Structural Studies of cullin-based ubiquitin ligases

描述(申请人提供)：剔除环连接酶(CRL)代表真核细胞中最大的多亚基E3泛素连接酶复合体超家族。CRL1-5复合体通过促进大量蛋白质底物的泛素化，以模块化的形式组织起来，由一个剔除环催化支架、一个接头和一个可互换的底物-受体亚单位组成，调节着广泛的细胞过程。CRL的调节失调和功能障碍与肿瘤的发生、癌症和其他多种人类疾病有关。本项目重点研究了几种CLR复合体在调节细胞生长、DNA复制、DNA修复、氧化应激反应和激素感知方面的结构和功能。在AIM1中，将使用结构生物学的方法来研究新发现的CLR家族CRL4 E3复合体的底物受体亚单位(DCAF)是如何被招募到泛素连接酶机制中的。这项研究旨在解决目前关于人类基因组中所有WD40重复蛋白中CRL4底物受体的身份的争议。在目标2中，将结合生化和结构生物学的方法来分析一个新的功能泛素E3变体蛋白在CRL4-DET1 E3复合体中的结构和功能作用，它泛化bZIP家族的转录因子，如c-Jun.在目标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感受器以及CRL3 E3复合体的二聚体底物受体亚单位发挥功能。在目标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结果，本研究旨在建立这些自然产生的小分子如何作为激动剂调节CRL E3的结构原理，并为目前和未来开发针对人CRL的抗癌药物奠定基础。与公共卫生相关的肿瘤发生、癌症和包括神经系统疾病和病毒感染在内的各种其他人类疾病与一种新发现的酶复合体家族的异常功能有关，该家族被称为cullin-ring泛素连接酶。这项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发挥作用的结构基础，从而可以得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.