Unveiling antimicrobial peptide–generating human proteases using PROTEASIX

Abstract

Extracting information from peptidomics data is a major current challenge, as endogenous peptides can result from the activity of multiple enzymes. Proteolytic enzymes can display overlapping or complementary specificity. The activity spectrum of human endogenous peptide-generating proteases is not fully known. Hence, the indirect study of proteolytic enzymes through the analysis of its substrates is largely hampered. Antimicrobial peptides (AMPs) represent a primordial set of immune defense molecules generated by proteolytic cleavage of precursor proteins. These peptides can be modulated by host and microorganismal stimuli, which both dictate proteolytic enzymes' expression and activity. Peptidomics is an attractive approach to identify peptides with a biological role and to assess proteolytic activity. However, bioinformatics tools to deal with peptidomics data are lacking. PROTEASIX is an excellent choice for the prediction of AMPsgenerating proteases based on the reconstitution of a substrate's cleavage sites and the crossing of such information with known proteases' specificity retrieved by several publicly available databases. Therefore, the focus of the present tutorial is to explore the potential of PROTEASIX when gather information concerning proteases involved in the generation of human AMPs and to teach the user how to make the most out of peptidomics results using PROTEASIX.

Sequential Precipitation and Delipidation Enables Efficient Enrichment of Low-Molecular Weight Proteins and Peptides from Human Plasma

“…This could be partially attributed to the function of delipidation provided by SPD, which could not only damage the membrane structure of HDL, LDL, and other nanoparticles to release more LMW species but also reduce the effects of lipids on the identification of proteins and peptides with LC–MS/MS. Moreover, SPD could be helpful for denaturing proteins or dissociating protein–protein/metabolite complexes since the MTBE/methanol/water solvent system has been widely used to dissociate and extract metabolites and proteins − for the study of protein–protein and protein–metabolite complexes. Furthermore, as an additional line of evidence, purified HDL has been reported to carry a complex range of small peptides, such as fragments from apolipoproteins, fibrinogen, the α1-proteinase inhibitor, and transthyretin .…”

“…Considering that endogenous peptides/proteins can act as substrates for proteases and the presence of their proteolytic fragments could accurately reflect the abundance or activity of specific in vivo proteases, , peptidomic signatures identified in blood can be mined for information pertaining to changes in protease expression and activity in different conditions, such as anti-cancer treatment. To determine which proteases might be involved in producing these peptides, the sequences of identified peptides were subjected to Proteasix-based analysis , by which the frequencies of cleavage sequences and associated proteases were calculated (Table S5). Proteases with more than 10 cleavage sequences were recorded.…”

Immunomodulatory and Allergenic Properties of Antimicrobial Peptides

“…A wide spectrum of antimicrobial activities of AMPs inspires researchers to search for new active peptides in biological material [ 252 , 253 ], and also serves as the basis for the design of new synthetic compounds [ 254 , 255 ]. To date, information about AMPs is presented in numerous databases describing the structural, functional, allergenic and toxicological properties of these peptide factors of innate immunity [ 256 , 257 , 258 , 259 , 260 ].…”

Digging Deep into Peptidomics Applied to Body Fluids

“…To overcome these limitations several tools have been proposed for peptide mapping (e.g., MS peptide–protein coverage) such as iPiG, EnzymePredictor, PatternLab, Peptigram, UStags, PepNovo, and MS‐Tag . For biological interpretation there are tools such as Proteasix, CutDB, SwePep, Erop‐Moscow, and PeptideDB (http://www.peptides.be). Similar functions might also be inferred based on homology comparisons with library sequences …”