Deciphering the poly-ubiquitin code with isopeptide selective antibodies

用同肽选择性抗体破译多聚泛素密码

• 批准号: 8714719
• 负责人: Mark Mason
• 金额: $ 22.37万
• 依托单位: LIFESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-06 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8714719
• 关键词: Active Sites; Address; Advertising; Affect; Affinity; Alzheimer' s Disease; Amino Acids; Antibodies; Antibody Affinity; Antibody Specificity; Antigens; Apoptosis; Architecture; Autoimmune Process; Autophagocytosis; Binding; Biochemistry; Biological; Biological Assay; Biology; C-terminal; Cell Cycle Progression; Cell physiology; Cellular biology; Chemicals; Clinical; Code; Communicable Diseases; Communities; Complex Mixtures; Coupled; DNA Damage; DNA Repair; Detection; Development; Diagnostic; Disease; Distal; Endocytosis; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Epitopes; Excision; Exhibits; Figs - dietary; Fluorescent Dyes; Glycine; Goals; Grant; Half-Life; Human Genome; Image; Immune response; Immunoglobulin Somatic Hypermutation; Inflammation; Label; Length; Libraries; Link; Lysine; Malignant Neoplasms; Marketing; Measures; Mediating; Modification; Molecular Weight; Nature; Nerve Degeneration; Pathway interactions; Phase; Phospho-Specific Antibodies; Phosphorylation; Play; Polymers; Polyubiquitin; Population; Post-Translational Protein Processing; Protein Chemistry; Protein Microchips; Proteins; Proteomics; Reagent; Regulation; Research; Role; Sepharose; Signal Pathway; Signal Transduction; Site; Small Business Innovation Research Grant; Specificity; Staining method; Stains; Surface; Surface Plasmon Resonance; System; Techniques; Technology; Time; UBD protein; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitination; Western Blotting; Work; Yeasts; amino group; antigen binding; base; carboxylate; cell growth regulation; drug discovery; experience; innovation; isopeptidase; meetings; multicatalytic endopeptidase complex; protein degradation; protein function; protein structure function; protein transport; public health relevance; receptor; receptor recycling; response; synthetic peptide; tool; ubiquitin-protein ligase; yeast genetics

DESCRIPTION (provided by applicant): Ubiquitin (Ub), a 76 amino acid protein, is implicated in protein degradation and a myriad of cell signaling pathways, including DNA damage response, protein trafficking, cell-cycle progression, inflammation, immune response and regulation of apoptosis. Ubiquitylation occurs through formation of an isopeptide bond between the C-terminus of Ub and the ?-amino group of a lysine (Lys) residue on target proteins. Ub itself has seven Lys residues (K6, K11, K27, K29, K33, K48, and K63), each of which can be further ubiquitylated, generating poly-Ub chains ranging from 2 to 7 Ubs in length. The ability of Ub to form polymers through various lysines appears to be central to the versatility of this system in regulating cell processes. Research in the ubiquitin field is hampered by a lack of antibodies or other tools that selectively recognize poly-ubiquitylated proteins. To address this issue we have combined our experience of generating isopeptide selective poly-Ubs and yeast genetics to generate diverse single chain antibodies that will advance the field of cell biology. I this grant we propose to use yeast display of single domain camelid antibodies to select for linkage specific binders. We will employ synthetic peptides which encompass both the isopeptide bond and the sequence surrounding the lysine attachment site in ubiquitin as well as a complete set of di-ubiquitins comprising each of the 7 isopeptide linkages. Both synthetic peptide antigens and diUbs will be used individually to pan for antibodies. LifeSensors and the scientific community will use these tools to chart new pathways in cell biology and biochemistry. These tools will be used as detection agents in protein chemistry, clinical diagnostic applications and imaging studies. These tools will unmask ubiquitomics of the cellular proteins and uncover new roles of unique poly-ubiquitylated proteins. Given the poor nature of tools available for K48- and K63- poly-Ub linkages, as a proof-of- principle, the phase I grant will focus on K48- and K63-linkages and expand to the remaining linkages in Phase II. We believe that development of linkage selective tools will have as great an impact on cell biology as the development of phospho-specific antibodies had on cellular phosphorylation.

描述（由申请人提供）：泛素（Ub）是一种76个氨基酸的蛋白质，涉及蛋白质降解和无数细胞信号传导途径，包括DNA损伤反应、蛋白质运输、细胞周期进程、炎症、免疫反应和细胞凋亡调节。泛素化通过在Ub的C-末端和？-末端之间形成异肽键而发生。靶蛋白上赖氨酸（Lys）残基的氨基。Ub本身有7个Lys残基（K6、K11、K27、K29、K33、K48和K63），每个残基都可以进一步泛素化，产生长度为2至7个Ub的聚Ub链。Ub通过各种赖氨酸形成聚合物的能力似乎是该系统在调节细胞过程中的多功能性的核心。泛素领域的研究由于缺乏选择性识别多聚泛素化蛋白的抗体或其他工具而受到阻碍。为了解决这个问题，我们结合了我们的经验，产生异肽选择性poly-Ubs和酵母遗传学，以产生不同的单链抗体，这将推进细胞生物学领域。在这项授权中，我们建议使用酵母展示的单域骆驼抗体来选择连接特异性结合剂。我们将使用合成肽，其包含泛素中的异肽键和赖氨酸连接位点周围的序列，以及包含7个异肽键中的每一个的一整套双泛素。合成肽抗原和diUb将单独用于淘选抗体。LifeSensors和科学界将使用这些工具来绘制细胞生物学和生物化学的新途径。这些工具将被用作蛋白质化学、临床诊断应用中的检测剂 和成像研究。这些工具将揭开细胞蛋白质的泛素组学，并揭示独特的多聚泛素化蛋白质的新作用。鉴于K48-和K63- poly-Ub连接可用工具的性质较差，作为原则证明，第一阶段赠款将集中于K48-和K63-连接，并在第二阶段扩展到其余连接。我们相信，连接选择性工具的发展将对细胞生物学产生巨大的影响，就像磷酸化特异性抗体的发展对细胞磷酸化的影响一样。