Structural studies of substrate recognition and specificity by the SCF ubiquitin

SCF 泛素底物识别和特异性的结构研究

• 批准号: 8304730
• 负责人: Bing Hao
• 金额: $ 29.12万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2017-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8304730
• 关键词: Adaptor Signaling Protein; Address; Aging; Amino Acid Sequence; Amino Acids; Binding; Biochemical; Biological Assay; Biological Models; Biological Sciences; Biology; C-terminal; Calorimetry; Cell Cycle; Cells; Complex; Crystallins; Cues; Cyclin D1; DNA Damage; DNA-Binding Proteins; Data; Development; Dimerization; Disease; Dissection; Enzymatic Biochemistry; Event; F Box Domain; F-Box Motifs; F-Box Proteins; Goals; Human; In Vitro; Individual; Knowledge; Length; Ligase; Logic; Lysine; Malignant Neoplasms; Mediating; Methodology; Methods; Modification; Molecular; Mutagenesis; Mutation; N-terminal; New Agents; Oncogene Proteins; Pathway interactions; Pharmaceutical Preparations; Phosphopeptides; Phosphorylation; Play; Process; Property; Protein Biochemistry; Proteins; Recruitment Activity; Regulation; Regulatory Pathway; Research; Research Project Grants; Resolution; Role; Series; Side; Signal Pathway; Specificity; Structure; Substrate Interaction; Substrate Specificity; System; TERF1 gene; Telomeric Repeat Binding Protein 1; Testing; Time; Tumor Suppressor Proteins; Ubiquitin; Ubiquitination; Virus Diseases; base; design; human disease; interfacial; multicatalytic endopeptidase complex; mutant; nervous system disorder; novel; novel therapeutics; protein B; protein complex; protein degradation; protein protein interaction; protein structure; reconstitution; research study; response; structural biology; telomere; ubiquitin ligase; ubiquitin-protein ligase

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. PUBLIC HEALTH RELEVANCE: The SCF ubiquitin ligases are key components of the ubiquitin-proteasome pathways and dysregulation of these multi-component protein complexes has been associated with multiple human disorders including cancer, neurological diseases, and viral infection. This research project will define the structural and regulatory features of specific interactions between the F-box subunits of the SCF ligases and their substrates. This research will derive fundamental principles of life science at molecular level and have a direct impact in biomedicine by accelerating the discovery and development of novel therapeutic drugs.

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主要通过SCF泛素E3连接酶（E3）实现，所述SCF泛素E3连接酶通过不同的可互换的F盒亚基结合底物，从而控制底物丰度和/或活性。许多F-box蛋白的一个显著特征是它们通常具有模块化结构，并且每个蛋白质可以将靶蛋白的特定子集募集到SCF E3用于泛素化。例如，F-box蛋白Fbx 4识别原癌蛋白细胞周期蛋白D1和端粒DNA结合蛋白TRF 1，而Fbxo 31介导DNA损伤后细胞周期蛋白D1的降解。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。我们的总体假设是SCF E3的靶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性质。在这个项目中，我们的目标是描绘的结构机制和分子逻辑的选择性Fbx 4和Fbxo 31对各自的底物细胞周期蛋白D1和TRF 1。这些知识将有助于设计以特异性和治疗有益的方式与SCF E3相互作用的新药物。具体目标是：1。确定Fbx 4-TRF 1相互作用的分子机制和Fbx 4二聚化的调控。我们将a）详细描述的结构特性， Skp 1-Fbx 4-TRF 1复合物，并鉴定参与E3-底物相互作用的特定氨基酸侧链; B）产生一系列合理设计的Fbx 4突变体，并评估它们对Fbx 4-TRF 1相互作用、Fbx 4二聚化和TRF 1降解的影响;和c）确定突变的结构和功能影响之间的相关性。2.确定Fbx 4与<$B-crystallin和cyclin D1的相互作用和伙伴机制。我们将a）确定Skp 1-Fbx 4-<$B-晶状体蛋白-细胞周期蛋白D1磷酸肽复合物的晶体结构，并使用NMR和其他生物物理方法定义Fbx 4底物对细胞周期蛋白D1的选择性超过TRF 1的结构原理; b）进行Fbx 4/<$B-晶状体蛋白/细胞周期蛋白D1相互作用的结构-活性研究，并确定<$B-晶状体蛋白/细胞周期蛋白D1相互作用的作用。B-晶体蛋白在细胞周期蛋白D1泛素化及其调控中的作用--使用完全重建的体外细胞周期蛋白D1泛素化系统和基于细胞的测定。3.为了阐明Fbxo 31的结构特性，无论是游离的还是与细胞周期蛋白D1结合的。我们将a）表征Fbxo 31的详细结构特性并鉴定Fbxo 31-细胞周期蛋白D1相互作用的决定因素; B）确定Fbxo 31-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;和c）确定Fbxo 31/Fbx 4-细胞周期蛋白D1相互作用靶向特定赖氨酸残基的程度，用于泛素化，从而调节细胞周期蛋白D1的蛋白酶体降解。 公共卫生相关性：SCF泛素连接酶是泛素-蛋白酶体途径的关键组分，并且这些多组分蛋白质复合物的失调与多种人类疾病（包括癌症、神经系统疾病和病毒感染）相关。本研究计划将定义SCF连接酶的F-box亚基与其底物之间特异性相互作用的结构和调控特征。这项研究将在分子水平上得出生命科学的基本原理，并通过加速新型治疗药物的发现和开发对生物医学产生直接影响。