Table_2_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.

在阐释泛素化反应中动态蛋白-蛋白相互作用的研究领域是一项颇具挑战性的工作。构建针对泛素本身、E1、E2或E3等组分的小肽适配体，有望提供剖析酶联级联反应新颖特征的实验工具。本研究采用了下一代深度测序平台，从针对泛素及其同源物NEDD8筛选的噬菌体-肽库中分离出肽序列。在经过三轮不同洗涤标准的筛选后，成功获取超过13,000个针对泛素的肽段，以及超过10,000个针对NEDD8的肽段。这两组肽段之间的重叠度低于5%，表明泛素或NEDD8对线性肽基序具有高度的特异性。其中两种识别泛素的肽段被发现能够抑制E3泛素连接酶MDM2和CHIP。核磁共振分析突显了两种不同肽段适配体独特的结合模式。这些数据凸显了利用下一代测序技术对组合噬菌体-肽库进行测序，以分离针对蛋白质靶点的肽段适配体的效用，这些肽段适配体可作为化学工具应用于复杂的多酶反应中。