Development Of Arginine Linkage-Specific Antibodies

ASBTRACT。精氨酸基转移酶(ATE1)介导的精氨酸化是一种翻译后修饰 在哺乳动物中扮演着全球角色。精氨酸化对于哺乳动物胚胎的发生和调控是必不可少的。 细胞骨架在细胞迁移中的作用，这一过程在组织形态形成和癌症中发挥关键作用 转移瘤。精氨酸化是通过与E或D氨基酸残基形成精氨酸连接而发生的 位于目标蛋白质的内部或N-末端。将N-末端精氨酸添加到蛋白质中可能 通过N-端规则通路诱导其泛素化和降解，然而最近的研究表明 精氨酸化的生物学效应范围要广得多，这让人想起蛋白质的效应。 磷酸化。最近激增的研究已经把精氨酸化放在了细胞内代谢的地图上 途径和生物学过程，并展示了它在细胞中心事件中的关键参与 代谢，多个生理系统的正常功能，以及细胞向转移性癌症的转变。 然而，由于缺乏抗体或其他工具，蛋白质精氨酸化领域的研究受到阻碍 选择性地识别精氨酸化的蛋白质。为了解决这个问题，我们建议利用我们的联合专业知识 在产生精氨酸化连接肽和酵母遗传学以产生一系列不同的抗体时， 推进细胞生物学领域。 在这项应用中，我们建议使用创新的酵母展示高度多样化的人抗体Fab库 具有选择精氨酸化特定结合剂的自我多样化能力。我们将使用合成多肽， 包含α中的异肽键和精氨酸连接位点周围的序列- 突触核蛋白(第一阶段)，并将扩大这一方法，以包括开发精氨酸化抗体 全球特异性，这可以作为生物医学研究的主要工具，包括 精氨酸化和衰老、神经变性和临床诊断(第二阶段)。作为这个项目的结果，一个 将开发一套位点特异性精氨酸化和泛精氨酸化抗体。科学界将会 使用这些工具来绘制细胞生物学和生物化学的新途径。这些工具将被用作检测 在蛋白质化学、临床诊断应用和成像研究中的代理。这些工具将揭开 细胞蛋白质的精氨酸化，并发现精氨酸化蛋白质独有的新作用。鉴于……的恶劣性质 可用于精氨酸化领域的工具，作为一项原则证明，第一阶段的赠款将侧重于E46和E83- 连接在突触核蛋白中，随后在第二阶段扩展到一系列精氨酸化的蛋白质靶标与Side 链式和N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。

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.