Development Of Arginine Linkage-Specific Antibodies

精氨酸连接特异性抗体的开发

• 批准号: 9344737
• 负责人: Hiep T Tran
• 金额: $ 22.49万
• 依托单位: ABZYME THERAPEUTICS, LLC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9344737
• 关键词: Actins; Address; Affect; Affinity; Aging; Amino Acids; Antibodies; Antibody Formation; Antigens; Arginine; Binding; Biochemistry; Biological; Biological Markers; Biological Process; Biomedical Research; Brain; Brain Mass; Cardiac Myosins; Catalytic Antibodies; Cells; Cellular Metabolic Process; Cellular biology; Clinical; Communities; Cytoskeleton; Data; Detection; Development; Diagnostic; Disease; Disseminated Malignant Neoplasm; Embryonic Development; Engineering; Enzyme-Linked Immunosorbent Assay; Event; Explosion; Generations; Grant; Heart failure; Human; Immunoassay; Impairment; Knockout Mice; Laboratories; Libraries; Light; Malignant Neoplasms; Mammals; Maps; Mass Spectrum Analysis; Mediating; Metabolic Pathway; Metabolism; Metastatic to; Molecular; Morphogenesis; Mus; Muscular Dystrophies; N-terminal; Nature; Neoplasm Metastasis; Nerve Degeneration; Partner in relationship; Pathway interactions; Patients; Pennsylvania; Peptides; Phase; Phospho-Specific Antibodies; Phosphorylation; Physiological; Physiology; Play; Post-Translational Protein Processing; Process; Protein Chemistry; Proteins; Proteome; Reaction; Regulation; Research; Role; Saccharomyces cerevisiae; Sampling; Side; Site; Specificity; System; Therapeutic; Tissues; Ubiquitination; Universities; Yeasts; alpha synuclein; base; cell motility; clinical diagnostics; connectin; human disease; imaging study; in vivo; innovation; interest; mouse model; novel; polypeptide; protein aminoacid sequence; skeletal; synthetic peptide; synuclein; therapeutic target; tool; virtual; yeast genetics

ASBTRACT. Arginylation mediated by arginyltransferase (ATE1) is a posttranslational modification that plays a global role in mammals. Arginylation is essential for mammalian embryogenesis and regulation of the cytoskeleton function in cell migration, a process that plays key roles during tissue morphogenesis and cancer metastases. Arginylation occurs through formation of arginine linkage to E or D amino acid residues that are located either internally or at the N-terminus of target proteins. Addition of N-terminal Arg to proteins may induce their ubiquitination and degradation by the N-end rule pathway, however recent studies suggest that the scope of the biological effects of arginylation is much broader, reminiscent of the effects of protein phosphorylation. A recent explosion of studies has put arginylation on the map of intracellular metabolic pathways and biological processes and demonstrated its key involvement in the central events of cell metabolism, normal function of multiple physiological systems, and cell transition to metastatic cancers. Research in the protein arginylation field is however hampered by the lack of antibodies or other tools that selectively recognize arginylated proteins. To address this issue we propose utilizing our combined expertise in generating arginylated linkage peptides and yeast genetics to generate a diverse array of antibodies that will advance the field of cell biology. In this application, we propose to use innovative yeast display of a highly diverse human antibody Fab library with self-diversifying ability to select for arginylation specific binders. We will employ synthetic peptides which encompass both the isopeptide bond and the sequence surrounding the arginine attachment sites in α- synuclein (Phase I) and will expand this approach to include the development of arginylation antibodies of global specificity, which can serve as a major tools for biomedical research including inverse correlation of arginylation and ageing, neurodegeneration and clinical diagnostics (Phase II). As the result of this project, a set of site-specific arginylation and pan-arginylation antibodies will be developed. 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 arginylation of cellular proteins and uncover new roles unique to arginylated proteins. Given the poor nature of tools available for the arginylation field, as a proof-of- principle, the phase I grant will focus on E46- and E83- linkages in -synuclein and subsequently in Phase II expand to a range of arginylated protein targets with side chain and N-terminal linkages. We believe that development of arginine linkage selective tools will have as great an impact on cell biology as the development of phospho-specific antibodies had on cellular phosphorylation.

ASBTRACT。精氨酰转移酶（ATE 1）介导的精氨酸化是一种翻译后修饰， 在哺乳动物中的全球性作用。精氨酸化对于哺乳动物胚胎发生和细胞周期的调节是必需的。 细胞骨架在细胞迁移中起作用，这一过程在组织形态发生和癌症中起关键作用。 转移精氨酸化通过形成精氨酸连接至与精氨酸连接的E或D氨基酸残基而发生。 位于靶蛋白的内部或N末端。向蛋白质添加N-末端Arg可 通过N端规则途径诱导其泛素化和降解，然而最近的研究表明， 乙酰化的生物学效应的范围要广得多，使人联想到蛋白质的作用。 磷酸化最近大量的研究已经把乙酰化放在细胞内代谢的地图上 途径和生物过程，并证明了其在细胞的核心事件的关键参与 代谢、多个生理系统的正常功能和细胞向转移性癌症的转变。 然而，蛋白质去乙酰化领域的研究受到缺乏抗体或其他工具的阻碍， 选择性识别乙酰化蛋白质。为了解决这个问题，我们建议利用我们的综合专业知识 在产生腺苷酰化连接肽和酵母遗传学中， 推进细胞生物学领域。 在这个应用中，我们建议使用创新的酵母展示高度多样化的人抗体Fab文库 具有自我多样化的能力来选择乙酰化特异性结合剂。我们将使用合成肽， 包括异肽键和α-精氨酸连接位点周围的序列。 突触核蛋白（第一阶段），并将扩大这种方法，包括发展的α-淀粉样蛋白的抗体， 全球特异性，它可以作为生物医学研究的主要工具，包括逆相关性， 乙酰化和老化，神经变性和临床诊断（第二阶段）。由于该项目， 将开发一组位点特异性β-淀粉酰化和泛β-淀粉酰化抗体。科学界将 使用这些工具绘制细胞生物学和生物化学的新途径。这些工具将被用作检测 蛋白质化学、临床诊断应用和成像研究中的试剂。这些工具将揭露 细胞蛋白质的乙酰化，并发现乙酰化蛋白质独特的新作用。鉴于贫穷的性质， 作为一种原理证明，第一阶段赠款将集中在E46和E83上， 突触核蛋白中的连接，随后在第二阶段扩展到一系列的腺苷酸化蛋白质靶点， 链和N-末端连接。我们相信，精氨酸连接选择性工具的开发将具有 磷酸化特异性抗体的发展对细胞生物学产生了巨大的影响， 磷酸化