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: 10180287
• 负责人: Gary L. Kleiger
• 金额: $ 46万
• 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/10180287
• 关键词: Ablation; Affect; Architecture; Area; Biological; Biological Assay; Biology; Biotechnology; Cell Line; Cells; Clinic; Clinical Trials; Code; Collaborations; Communities; Complement; Complex; Cullin Proteins; Development; Disease; 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; Pharmacology; Phosphorylation; Phosphotransferases; Physiological; Polyubiquitin; Protac; 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/antagonist; interest; malignant breast neoplasm; member; multicatalytic endopeptidase complex; novel; novel therapeutics; p38 Mitogen Activated Protein Kinase; protein degradation; protein protein interaction; receptor; reconstitution; recruit; scaffold; small molecule; therapy development; 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基石的特征。蛋白质周期 数十年来，哲学和去磷酸化已被赞赏，以调节酶，并且 蛋白激酶和磷酸酶的药理抑制作用为生物技术行业提供了意图 几乎可以治疗各种人类疾病。该策略的一个主要缺点是酶 调节蛋白质磷酸化大概是人类蛋白质组的5％，因此绝大多数 负责人类疾病的异常蛋白质仍然不可能。最近，药物已经 发明了诱导引起疾病的蛋白质与称为泛素连接酶的酶之间的邻近性 促进有问题蛋白质的破坏。确实，这种新药物的方式正在喂养10亿美元 年度行业推动使用泛素连接酶治疗包括乳房和前列腺在内的各种人类疾病 癌症和多发性骨髓瘤等等。这些努力中的大多数一直在利用一个酶家族 称为Cullin环连接酶（CRLS）。 CRL共同控制了人类中约200名成员 细胞中约20％的泛素依赖性蛋白质降解。因此，对这些方式的赞赏 对科学界广泛的受众群体的兴趣非常重要。类似于 蛋白质磷酸化的范式，CRL的控制被认为主要通过 它们使用称为NEDD8的蛋白质可逆修饰。虽然人类CRL众所周知与AT合作 至少7种额外的酶，我们称为泛素载体酶（UCES），有助于促进CRL- 依赖性蛋白质底物降解，也认为uces对CRL进行了混杂的作用 将排除CRL相互作用水平的CRL调节。但是，CRL的特异性很强 被活动CRL的最新结构所暗示。这里的初步结果表明，特异性 赋予这些对具有泛素转移的快速速率，并具有产生CRL的能力 用独特的多泛素链体系结构修饰的底物，有可能提供额外的层 控制CRL功能超出Neddylation。考虑到这些观察，本申请试图测试 UCE通常显示出对Crl-Substrate复合物的特异性的假设，并且生物学 这些特定CRL-COLCOL对的目的是提高泛素从UCES到CRL的转移速率 底物以及唯一地编码多泛素链以促进包括蛋白质的结果 退化或本地化。拟议的研究将探讨CRL生物学的一个全新的领域，即 UCES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。这些研究将阐明如何调节CRL活动并支持新药物形态 这利用了CRL的力量降解引起疾病的蛋白质。