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学和人类疾病的机制，并确定新的治疗策略。蛋白质泛素化的调节很大程度上是通过 SCF 泛素 E3 连接酶 (E3) 实现的，E3 连接酶通过不同的、可互换的 F-box 亚基结合底物，从而控制底物丰度和/或活性。许多 F-box 蛋白的一个显着特征是它们通常具有模块化结构，并且每种蛋白都可以将特定的靶蛋白子集招募到 SCF E3 进行泛素化。例如，F-box 蛋白 Fbx4 识别原癌蛋白细胞周期蛋白 D1 和端粒 DNA 结合蛋白 TRF1，而 Fbxo31 在 DNA 损伤后介导细胞周期蛋白 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 的目标特异性和多样性是由结构决定的 以及 F-box 亚基与其靶标的相互作用特性。在这个项目中，我们的目标是描述 Fbx4 和 Fbxo31 对各自的底物细胞周期蛋白 D1 和 TRF1 的选择性背后的结构机制和分子逻辑。这些知识将有助于设计以特定且有益于治疗的方式与 SCF E3 相互作用的新药物。具体目标是： 1. 确定Fbx4-TRF1相互作用和Fbx4二聚化调节的分子机制。我们将a）详细描述结构特性 Skp1-Fbx4-TRF1 复合物并识别参与 E3-底物相互作用的特定氨基酸侧链； b) 生成一系列合理设计的Fbx4突变体并评估它们对Fbx4-TRF1相互作用、Fbx4二聚化和TRF1降解的影响； c) 确定突变的结构和功能效应之间的相关性。 2. 确定Fbx4与¿B-晶状体蛋白和细胞周期蛋白D1的相互作用和配对机制。我们将 a) 使用 NMR 和其他生物物理方法确定 Skp1-Fbx4-¿B-晶状体蛋白-细胞周期蛋白 D1 磷酸肽复合物的晶体结构，并定义 Fbx4 底物对细胞周期蛋白 D1 相对于 TRF1 的选择性的结构原理； b) 对 Fbx4/¿B-晶状体蛋白/细胞周期蛋白 D1 相互作用进行结构-活性研究，并使用完全重建的玻璃周期蛋白 D1 泛素化系统和基于细胞的测定确定 ¿B-晶状体蛋白在细胞周期蛋白 D1 泛素化中的作用及其调节。 3. 阐明游离的和与细胞周期蛋白D1结合的Fbxo31的结构特性。我们将 a) 表征 Fbxo31 的详细结构特性并确定 Fbxo31-cyclin D1 相互作用的决定因素； b)确定Fbxo31-细胞周期蛋白D1复合物中相互作用的界面残基在细胞周期蛋白D1的结合和降解中的作用； c) 确定 Fbxo31/Fbx4-细胞周期蛋白 D1 相互作用针对特定赖氨酸残基进行泛素化的程度，从而调节细胞周期蛋白 D1 的蛋白酶体降解。

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.