PT  - JOURNAL ARTICLE
AU  - Bart Claushuis
AU  - Robert A. Cordfunke
AU  - Arnoud H. de Ru
AU  - Annemarie Otte
AU  - Hans C. van Leeuwen
AU  - Oleg I. Klychnikov
AU  - Peter A. van Veelen
AU  - Jeroen Corver
AU  - Jan W. Drijfhout
AU  - Paul J. Hensbergen
TI  - In-depth specificity profiling of Pro-Pro endopeptidases (PPEPs) using combinatorial synthetic peptide libraries
AID  - 10.1101/2022.11.15.516584
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.11.15.516584
4099  - http://biorxiv.org/content/early/2022/11/15/2022.11.15.516584.short
4100  - http://biorxiv.org/content/early/2022/11/15/2022.11.15.516584.full
AB  - Proteases comprise the class of enzymes that catalyze the hydrolysis of peptide bonds, thereby playing a pivotal role in many aspects of life. The amino acids surrounding the scissile bond determine the susceptibility towards protease-mediated hydrolysis. A detailed understanding of the cleavage specificity of a protease can lead to the identification of its endogenous substrates, while it is also essential for the design of inhibitors. We developed a new method which combines the high diversity of a combinatorial synthetic peptide library with the sensitivity and detection power of mass spectrometry to determine protease cleavage specificity. We applied this method to study a group of bacterial metalloproteases that have the unique specificity to cleave between two prolines, i.e. Pro-Pro endopeptidases (PPEPs). We not only confirmed the prime-side specificity of PPEP-1 and PPEP-2, but also revealed some new unexpected peptide substrates. Moreover, we have characterized a new PPEP (PPEP-3) which has a prime-side specificity that is very different from that of the other two PPEPs. Importantly, the approach that we present in this study is generic and can be extended to investigate the specificity of other proteases.Competing Interest StatementThe authors have declared no competing interest.