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

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