Structural studies of substrate recognition and specificity by the SCF ubiquitin

描述（由申请人提供）：迫切需要了解蛋白质泛素化控制真核生物和人类疾病的机制，并确定新的治疗策略。蛋白质泛素化的调控主要是通过SCF泛素E3连接酶（E3s）实现的，它通过不同的、可互换的F-box亚基结合底物，从而控制底物的丰度和/或活性。许多F-box蛋白的一个显著特征是它们通常具有模块化结构，并且每个蛋白可以招募特定的靶蛋白子集到SCF E3进行泛素化。例如，F-box蛋白Fbx4识别原癌蛋白cyclin D1和端粒DNA结合蛋白TRF1，而Fbxo31介导DNA损伤后的cyclin D1降解。该研究计划的广泛、长期目标是了解单个F-box亚基如何识别和结合氨基酸序列和三级结构不同的底物蛋白，以及多个F-box蛋白如何作用于单个靶标。我们的首要假设是，SCF e3的靶特异性和多样性是由结构决定的

DESCRIPTION (provided by applicant): There is an urgent need to understand the mechanisms by which protein ubiquitination controls eukaryotic biology and human diseases and to identify novel therapeutic strategies. The regulation of protein ubiquitination is largely achieved by SCF ubiquitin E3 ligases (E3s) that bind substrates through different, interchangeable F-box subunits, thereby controlling substrate abundance and/or activity. A remarkable feature of many F-box proteins is that they often have a modular structure and that each protein can recruit a specific subset of target proteins to an SCF E3 for ubiquitination. For example, the F-box protein Fbx4 recognizes the proto-oncoprotein cyclin D1 and the telomeric DNA-binding protein TRF1, while Fbxo31 mediates cyclin D1 degradation after DNA damage. The broad, long-term objective of this research plan is to understand how individual F-box subunits can recognize and bind substrate proteins that vary in amino-acid sequence and tertiary structure, and how multiple F-box proteins can act on a single target. Our overarching hypothesis is that the target specificity and diversity of SCF E3s are determined by the structural and interaction properties of F-box subunits with their targets. In this project we aim to delineat the structural mechanisms and molecular logic underlying the selectivity of Fbx4 and Fbxo31 towards their respective substrates cyclin D1 and TRF1. This knowledge will facilitate efforts to design new agents that interact with SCF E3s in specific and therapeutically beneficial manners. The Specific Aims are: 1. To determine the molecular mechanism of the Fbx4-TRF1 interaction and regulation of Fbx4 dimerization. We will a) characterize in detail the structural properties of the Skp1-Fbx4-TRF1 complex and identify specific amino-acid side chains involved in the E3-substrate interaction; b) generate a series of rationally designed Fbx4 mutants and evaluate their effects on the Fbx4-TRF1 interaction, Fbx4 dimerization and TRF1 degradation; and c) determine correlations between the structural and functional effects of the mutations. 2. To determine the interaction and partnering mechanism of Fbx4 with ¿B-crystallin and cyclin D1. We will a) determine the crystal structure of the Skp1-Fbx4-¿B-crystallin-cyclin D1 phosphopeptide complex and define the structural principles underlying the Fbx4 substrate selectivity for cyclin D1 over TRF1 using NMR and other biophysical methods; b) perform structure-activity studies of the Fbx4/¿B-crystallin/cyclin D1 interactions and determine the role of ¿B-crystallin in cyclin D1 ubiquitination and its regulation using a fully reconstituted in vitr cyclin D1 ubiquitination system and cell-based assays. 3. To elucidate the structural properties of Fbxo31 both free and bound to cyclin D1. We will a) characterize the detailed structural properties of Fbxo31 and identify the determinants of the Fbxo31-cyclin D1 interaction; b) determine the role of interacting interfacial residues in the Fbxo31-cyclin D1 complex in the binding and degradation of cyclin D1; and c) determine the extent to which the Fbxo31/Fbx4-cyclin D1 interaction targets specific lysine residue(s) for ubiquitination and thus regulates the proteasomal degradation of cyclin D1.