Characterization of Cell-Envelope Proteinases from Two Lacticaseibacillus casei Strains Isolated from Parmigiano Reggiano Cheese

In the present work, two cell-envelope proteinases (CEPs) from Lacticaseibacillus casei strains PRA205 and 2006 were characterized at both the biochemical and genetic levels. The genomes of both L. casei strains included two putative CEPs genes prtP2 and prtR1, but only prtR1 was transcribed. The extracted PrtR1 proteinases were serine proteinases with optimal activity at 40 °C and pH 7.5, and were activated by Ca2+ ions. Interestingly, PrtR1 from L. casei PRA205 exhibited high residual activity at pH 4 and at 5 °C, suggesting its possible exploitation for fermented food production. The caseinolytic activity against αS1- and β-casein indicated that both PrtR1s belonged to the PI/PIII type. These PrtR1s cleaved β-casein peptide bonds preferentially when amino acid M or N was present at the P1 subsite and amino acids A and D were at the P1′ subsite. Several bioactive peptides were found to be released from PrtR1 after αs1- and β-casein hydrolysis.

Serpins in cartilage and osteoarthritis: what do we know?

Serpins (serine proteinase inhibitors) are an ancient superfamily of structurally similar proteins, the majority of which use an elegant suicide inhibition mechanism to target serine proteinases. Despite likely evolving from a single common ancestor, the 36 human serpins have established roles regulating diverse biological processes, such as blood coagulation, embryonic development and extracellular matrix (ECM) turnover. Genetic mutations in serpin genes underpin a host of monogenic disorders — collectively termed the ‘serpinopathies’ — but serpin dysregulation has also been shown to drive pathological mechanisms in many common diseases. Osteoarthritis is a degenerative joint disorder, characterised by the progressive destruction of articular cartilage. This breakdown of the cartilage is driven by the metalloproteinases, and it has long been established that an imbalance of metalloproteinases to their inhibitors is of critical importance. More recently, a role for serine proteinases in cartilage destruction is emerging; including the activation of latent matrix metalloproteinases and cell-surface receptors, or direct proteolysis of the ECM. Serpins likely regulate these processes, as well as having roles beyond serine proteinase inhibition. Indeed, serpins are routinely observed to be highly modulated in osteoarthritic tissues and fluids by ‘omic analysis, but despite this, they are largely ignored. Confusing nomenclature and an underappreciation for the role of serine proteinases in osteoarthritis (OA) being the likely causes. In this narrative review, serpin structure, biochemistry and nomenclature are introduced, and for the first time, their putative importance in maintaining joint tissues — as well as their dysregulation in OA — are explored.

Proteinase-Mediated Macrophage Signaling in Psoriatic Arthritis

ObjectiveMultiple proteinases are present in the synovial fluid (SF) of an arthritic joint. We aimed to identify inflammatory cell populations present in psoriatic arthritis (PsA) SF compared to osteoarthritis (OA) and rheumatoid arthritis (RA), identify their proteinase-activated receptor 2 (PAR2) signaling function and characterize potentially active SF serine proteinases that may be PAR2 activators.MethodsFlow cytometry was used to characterize SF cells from PsA, RA, OA patients; PsA SF cells were further characterized by single cell 3’-RNA-sequencing. Active serine proteinases were identified through cleavage of fluorogenic trypsin- and chymotrypsin-like substrates, activity-based probe analysis and proteomics. Fluo-4 AM was used to monitor intracellular calcium cell signaling. Cytokine expression was evaluated using a multiplex Luminex panel.ResultsPsA SF cells were dominated by monocytes/macrophages, which consisted of three populations representing classical, non-classical and intermediate cells. The classical monocytes/macrophages were reduced in PsA compared to OA/RA, whilst the intermediate population was increased. PAR2 was elevated in OA vs. PsA/RA SF monocytes/macrophages, particularly in the intermediate population. PAR2 expression and signaling in primary PsA monocytes/macrophages significantly impacted the production of monocyte chemoattractant protein-1 (MCP-1). Trypsin-like serine proteinase activity was elevated in PsA and RA SF compared to OA, while chymotrypsin-like activity was elevated in RA compared to PsA. Tryptase-6 was identified as an active serine proteinase in SF that could trigger calcium signaling partially via PAR2.ConclusionPAR2 and its activating proteinases, including tryptase-6, can be important mediators of inflammation in PsA. Components within this proteinase-receptor axis may represent novel therapeutic targets.

Study of proteolytic activity in rats kidney and liver during the development of chronic alcoholic intoxication

The study of biochemical processes in chronic alcohol intoxication is a topical issue of our time. A deeper understanding of the mechanism of action of proteinases in this pathological condition will help in the development and search for non-invasive diagnostic methods, thereby minimizing the risk of harming human health during complex diagnostic procedures. In our experiment, we investigated the general proteolytic activity, the activity of metalloproteinases and serine proteinases in the liver and kidneys of rats on an experimental model of chronic alcohol intoxication on days 1, 3, 7, and 11 of the experiment, as well as on days 21 and 28 after the cessation of ethanol administration. Male rats weighing 180–200 g were modeled for chronic alcohol intoxication by intragastric administration of 30% ethyl alcohol solution for 10 days on an empty stomach, at the rate of 2 ml per 100 g of animal weight. Liver and kidney homogenate by well-known methods. The concentration was determined by the Bradford method. The total proteolytic activity, the activity of metalloproteinases and serine proteinases were determined by the caseinolytic method with modifications. The total proteolytic activity, the activity of metalloproteinases and serine proteinases were determined by the caseinolytic method with modifications. It was shown that on the 3rd, 7th and 11th days of the experiment in the liver there was an increase in the total proteolytic activity and the activity of metalloproteinases. The activity of serine proteinases significantly increased on days 3 and 7 of the study. In the kidneys, a significant increase in all studied activities was observed only on the 3rd day. Such differences in the activities of metalloproteinases and serine proteinases can be associated with the different roles of these enzymes in physiological processes. Thus, we observed an increase in the activity of serine proteinases in acute intoxication, and in metalloproteinases in chronic intoxication.

Priming of SARS-CoV-2 S protein by several membrane-bound serine proteinases could explain enhanced viral infectivity and systemic COVID-19 infection

The ongoing COVID-19 pandemic has already caused over a million deaths worldwide, and this death toll will be much higher before effective treatments and vaccines are available. The causative agent of the disease, the coronavirus SARS-CoV-2, shows important similarities with the previously emerged SARS-CoV-1, but also striking differences. First, SARS-CoV-2 possesses a significantly higher transmission rate and infectivity than SARS-CoV-1 and has infected in a few months over 60 million people. Moreover, COVID-19 has a systemic character, as in addition to the lungs it also affects heart, liver, and kidneys among other organs of the patients, and causes frequent thrombotic and neurological complications. In fact, the term “viral sepsis” has been recently coined to describe the clinical observations. Here I review current structure-function information on the viral spike proteins and the membrane fusion process to provide plausible explanations for these observations. I hypothesize that several membrane-associated serine proteinases (MASPs), in synergy with or in place of TMPRSS2, contribute to activate the SARS-CoV-2 spike protein. Relative concentrations of the attachment receptor, ACE2, MASPs, their endogenous inhibitors (the Kunitz-type transmembrane inhibitors, HAI-1/SPINT1 and HAI-2/SPINT2, as well as major circulating serpins) would determine the infection rate of host cells. The exclusive or predominant expression of major MASPs in specific human organs suggests a direct role of these proteinases in e.g. heart infection and myocardial injury, liver dysfunction, kidney damage, as well as neurological complications. Thorough consideration of these factors could have a positive impact on the control of the current COVID-19 pandemic.

The effect of melanin on the proteolytic potential of blood under alkali esophageal burn

Caustic esophageal burns are among serious medical problems of global significance. Due to a key role in biological processes proteolytic enzymes actively involved in the pathological mechanisms underpinning the development and progression of burn-related complications. Since melanin possesses a broad spectrum of biological activities we have investigated whether this compound can influence the proteolytic activity and level of proteinase inhibitors in the blood of rats with an alkali esophageal burn. Alkaline esophageal burns, which correspond to the first and second degree of burn, were induced by 10% and 20% NaOH, respectively. White, nonlinear, immature (4 weeks old) rats were used in the experiment. Total proteolytic activity was measured using casein as a substrate. The activities of α1- antitrypsin and α2-macroglobulin were measured considering the degree of inhibition of hydrolysis of N-benzoyl-L-arginineethyl ester. The fraction of serine proteinases was obtained by affinity chromatography on a benzamidine sepharose column. The qualitative composition of serine proteinases fraction was analyzed by zymography technique. According to the data obtained, the pathogenesis of alkaline esophageal burn is accompanied by a significant increase in the total proteolytic activity, activity of serine proteinases, and activity of α1-antitrypsin and α2-macroglobulin compared with the control rats. The present results clearly indicated that melanin is able to normalize the proteolytic homeostasis by affecting the activity of serine proteinases and the level of proteinase inhibitors in the plasma of rats with alkali esophageal burns.

Novel Bradykinin-Potentiating Peptides and Three-Finger Toxins from Viper Venom: Combined NGS Venom Gland Transcriptomics and Quantitative Venom Proteomics of the Azemiops feae Viper

Feae’s viper Azemipos feae belongs to the Azemiopinae subfamily of the Viperidae family. The effects of Viperidae venoms are mostly coagulopathic with limited neurotoxicity manifested by phospholipases A2. From A. feae venom, we have earlier isolated azemiopsin, a novel neurotoxin inhibiting the nicotinic acetylcholine receptor. To characterize other A. feae toxins, we applied label-free quantitative proteomics, which revealed 120 unique proteins, the most abundant being serine proteinases and phospholipases A2. In total, toxins representing 14 families were identified, among which bradykinin-potentiating peptides with unique amino acid sequences possessed biological activity in vivo. The proteomic analysis revealed also basal (commonly known as non-conventional) three-finger toxins belonging to the group of those possessing neurotoxic activity. This is the first indication of the presence of three-finger neurotoxins in viper venom. In parallel, the transcriptomic analysis of venom gland performed by Illumina next-generation sequencing further revealed 206 putative venom transcripts. Together, the study unveiled the venom proteome and venom gland transciptome of A. feae, which in general resemble those of other snakes from the Viperidae family. However, new toxins not found earlier in viper venom and including three-finger toxins and unusual bradykinin-potentiating peptides were discovered.