Table_1_Next-generation sequencing of a combinatorial peptide phage library screened against ubiquitin identifies peptide aptamers that can inhibit the in vitro ubiquitin transfer cascade.XLSX

Defining dynamic protein–protein interactions in the ubiquitin conjugation reaction is a challenging research area. Generating peptide aptamers that target components such as ubiquitin itself, E1, E2, or E3 could provide tools to dissect novel features of the enzymatic cascade. Next-generation deep sequencing platforms were used to identify peptide sequences isolated from phage-peptide libraries screened against Ubiquitin and its ortholog NEDD8. In over three rounds of selection under differing wash criteria, over 13,000 peptides were acquired targeting ubiquitin, while over 10,000 peptides were selected against NEDD8. The overlap in peptides against these two proteins was less than 5% suggesting a high degree in specificity of Ubiquitin or NEDD8 toward linear peptide motifs. Two of these ubiquitin-binding peptides were identified that inhibit both E3 ubiquitin ligases MDM2 and CHIP. NMR analysis highlighted distinct modes of binding of the two different peptide aptamers. These data highlight the utility of using next-generation sequencing of combinatorial phage-peptide libraries to isolate peptide aptamers toward a protein target that can be used as a chemical tool in a complex multi-enzyme reaction.

解析泛素缀合反应中的动态蛋白质-蛋白质相互作用，是极具挑战性的研究领域。靶向泛素本身、E1、E2或E3等组分的肽适配体（peptide aptamers），可为解析该酶促级联反应的全新特征提供研究工具。本研究借助下一代深度测序平台，对针对泛素（Ubiquitin）及其同源蛋白NEDD8进行筛选的噬菌体肽库中分离得到的肽序列进行鉴定。经三轮不同洗脱条件下的筛选，共获得靶向泛素的肽段13000余条，靶向NEDD8的肽段则达10000余条。针对这两种蛋白的肽段重叠率不足5%，提示泛素与NEDD8对线性肽基序具有极高的结合特异性。本研究筛选到两条结合泛素的肽段，可同时抑制E3泛素连接酶MDM2与CHIP。核磁共振波谱（NMR）分析揭示了这两种不同肽适配体的独特结合模式。本研究数据证实，利用组合噬菌体肽库的下一代测序技术分离靶向特定蛋白的肽适配体，可作为复杂多酶反应中的化学工具使用。