Glycoregulation of Skp1 in the cytoplasm and nucleus

Skp1 在细胞质和细胞核中的糖调节

• 批准号: 8839588
• 负责人: Ira J Blader
• 金额: $ 40.91万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8839588
• 关键词: 26S proteasome; Acquired Immunodeficiency Syndrome; Address; Aerobic; Affect; Affinity; American; Amino Acid Substitution; Amoeba genus; Animal Model; Animals; Attention; Binding; Biochemical; Biological Assay; Carbon Dioxide; Cell Culture Techniques; Cell Nucleus; Cell physiology; Co-Immunoprecipitations; Complement; Complex; Cytoplasm; Data; Defect; Development; Dictyostelium; Dictyostelium discoideum; Dioxygenases; Drug Targeting; Enzymes; F-Box Proteins; Fetus; Fibroblasts; Funding; Future; Genes; Genetic; Growth; Health; Human; Hydroxylation; Hydroxyproline; Hypoxia; Hypoxia Inducible Factor; Immune system; Immunosuppression; Individual; Infection; Knock-out; Lead; Libraries; Ligase; Mediating; Medical; Metabolic; Metabolic stress; Modeling; Modification; Monitor; Organism; Oxygen; Parasites; Pathway interactions; Pharmacotherapy; Physiological; Polysaccharides; Polyubiquitin; Polyubiquitination; Pregnant Women; Procollagen-Proline Dioxygenase; Proline; Publishing; Research; Research Personnel; Role; Series; Signal Transduction; Specialist; Structure; Succinates; Testing; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transplantation; Virulence; Work; alpha ketoglutarate; base; comparative; design; drug development; experience; genome analysis; glycosylation; glycosyltransferase; in vivo; inhibitor/antagonist; member; mutant; novel; pathogen; response; sensor; small molecule libraries; social; transcription factor

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.

描述（由适用提供）：对环境O2的变化进行感应和响应对于有氧生物至关重要。后生动物通过破坏了O2安全性中称为低氧诱导因子（HIF）的转录因子来实现这一目标。这是通过O2依赖性丙酰4-羟化酶（PHD）来实现的，该脯氨酸在HIF-1α亚基中保留了脯氨酸，使其成为E3多偶素蛋白连接酶的靶标，并最终将其派遣到26S-果皮中以降解。 PHD是非血红素二氧酶，使用O2和Ketoglutarate用于羟基盐水脯氨酸的住所并形成琥珀酸酯和CO2。原生动物具有PHD，但缺乏HIF，因此对氧气有所不同。在社交变形虫dictyostelium中，我们发现它的博士学位修饰了SKP1中的脯氨酸，这是SCF（SKP1/Cullin1/f-box蛋白/RBX1）多泛素连接酶复合物的组成部分。 SKP1羟基羟基化不会影响其稳定性，但可以通过一系列糖基转移酶进行修饰。反过来，糖基化稳定了SKP1与F-box蛋白的结合对于组装SCF多偶联蛋白连接酶必不可少的。基因组分析和生化测定表明，这种SKP1修饰途径在原生动物寄生虫弓形虫弓形虫中保守，但在其人类宿主中不保守。此外，托克斯玛博士学位的丧失或两个糖基转移酶中的任何一个导致寄生虫复制的缺陷在低但物理氧水平下以及其SKP1相互作用的变化。由于其医学重要性，我们将专注于弓形虫，并购买三个具体目标：i）SKP1是弓形虫博士学位的关键底物，新型糖基化对Phya激活的贡献是什么？ ii）SKP1和多偶联蛋白连接酶的组装如何受羟基化影响？ iii）如何调节寄生虫的PHD，这些数据如何用于开发寄生虫特异性的药物抑制剂？ 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.