Structural Studies of cullin-based ubiquitin ligases

描述（由申请人提供）：cullin-RING连接酶（CRL）代表真核细胞中最大的多亚基E3泛素连接酶复合物超家族。CRL 1 -5复合物以模块化形式与cullin-RING催化支架、适配器和可互换的底物-受体亚基一起组织，通过促进大量蛋白质底物的泛素化来调节各种各样的细胞过程。CRL的失调和功能障碍与肿瘤发生、癌症和多种其他人类疾病有关。该项目的重点是几个的结构和功能的研究在细胞生长，DNA复制，DNA修复，氧化应激反应和激素的感知调节的复合物。在Aim 1中，将使用结构生物学方法来研究新鉴定的CLR家族的底物受体亚基（DCAF），即CRL 4 E3复合物，如何被招募到泛素连接酶机制中。这项研究的目的是解决目前的争议中的所有WD 40重复蛋白在人类基因组中的CRL 4底物受体的身份。在目标2中，将采用生物化学和结构生物学方法的组合来分析新型功能性泛素E3变体蛋白在CRL 4-Det 1 E3复合物中的结构和功能作用，该复合物泛素化转录因子的bZIP家族，如c-Jun。将确定全长Keap 1二聚体的晶体结构，以阐明BTB-结构域蛋白作为氧化应激传感器以及CRL 3 E3复合物的二聚底物受体亚基发挥功能。在目的4中，将研究植物COI 1 F-box蛋白作为茉莉酸植物激素受体发挥功能的结构机制。结合最近发表的另一种植物F-box蛋白TIR 1如何识别植物激素生长素的结果，本研究旨在建立这些天然存在的小分子如何作为激动剂调节CRL E3的结构原理，并为正在进行和未来开发针对人类CRL的抗癌药物奠定基础。肿瘤发生、癌症和包括神经系统疾病和病毒感染在内的多种其他人类疾病都与一种新发现的酶复合物家族的异常功能有关，该酶复合物家族被称为cullin-RING泛素连接酶。该提案旨在研究这些酶机制如何在人类和其他生物体中组装和发挥作用的结构基础，以便可以导出新的策略来开发针对这种疾病相关酶的新药。

DESCRIPTION (provided by applicant): The cullin-RING ligases (CRLs) represent the largest super-family of multi-subunit E3 ubiquitin ligase complexes in eukaryotic cells. Organized in a modular form with a cullin-RING catalytic scaffold, an adaptor, and an interchangeable substrate-receptor subunit, the CRL1-5 complexes regulate a wide variety of cellular processes by promoting ubiquitination of a large number of protein substrates. Dysregulation and malfunction of CRLs are implicated in tumorigenesis, cancers, and a variety of other human diseases. This project focuses on structure-function studies of several CLR complexes in the regulation of cell growth, DNA replication, DNA repair, oxidative stress response, and hormone perception. In Aim1, a structural biology approach will be used to investigate how the substrate receptor subunits (DCAFs) of a newly identified family of CLRs, the CRL4 E3 complexes, are recruited to the ubiquitin ligase machinery. This study is aimed at resolving the current controversy over the identity of the CRL4 substrate receptors among all WD40-repeat proteins in the human genome. In Aim 2, a combination of biochemical and structural biology approaches will be employed to analyze the structural and functional roles of a novel functional ubiquitin E3 variant protein in the CRL4-Det1 E3 complex, which ubiquitinates the bZIP family of transcription factors such as c-Jun. In Aim 3, the crystal structure of the full length Keap1 dimer will be determined to elucidate how the BTB-domain protein functions as an oxidative stress sensor as well as a dimeric substrate receptor subunit of the CRL3 E3 complex. In Aim 4, the structural mechanism by which the plant COI1 F-box protein functions as the jasmonate plant hormone receptor will be investigated. Together with the recently published results of how another plant F-box protein TIR1 recognizes the plant hormone auxin, this study is aimed at establishing the structural principles of how these naturally occurring small molecules regulate the CRL E3s as agonists and laying the foundation of ongoing and future efforts of developing anti-cancer drugs targeting human CRLs. PUBLIC HEALTH RELEVANCE Tumorigenesis, cancer, and a variety of other human diseases including neurological disorders and viral infections are associated with the abnormal functions of a newly identified family of enzymatic complexes, known as the cullin-RING ubiquitin ligases. This proposal is aimed at studying the structural basis of how these enzyme machineries assemble and function in human and other organisms so that new strategies can be derived to develop novel drugs targeting this disease-associated enzymes.