Dissecting ubiquitin pathway selectivity with Ub-isopeptide microarrays

使用 Ub 异肽微阵列剖析泛素通路选择性

• 批准号: 7994252
• 负责人: James Strickler
• 金额: $ 27.82万
• 依托单位: LIFESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7994252
• 关键词: Active Sites; Alzheimer' s Disease; Amines; Amino Acids; Arthritis; Binding; Biological Assay; C-terminal; Caspase; Cathepsins; Cell physiology; Cells; Cleaved cell; Cysteine; Data; Deubiquitinating Enzyme; Deubiquitination; Development; Disease; Drug Design; Enzymes; Etiology; Event; Exhibits; Genes; Glass; Glycine; Goals; Grant; Homeostasis; Human; Human Genome; Human Pathology; Hydrolysis; In Vitro; Individual; Inflammation; Inflammatory; Label; Libraries; Ligase; Link; Lysine; Maintenance; Malignant Neoplasms; Measures; Mediating; Membrane; Methodology; Methods; Modification; Nature; Neurodegenerative Disorders; Organism; Parkinson Disease; Pathway interactions; Pattern; Peptide Hydrolases; Peptide Library; Peptides; Phase; Phosphorylation; Play; Process; Protein Dephosphorylation; Proteins; Proteome; Regulation; Research; Role; Series; Side; Site; Specificity; Structure; Substrate Specificity; System; Technology; Testing; Therapeutic Agents; Transcriptional Activation; Transcriptional Regulation; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; base; cell growth regulation; drug discovery; enzyme pathway; inhibitor/antagonist; innovation; insight; meetings; member; multicatalytic endopeptidase complex; novel; novel strategies; preference; protein degradation; public health relevance; receptor; receptor recycling; secretase; small molecule; thioester; tool; ubiquitin ligase; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): The network of enzymes involved in the ubiquitination (ligases) and deubiquitination (deubiquitinases or DUbs) of cellular proteins is one of the largest in the human proteome and plays a major role in the regulation of cellular homeostasis. This network interacts with other networks in the cell, especially the kinome, in regulating transcriptional activation and protein degradation among other functions. Finally, dysregulation of this network has been linked to a variety of diseases including neurodegenerative diseases, cancer, and arthritis and inflammation. One of the most powerful tools for determining the specificity of proteases has been the use of peptide libraries to define substrate preferences and determine the influence of amino acids flanking the scissile bond on cleavage efficiency. These studies also provide insights into the nature of side chains interacting with binding subsites within the enzyme's active site. Because deubiquitinases hydrolyze isopeptide bonds between ubiquitin (Ub) and the protein substrate, this type of analysis cannot be performed with such linear peptide libraries which do not mimic the geometry of an isopeptide linkage. In this application, we propose to construct a peptide library linked to Ub through a physiologically relevant isopeptide bond between the carboxyl of the C-terminal Gly of Ub and the 5-amine of a lysine residue in the peptide. A microarray of the library will be constructed on a glass substrate to allow facile analysis of the activity of individual DUbs against each Ub-isopeptide in the library. The data generated in this system will allow us to begin to define the substrate selectivity of different DUbs and the influence of different amino acids flanking the isopeptide lysine on catalytic efficiency of the DUbs. These results will help refine the pattern of interactions in the ubiquitin network and their role in regulating cellular physiology. PUBLIC HEALTH RELEVANCE: LifeSensors proposes to develop an innovative series of new tools that will impact the characterization of the ubiquitination/deubiquitination network. This network is intimately involved - both directly and indirectly through its interactions with other regulatory networks - in the maintenance of cellular homeostasis. Dysregulation of specific members of this network has been implicated in the etiology of a variety of human pathologies including neurodegenerative diseases, cancer, and arthritis and inflammation. The tools developed under this grant will provide new methods for understanding the functioning of ubiquitin pathway enzymes and new approaches to the development of therapeutic agents targeting these enzymes.

描述（由申请人提供）：参与细胞蛋白质泛素化（连接酶）和去泛素化（去泛素酶或 DUb）的酶网络是人类蛋白质组中最大的酶网络之一，在细胞稳态的调节中发挥着重要作用。该网络与细胞中的其他网络（尤其是激酶组）相互作用，调节转录激活和蛋白质降解等功能。最后，该网络的失调与多种疾病有关，包括神经退行性疾病、癌症、关节炎和炎症。确定蛋白酶特异性的最强大工具之一是使用肽库来定义底物偏好并确定易断键两侧的氨基酸对裂解效率的影响。这些研究还提供了对侧链与酶活性位点内的结合亚位点相互作用的性质的见解。由于去泛素酶会水解泛素 (Ub) 和蛋白质底物之间的异肽键，因此不能使用不模拟异肽键几何形状的线性肽文库进行此类分析。在本申请中，我们建议通过 Ub C 端 Gly 的羧基和肽中赖氨酸残基的 5-胺之间的生理相关异肽键构建与 Ub 连接的肽库。文库的微阵列将构建在玻璃基板上，以便轻松分析文库中各个 DUb 对每种 Ub 异肽的活性。该系统中生成的数据将使我们能够开始定义不同 DUb 的底物选择性以及异肽赖氨酸侧翼的不同氨基酸对 DUb 催化效率的影响。这些结果将有助于完善泛素网络中的相互作用模式及其在调节细胞生理学中的作用。 公共健康相关性：LifeSensors 提议开发一系列创新的新工具，这些工具将影响泛素化/去泛素化网络的表征。该网络通过与其他调节网络的相互作用直接或间接地密切参与细胞稳态的维持。该网络特定成员的失调与多种人类病理的病因学有关，包括神经退行性疾病、癌症、关节炎和炎症。这笔赠款下开发的工具将为了解泛素途径酶的功能提供新方法，并为开发针对这些酶的治疗药物提供新方法。