Data_Sheet_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.docx

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.

界定泛素化反应中动态的蛋白质-蛋白质相互作用是一块极具挑战性的研究领域。生成针对泛素本身、E1、E2或E3等成分的肽类适配体，可提供剖析酶促级联反应新特征的工具。通过利用下一代深度测序平台，从针对泛素及其同源蛋白NEDD8筛选的噬菌体-肽库中鉴定出肽序列。在多轮不同洗涤标准的筛选过程中，共获得超过13,000种针对泛素的肽，以及超过10,000种针对NEDD8的肽。这两种蛋白的肽重叠率低于5%，表明泛素或NEDD8对线性肽基序具有高度的特异性。其中两种泛素结合肽被鉴定出来，它们能够抑制E3泛素连接酶MDM2和CHIP。核磁共振分析突出了两种不同肽类适配体独特的结合模式。这些数据突显了使用组合噬菌体-肽库的下一代测序技术来分离针对蛋白质靶标的肽类适配体的实用性，这些适配体可作为复杂多酶反应中的化学工具。