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

基本信息

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
项目类别：
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

项目摘要

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生命得以存在的基石特征之一。蛋白质的循环磷酸化和去磷酸化几十年来一直被认为可以调节酶，并且蛋白激酶和磷酸酶的药理抑制已成为生物技术行业的目标治疗各种人类疾病的时间几乎相同。这一策略的一个主要缺点是酶调节蛋白磷酸化可能占人类蛋白质组的 5%，因此绝大多数导致人类疾病的异常蛋白质仍然无法用药物治疗。最近，药物已被发明了诱导致病蛋白质和称为泛素连接酶的酶之间的接近促进有问题的蛋白质的破坏。事实上，这种新的药物模式每人每年可带来 10 亿美元的收入。今年行业推动使用泛素连接酶来治疗各种人类疾病，包括乳腺癌和前列腺疾病癌症以及多发性骨髓瘤等。大多数这些努力都是利用一系列酶称为 Cullin-RING 连接酶 (CRL)。 CRL 拥有约 200 名人类成员，共同控制着细胞中大约 20% 的泛素依赖性蛋白质降解。因此，对这些如何酶的调控引起了科学界广大读者的极大兴趣。类似于根据蛋白质磷酸化的范式，CRL 的控制被认为主要是通过他们用一种叫做 NEDD8 的蛋白质进行可逆修饰。尽管众所周知，人类 CRL 会与至少 7 种额外的酶，我们称之为泛素携带酶 (UCE)，有助于促进 CRL- 由于依赖于蛋白质底物降解，UCE 也被认为对 CRL 产生混杂的作用，将排除 CRL-UCE 相互作用水平上的 CRL 监管。然而，CRL-UCE 特异性很强活动 CRL 的最新结构暗示了这一点。初步结果表明 CRL-UCE 特异性赋予这些配对以极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底物，可能提供额外的一层控制 CRL 功能，超越 neddylation。考虑到这些观察结果，本申请旨在测试假设 UCE 通常对 CRL-底物复合物表现出特异性，并且生物这些特定的 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地编码多聚泛素链以促进包括蛋白质在内的结果退化或本地化。拟议的研究将探索 CRL 生物学的一个全新领域，即 UCE 对 CRL 的特异性，利用蛋白质组学和细胞生物学检测来补充强大的定量分析动力学平台。这些研究将阐明 CRL 活动如何受到监管并支持新的药物模式利用 CRL 的力量来降解致病蛋白质。