Structural Studies of cullin-based ubiquitin ligases

描述（由申请人提供）：cullin-RING连接酶（CRLs）是真核细胞中最大的多亚基E3泛素连接酶复合物超家族。CRL1-5复合物由cullin-RING催化支架、适配器和可互换的底物受体亚基组成，以模块化形式组织，通过促进大量蛋白质底物的泛素化来调节多种细胞过程。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。本项目主要研究几种CLR复合物在细胞生长、DNA复制、DNA修复、氧化应激反应和激素感知等方面的结构功能调控。在Aim1中，将使用结构生物学方法来研究新发现的clr家族（CRL4 E3复合物）的底物受体亚基（DCAFs）如何被招募到泛素连接酶机制中。本研究旨在解决目前关于人类基因组中所有WD40-repeat蛋白中CRL4底物受体身份的争议。在Aim 2中，将采用生物化学和结构生物学相结合的方法来分析CRL4-Det1 E3复合物中一个新的功能性泛素E3变体蛋白的结构和功能作用，该蛋白泛素化bZIP家族的转录因子，如c-Jun。在Aim 3中，将确定全长Keap1二聚体的晶体结构，以阐明btb结构域蛋白如何作为氧化应激传感器以及CRL3 E3复合物的二聚物底物受体亚基。在Aim 4中，将研究植物COI1 F-box蛋白作为茉莉酸植物激素受体的结构机制。结合最近发表的另一种植物F-box蛋白TIR1如何识别植物激素生长素的研究结果，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 E3s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与新发现的酶复合物家族（称为cullin-RING泛素连接酶）功能异常有关。该建议旨在研究这些酶机制如何在人类和其他生物体中组装和功能的结构基础，以便可以衍生出新的策略来开发针对这些疾病相关酶的新药。

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.