Block-based characterization of protease specificity from substrate sequence profile

Abstract

BackgroundThe mechanism of action of proteases has been widely studied based on substrate specificity. Prior research has been focused on the amino acids at a single amino acid site, but rarely on combinations of amino acids around the cleavage bond.ResultsWe propose a novel block-based approach to reveal the potential combinations of amino acids which may regulate the action of proteases. Using the entropies of eight blocks centered at a cleavage bond, we created a distance matrix for 61 proteases to compare their specificities. After quantitative analysis, we discovered a number of prominent blocks, each of which consists of successive amino acids near a cleavage bond, intuitively characterizing the site cooperation of the substrate sequences.ConclusionThis approach will help in the discovery of specific substrate sequences which may bridge between proteases and cleavage substrate as more substrate information becomes available.Electronic supplementary materialThe online version of this article (10.1186/s12859-017-1851-1) contains supplementary material, which is available to authorized users.

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen

“…2d ), while decreasing analysis time (Supplementary Data 3 ). Next, positional frequency of amino acids, the cleavage entropy 34 (a quantitative measure of protease specificity) and block entropy 35 (a measure of protease sub-site cooperativity) were calculated with a series of scripts that we developed for the study and that were extensively annotated and deposited in GitHub ( https://github.com/anfoss/HTPS_workflow , 10.5281/zenodo.4484341). Protease specificity is a direct result of analyzing the peptide pool generated in a cleavage assay and mapping the determined termini onto the protein sequence.…”

“…The calculation of cleavage entropy was performed as a Shannon entropy calculation 34 . The block entropy calculation was performed as described elsewhere 35 .…”

Mapping specificity, entropy, allosteric changes and substrates in blood proteases by a high-throughput protease screen

“…This increased the ratio of matched MS/MS spectra over all MS/MS spectra and the number of identified peptides ( Figure S2D), while decreasing analysis time (Table S3). Next, positional frequency of amino acids, the cleavage entropy 34 (a quantitative measure of protease specificity) and block entropy 35 (a measure of protease sub-site cooperativity) were calculated with a series of scripts that we developed for the study and that were extensively annotated and deposited in GitHub (https://github.com/anfoss/HTPS_workflow). Briefly, after filtering the MQ peptide results (contaminants, decoys and low-score peptides), the cleavage sequences of the identified peptides were converted to a frequency matrix covering 8 amino acids upstream and 8 downstream the cleavage site (P8-P1 and P1'-P8', respectively).…”

“…Data analysis was performed in R (version 3.4.3) using the workflow deposited on Github The calculation of cleavage entropy was performed as a Shannon entropy calculation 34 . The block entropy calculation was performed as described elsewhere 35 .…”

Sensitive Identification of Known and Unknown Protease Activities by Unsupervised Linear Motif Deconvolution

“…Moreover, the amino acids found adjacent to proteolytic cleavage sites are characterized by a large variability in composition and properties . In addition to this, the cooperative relationship of successive positions (P4–P4′) on substrate sequences has been established for specific proteases, , strongly highlighting the need to consider the interaction of adjacent amino acids in the study of protease–substrate cleavage specificity. Proteolytic data sets therefore lack the fixed central residue required by these tools, even if the sequence alignment is shifted in order to center the sequences around a residue position rather than the cleavage site.…”