Analysis Of Proteolytic Activity Research Articles

Identification and quantification of degradome components in human synovial fluid reveals an increased proteolytic activity in knee osteoarthritis patients vs controls.

Synovial fluid (SF) may contain cleavage products of proteolytic activities. Our aim was to characterize the degradome through analysis of proteolytic activity and differential abundance of these components in a peptidomic analysis of SF in knee osteoarthritis (OA) patients versus controls (n=23). SF samples from end-stage knee osteoarthritis patients undergoing total knee replacement surgery and controls, that is, deceased donors without known knee disease were previously run using liquid chromatography mass spectrometry (LC-MS). This data was used to perform new database searches generating results for non-tryptic and semi-tryptic peptides for studies of degradomics in OA. We used linear mixed models to estimate differences in peptide-level expression between the two groups. Known proteolytic events (from the MEROPS peptidase database) were mapped to the dataset, allowing the identification of potential proteases and which substrates they cleave. We also developed a peptide-centric R tool, proteasy, which facilitates analyses that involve retrieval and mapping of proteolytic events. We identified 429 differentially abundant peptides. We found that the increased abundance of cleaved APOA1 peptides is likely a consequence of enzymatic degradation by metalloproteinases and chymase. We identified metalloproteinase, chymase, and cathepsins as the main proteolytic actors. The analysis indicated increased activity of these proteases irrespective of their abundance.

Peptide probes for proteases - innovations and applications for monitoring proteolytic activity.

Proteases are excellent biomarkers for a variety of diseases, offer multiple opportunities for diagnostic applications and are valuable targets for therapy. From a chemistry-based perspective this review discusses and critiques the most recent advances in the field of substrate-based probes for the detection and analysis of proteolytic activity both in vitro and in vivo.

Functional Characterization of the Proteolytic System ofLactobacillus sanfranciscensisDSM 20451Tduring Growth in Sourdough

Protein hydrolysis and amino acid metabolism contribute to the beneficial effects of sourdough fermentation on bread quality. In this work, genes of Lactobacillus sanfranciscensis strain DSM 20451 involved in peptide uptake and hydrolysis were identified and their expression during growth in sourdough was determined. Screening of the L. sanfranciscensis genome with degenerate primers targeting prt and analysis of proteolytic activity in vitro provided no indication for proteolytic activity. Proteolysis in aseptic doughs and sourdoughs fermented with L. sanfranciscensis was inhibited upon the addition of an aspartic protease inhibitor. These results indicate that proteolysis was not linked to the presence of L. sanfranciscensis DSM 20451 and that this strain does not harbor a proteinase. Genes encoding the peptide transport systems Opp and DtpT and the intracellular peptidases PepT, PepR, PepC, PepN, and PepX were identified. Both peptide uptake systems and the genes pepN, pepX, pepC, and pepT were expressed by L. sanfranciscensis growing exponentially in sourdough, whereas pepX was not transcribed. The regulation of the expression of Opp, DtpT, and PepT during growth of L. sanfranciscensis in sourdough was investigated. Expression of Opp and DtpT was reduced approximately 17-fold when the peptide supply in dough was increased. The expression of PepT was dependent on the peptide supply to a lesser extent. Thus, the accumulation of amino nitrogen by L. sanfranciscensis in dough is attributable to peptide hydrolysis rather than proteolysis and amino acid metabolism by L. sanfranciscensis during growth in sourdough is limited by the peptide availability.