Deciphering the poly-ubiquitin code with isopeptide selective antibodies

描述（由申请人提供）：泛素（Ub）是一种76个氨基酸的蛋白质，与蛋白质降解和无数细胞信号通路有关，包括DNA损伤反应、蛋白质运输、细胞周期进展、炎症、免疫反应和细胞凋亡调节。泛素化是通过在Ub的c端和？赖氨酸（Lys）残基在目标蛋白上的氨基。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，生成长度为2至7 Ubs的多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。由于缺乏选择性识别多泛素化蛋白的抗体或其他工具，泛素领域的研究受到阻碍。为了解决这个问题，我们结合了我们的经验，产生异肽选择性多ubs和酵母遗传学产生多样化的单链抗体，将推动细胞生物学领域。在本研究中，我们建议使用酵母单域骆驼抗体来选择连锁特异性结合物。我们将使用合成肽，既包含异肽键，也包含泛素中赖氨酸附着位点周围的序列，以及一套完整的二泛素，包括7个异肽键中的每一个。合成肽抗原和diUbs都将单独用于筛选抗体。生命传感器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。这些工具将作为检测试剂在蛋白质化学、临床诊断等方面的应用

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.