这是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，该Skp1修饰通路在弓形虫原虫中保守，但在其人类宿主中不保守。此外，弓形虫PHD或两种糖基转移酶中的任何一种的缺失，会导致寄生虫在低但生理的氧水平下复制缺陷，并导致其Skp1相互作用组的改变。由于其在医学上的重要性，我们将关注弓形虫，并追求三个具体目标：i) Skp1是弓形虫PHD的关键底物吗？新型糖基化对PhyA激活的贡献是什么？ii)脯氨酸羟基化如何影响Skp1和多泛素连接酶的组装？iii)寄生虫的PHD是如何调控的，这些数据如何用于开发寄生虫特异性的PHD药理学抑制剂？为了最大限度地提高这一更新应用的进展，我们汇集了3位独立研究者的专业知识：1)一位糖生物学家专门研究原生动物Skp1和多泛素连接酶，2)一位在缺氧研究方面经验丰富的弓形虫专家，3)一位有机化学家专门研究非血红素α酮戊二酸依赖的双加氧酶及其抑制。

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-1α subunit 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 α ketoglutarate-dependent dioxygenases and their inhibition.