Investigation of Cdc34 Molecular Recognition by its Enzyme Partners, Uba1 and SCF Ligases.

通过其酶合作伙伴、Uba1 和 SCF 连接酶对 Cdc34 分子识别进行研究。

• 批准号: 9327292
• 负责人: Katelyn Williams
• 金额: $ 4.6万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9327292
• 关键词: Active Sites; Address; Antibodies; Binding; Biochemical; Biochemical Genetics; Biological Assay; Cancer cell line; Cell Cycle; Cell Cycle Progression; Cell Cycle Proteins; Charge; Complex; Crystallization; Cullin Proteins; Cyclin-Dependent Kinase Inhibitor; Data; Development; Electrostatics; Enzymes; F Box Domain; Family; Genetic; Genetic Techniques; Goals; Human; In Vitro; Investigation; Knowledge; Laboratories; Libraries; Ligase; Literature; Malignant Neoplasms; Mass Spectrum Analysis; Modification; Molecular; Mutate; Mutation; Nature; Pattern; Phosphorylation; Phosphorylation Site; RBX1 gene; Regulation; Research; Research Personnel; Role; S Phase; Saccharomyces cerevisiae Proteins; Serine; Site; Site-Directed Mutagenesis; Structure; Surface; Techniques; Therapeutic; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; X-Ray Crystallography; base; cancer cell; cell growth; crosslink; in vivo; inhibitor/antagonist; interest; loss of function; molecular recognition; multicatalytic endopeptidase complex; novel; novel therapeutic intervention; screening; small molecule; small molecule inhibitor; structural biology; targeted treatment; ubiquitin-protein ligase; yeast genetics

PROJECT SUMMARY Cdc34 is a key regulator in cell cycle progression at the G1 to S phase checkpoint1-3. It acts as an E2 ubiquitin conjugating enzyme that is first charged with ubiquitin by E1 enzyme, Uba1, and then functions with the SCF family of E3 enzymes to modify other cell cycle proteins with ubiquitin for targeted degradation by the proteasome4,5. In years since its discovery, Cdc34 dysregulation has been implicated in several types of cancer6-8. Many downstream targets of Cdc34 are cyclin-dependent kinase inhibitors such as p27 and p40 that are critically important for cancer progression9-14. Together, these findings have led to investigations of Cdc34 as a target for cancer therapeutics but a more directed approach, such as specifically targeting Cdc34 interactions with its enzyme partners is needed. However, a detailed structural understanding for molecular recognition of Cdc34 by its E1 partner, Uba1, and E3 ligase partners is not available. Further, regulation of Cdc34 is not completely understood. Previous studies have shown positive regulation of Cdc34 by phosphorylation; however, we have exciting preliminary data that implicates phosphorylation of residue serine 10 as a negative regulator of Cdc34 activity and this is supported in the literature by a phosphoproteome mass spectrometry screen that identified this residue in vivo15-18. Further, based on structural predictions we believe Ser10 residues on the surface of Cdc34 that interacts with both E1 and E3 enzymes. Thus, we aim to identify key residues in the molecular recognition of Cdc34 by its enzyme partners and hypothesize that phosphorylation of Ser10 will negatively regulate this molecular recognition through steric clash and electrostatic repulsions. Our hypothesis will be addressed through the following Specific Aims. Aim 1 will identify key residues for Cdc34 interaction with E1 and E3 enzyme partners. We will use x-ray crystallography in concert with biochemical and genetic techniques to accomplish this aim. Aim 2 will elucidate the regulatory effects of Cdc34 phosphorylation at residue Ser10. We will confirm the phosphorylation of Ser10 in vivo and examine the effects of S10D phosphomimetic mutation on molecular recognition of Cdc34 by its enzyme partners in vitro with biochemical assays and in vivo using yeast genetics.

项目摘要 CDC34是在G1到S相检查点1-3处细胞周期进程中的关键调节剂。它充当E2泛素 E1酶，UBA1首先用泛素收取泛素的共轭酶，然后用SCF起作用 E3酶家族用泛素修饰其他细胞周期蛋白，以靶向降解 蛋白酶组4,5。自发现以来的几年中，CDC34失调涉及几种类型的 癌症6-8。 Cdc34的许多下游靶标是细胞周期蛋白依赖性激酶抑制剂，例如P27和P40 对于癌症进展至关重要9-14。这些发现一起导致了对CDC34的研究 作为癌症治疗剂的目标，但是一种更有指导的方法，例如专门针对CDC34 需要与其酶合作伙伴的互动。但是，对分子的详细结构理解 无法获得其E1合作伙伴UBA1和E3连接酶合作伙伴对CDC34的认可。此外，调节 CDC34尚未完全理解。先前的研究表明，通过 磷酸化；但是，我们有令人兴奋的初步数据，这意味着残基丝氨酸的磷酸化 10作为CDC34活性的负调节剂，文献中的磷酸蛋白酶质量支持这一点 光谱屏幕在Vivo15-18中鉴定出该残基。此外，根据我们相信的结构预测 与E1和E3酶相互作用的CDC34表面上的Ser10残基。因此，我们旨在确定 其酶伙伴对Cdc34分子识别的关键残基，并假设 Ser10的磷酸化将通过空间冲突和 静电排斥。 我们的假设将通过以下特定目标解决。 AIM 1将确定关键残留物 CDC34与E1和E3酶合作伙伴的相互作用。我们将与 实现这一目标的生化和遗传技术。 AIM 2将阐明CDC34的调节作用 残基Ser10处的磷酸化。我们将确认体内Ser10的磷酸化并检查效果 S10D磷酸化突变对Cdc34的分子识别的酶伴侣的体外酶伴侣 使用酵母遗传学的生化测定和体内。