How ubiquitin-carrying enzymes contribute to ubiquitin ligase specificity

泛素携带酶如何促进泛素连接酶特异性

• 批准号: 10583496
• 负责人: Gary L. Kleiger
• 金额: $ 35.32万
• 依托单位: UNIVERSITY OF NEVADA LAS VEGAS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10583496
• 关键词: Ablation; Affect; Architecture; Area; Binding; Biological; Biological Assay; Biology; Biotechnology; Cell Line; Cells; Clinic; Clinical Trials; Code; Collaborations; Communities; Complement; Complex; Cullin Proteins; Development; Disease; Endowment; Enzyme Interaction; Enzymes; Family; Grant; Human; In Vitro; Individual; Industry; Investigation; Kinetics; Knock-out; Life; Ligase; Literature; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Measures; Mediating; Modality; Modeling; Modification; Multiple Myeloma; Names; Outcome; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Polyubiquitin; Protac; Protein Dephosphorylation; Protein Inhibition; Protein Kinase; Protein phosphatase; Proteins; Proteome; Proteomics; Receptor Protein-Tyrosine Kinases; Regulation; Research; Resistance; Resolution; Role; Specificity; Structure; System; Techniques; Testing; Thalidomide; Therapeutic; Ubiquitin; Work; cancer therapy; enzyme activity; expectation; feeding; human disease; inhibitor; interest; invention; malignant breast neoplasm; member; multicatalytic endopeptidase complex; novel; novel therapeutics; p38 Mitogen Activated Protein Kinase; pharmacologic; protein degradation; protein protein interaction; receptor; reconstitution; recruit; scaffold; small molecule; ubiquitin ligase

The reversible control of enzyme activity is one of the cornerstone features enabling life. Cycles of protein phosophorylation and de-phosphorylation have been appreciated for decades to regulate enzymes, and the pharmacological inhibition of protein kinases and phosphatases has furnished a biotech industry intent on treating various human diseases for nearly as long. One major drawback to this strategy is that the enzymes that regulate protein phosphorylation represent perhaps 5 % of the human proteome, such that the vast majority of aberrant proteins responsible for human disease have remained undruggable. More recently, drugs have been invented that induce proximity between a disease-causing protein and an enzyme called a ubiquitin ligase that promotes the destruction of the problematic protein. Indeed, this new drug modality is feeding a billion dollar per year industry push to employ ubiquitin ligases to treat various human diseases including breast and prostate cancers as well as multiple myeloma, to name a few. Most of these efforts have been utilizing a family of enzymes called the Cullin-RING ligases (CRLs). With some 200 members in humans, the CRLs collectively control approximately 20 % of ubiquitin-dependent protein degradation in cells. As such, an appreciation for how these enzymes are regulated is of considerable interest to a wide audience from the scientific community. Similar to the paradigm of protein phosphorylation, the control of CRLs is believed to be determined predominantly through their reversible modification with a protein called NEDD8. And while human CRLs are known to partner with at least 7 additional enzymes, which we refer to as ubiquitin-carrying enzymes (UCEs), that help promote CRL- dependent protein substrate degradation, it also was believed that UCEs act promiscuously towards CRLs which would preclude CRL regulation at the level of CRL-UCE interaction. However, CRL-UCE specificity is strongly implied by the recent structure of an active CRL. Preliminary results here indicate that CRL-UCE specificity endows these pairs with exceptionally rapid rates of ubiquitin transfer and with the capability of producing CRL substrates modified with unique poly-ubiquitin chain architectures, potentially providing an additional layer of control of CRL function beyond neddylation. In consideration of these observations, this application seeks to test the hypothesis that UCEs generally display specificity for CRL-substrate complexes, and that the biological purpose of these specific CRL-UCE pairs is to both enhance the rates of ubiquitin transfer from UCEs to CRL substrates as well as to uniquely code the poly-ubiquitin chain to promote outcomes including protein degradation or localization. The proposed studies will explore an entirely novel area of CRL biology, the specificity of UCEs for CRLs, utilizing proteomic and cell biological assays to complement a powerful, quantitative kinetics platform. These studies will illuminate how CRL activities are regulated and support a new drug modality that harnesses the power of CRLs to degrade disease-causing proteins.

酶活性的可逆控制是使生命得以存在的基石特征之一。蛋白质循环 几十年来，磷酸化和去磷酸化已经被理解为调节酶， 蛋白激酶和磷酸酶的药理学抑制提供了生物技术工业， 治疗各种人类疾病的时间几乎一样长这种策略的一个主要缺点是， 调节蛋白质磷酸化的蛋白质可能占人类蛋白质组的5%，因此， 导致人类疾病的异常蛋白质仍然是不可治疗的。最近，药物已经 发明了一种诱导致病蛋白和一种叫做泛素连接酶的酶之间接近的方法， 促进有问题的蛋白质的破坏。事实上，这种新的药物模式正在为每个人提供10亿美元的资金。 2004年，业界推动利用泛素连接酶治疗各种人类疾病，包括乳腺癌和前列腺疾病 癌症以及多发性骨髓瘤，仅举几例。这些努力中的大多数都是利用酶家族 称为Cullin-RING连接酶（CRL）。在人类中有大约200个成员， 细胞中大约20%的泛素依赖性蛋白降解。因此，欣赏这些 酶的调节是科学界广泛关注的问题。类似于 作为蛋白质磷酸化的范式，CRL的控制被认为主要通过以下方式来确定： 它们被一种叫做NEDD 8的蛋白质可逆地修饰。虽然人类CRL已知与 至少7种额外的酶，我们称之为泛素携带酶（UCEs），有助于促进CRL- 依赖于蛋白质底物降解，还认为UCE对CRL作用混杂， 将在CRL-UCE交互水平上排除CRL监管。然而，CRL-UCE特异性强， 由活动CRL的最新结构暗示。初步结果表明，CRL-UCE特异性 赋予这些对非常快的泛素转移速率和产生CRL的能力， 用独特的多聚泛素链结构修饰的基底，潜在地提供了一个额外的 控制CRL功能，超越neddylation。考虑到这些观察结果，本申请寻求测试 假设UCE通常显示对CRL-底物复合物的特异性， 这些特定CRL-UCE对的目的是提高泛素从UCE转移到CRL的速率 底物以及独特编码多聚泛蛋白链以促进包括蛋白质在内的结果 降解或定位。拟议的研究将探索CRL生物学的一个全新领域， UCE对CRL的特异性，利用蛋白质组学和细胞生物学测定来补充强大的定量 动力学平台这些研究将阐明CRL活动是如何调节的，并支持一种新的药物模式 利用CRL的力量来降解致病蛋白质。