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.

界定泛素化反应中动态蛋白-蛋白相互作用是一个极具挑战性的研究领域。生成针对泛素本身、E1、E2或E3等组分的小肽适配体，可为剖析酶促级联反应的新特性提供工具。利用下一代深度测序平台，从针对泛素及其同源蛋白NEDD8进行筛选的噬菌体肽库中分离出肽序列。经过三轮以上不同洗涤标准下的筛选，获取了超过13,000种针对泛素的肽，同时选取了超过10,000种针对NEDD8的肽。这两种蛋白针对肽的交叉重叠小于5%，表明泛素或NEDD8对线性肽基序具有高度特异性。其中两种泛素结合肽被发现能够抑制MDM2和CHIP这两种E3泛素连接酶。核磁共振分析突出了两种不同肽适配体结合的特异性模式。这些数据突显了利用组合噬菌体肽库的下一代测序技术分离针对蛋白质靶标肽适配体的实用价值，这些肽适配体可用作复杂多酶反应中的化学工具。