Tailored Branched Polymer-Protein Bioconjugates for Tunable Sieving Performance.

Protein-polymer conjugates combine the unique properties of both proteins and synthetic polymers, making them important materials for biomedical applications. In this work, we synthesized and characterized protein-branched polymer bioconjugates that were precisely designed to retain protein functionality while preventing unwanted interactions. Using chymotrypsin as a model protein, we employed a controlled radical branching polymerization (CRBP) technique utilizing a water-soluble inibramer, sodium 2-bromoacrylate. The green-light-induced atom transfer radical polymerization (ATRP) enabled the grafting of branched polymers directly from the protein surface in the open air. The resulting bioconjugates exhibited a predetermined molecular weight, well-defined architecture, and high branching density. Conformational analysis by SEC-MALS validated the controlled grafting of branched polymers. Furthermore, enzymatic assays revealed that densely grafted polymers prevented protein inhibitor penetration, and the resulting conjugates retained up to 90% of their enzymatic activity. This study demonstrates a promising strategy for designing protein-polymer bioconjugates with tunable sieving behavior, opening avenues for applications in drug delivery and biotechnology.

Design and Synthesis of Novel Squaraine-Based Fluorescent Probe for Far-Red Detection of Chymotrypsin Enzyme.

Chymotrypsin, a crucial enzyme in human digestion, catalyzes the breakdown of milk proteins, underscoring its significance in both health diagnostics and dairy quality assurance. Addressing the critical need for rapid, cost-effective detection methods, we introduce a groundbreaking approach utilizing far-red technology and HOMO-Förster resonance energy transfer (FRET). Our novel probe, SQ-122 PC, features a unique molecular design that includes a squaraine dye (SQ), a peptide linker, and SQ moieties synthesized through solid-phase peptide synthesis. Demonstrating a remarkable quenching efficiency of 93.75% in a tailored H2O:DMSO (7:3) solvent system, our probe exhibits absorption and emission properties within the far-red spectrum, with an unprecedented detection limit of 0.130 nM. Importantly, our method offers unparalleled selectivity towards chymotrypsin, ensuring robust and accurate enzyme detection. This pioneering work underscores the immense potential of far-red-based homo-FRET systems in enabling the sensitive and specific detection of chymotrypsin enzyme activity. By bridging the gap between cutting-edge technology and biomedical diagnostics, our findings herald a new era of enzyme sensing, promising transformative advancements in disease diagnosis and dairy quality control.

Thermodynamic and functional changes of alpha-chymotrypsin after interaction with gallic acid.

In the present study, the interaction mechanism between gallic acid (GA) and α-Chymotrypsin (α-CT) was investigated by employing a series ofspectroscopic methods, computational docking and molecular dynamic (MD) simulation. Fluorescence spectra analysis indicated the formation of a stable complex between GA and α-CT, where the quenching of the fluorescence emission was predominantly characterized by a static mechanism. TheCA obtained binding constants for the α-CT-GA complex were in the order of 103 M-1, indicating the moderate binding affinity of GA for α-CT. The corresponding CD findings showed that the interaction between GA and α-CT resulted in an alteration of the protein's secondary structure. The findings of the enzyme activity investigation clearly showed that the presence of GA led to a notable decline in the enzymatic activity of α-CT, highlighting GA's function as an effective inhibitor for α-CT. The molecular docking simulations revealed the optimal binding site for the GA molecule within the α-CT structure and MD simulations confirmed the stability of the α-CT-GA complex. This research expands our comprehension regarding the behavior of enzymes in the presence of small-molecule ligands and opens avenues for food safety.

Thermal treatment of alkali lignin to eliminate its inhibition of pancreatic proteases in vitro.

This study aims to investigate how alkali lignin inhibits protein digestion and explore thermal treatment as a potential solution. Solid alkali lignin species pre-heated at different temperatures (150, 200, and 250 °C) and soluble acid-differentiated fractions are subjected to in vitro protein digestion. A range of techniques, including Thermogravimetric Analysis (TGA), Size-Exclusion Chromatography (SEC), Zeta Potential Analyzer, 1H NMR, Isothermal Titration Calorimetry (ITC), and Molecular Docking, were used to investigate the inhibitory mechanism of alkali lignin on pancreatic proteases hydrolysis. Our results suggest that soluble alkali lignin inhibits pancreatic trypsin and chymotrypsin, with the acid-differentiated soluble fraction (LgpH<1) displaying the strongest inhibition and proteases' binding affinity due to the abundance of polar groups (e.g., -OH, -CHO), which facilitate hydrogen-bond formation. Furthermore, pre-heating lignin (200 °C) was confirmed effective for removing LgpH<1 and its negative nutritional influence, providing a feasible strategy for overcoming the negative impact of alkali lignin on protein digestion.

Chymotrypsin and Trypsin Inhibitory Activity of Some Medicinal Plants Collected from Rize (Türkiye).

In this research, the evaluation of in vitro chymotrypsin and trypsin inhibitory activities of ten plant species collected from Rize were aimed, and fractions that showed strong activity were analyzed through HPLC. Daphne pontica L. and Mentha longifolia (L.) L. were found to have the highest chymotrypsin inhibitory activities (87.75 and 84.24 % inhibition). Similarly, the highest trypsin inhibitory activity was observed in D. pontica (%99.93 inhibition), followed by Sambucus ebulus L. flowers (87.47 % inhibition). Extracts showing strong enzyme inhibition were fractioned and subjected to activity tests. The highest chymotrypsin inhibitory activity was observed in the n-hexane fraction of D. pontica (%80.70 inhibition), while the highest trypsin inhibitory activity was found in the n-butanol fraction of S. ebulus (%86.81 inhibition). HPLC studies determined that the 80 % ethanol extract of D. pontica and its dichloromethane and ethyl acetate fractions contained umbelliferone. It was found that chlorogenic acid was present in the 80 % ethanol extracts of S. ebulus flowers. M. longifolia was found to contain chlorogenic acid, caffeic acid, luteolin-7-glucoside, and rosmarinic acid. M. longifolia has been identified as the plant exhibiting the highest antioxidant activity in ABTS and CUPRAC tests, consistent with its high phenolic and flavonoid content.

Rhodiola rosea L. improved intestinal digestive enzyme activities, inflammatory response, barrier and microbiota dysbiosis in Lateolabrax maculatus juveniles fed with high-carbohydrate diets.

A 56-d feeding trial was conducted to evaluate the influences of Rhodiola rosea L. on digestive enzyme activities, intestinal barrier, inflammatory response, and microbiota dysbiosis in Lateolabrax maculatus juveniles (9.37 ± 0.03 g) fed with high-carbohydrate diets. Six diets were designed: a control diet (20% corn starch, Control), high-carbohydrate diet (30% corn starch, HC1), and four high-carbohydrate diets supplemented with Rhodiola rosea L. at 30, 60, 90 and 120 mg/kg (HC2, HC3, HC4 and HC5, respectively). Compared with the control group, the HC1 diet remarkably increased α-amylase, lipase, and chymotrypsin activities in the intestine (p < 0.05), as well as the mRNA levels of Claudin-15, NF-κB, TNF-α, IL-1ß, and IL-8 (p < 0.05) and the relative abundance of Proteobacteria and Photobacterium in the intestine, which belong to the phylum and genus level, respectively. But the opposite trend was found in muscular thickness and villus lengths (p < 0.05), the mRNA levels of Occludin, ZO-1, and TGF-ß (p < 0.05), at the level of phylum and genus level in the HC1 group, and the relative abundance of Firmicutes, Bacteroidetes, and Bacillus in the intestine compared with the control group. Intestinal chymotrypsin activity was significantly higher in the HC3 group and intestinal muscular thickness and villus lengths were also significantly higher in the HC2, HC3, HC4, and HC5 groups compared to the HC1 group (p < 0.05). In addition, Occludin mRNA expression in the intestine was significantly increased in the HC2, HC4, and HC5 groups compared to the HC1 group. ZO-1 and TGF-ß mRNA expression in the intestine were significantly increased in the HC2, HC3, HC4, and HC5 groups compared to the HC1 group (p < 0.05). At the phylum level, the relative abundance of Firmicutes and Bacteroidetes was higher in the intestine in the HC2, HC3, HC4, and HC5 groups than that in the HC1 group. On the contrary, intestinal lipase and chymotrypsin activities were significantly decreased in the HC2 group compared to the HC1 group, respectively (p < 0.05). The Claudin-15, NF-κB, TNF-α, IL-1ß, and IL-8 mRNA expression in the intestine were significantly decreased in the HC2, HC3, HC4, and HC5 groups compared to the HC1 group (p < 0.05). Besides, at the genus level, compared to the HC1 group, the relative abundance of Photobacterium in the intestine and the diversity of the intestinal microbiota in the HC2, HC3, HC4, and HC5 groups were all decreased. In conclusion, these results demonstrated that the addition of Rhodiola rosea L. in high-carbohydrate diets can improve intestinal digestive enzyme activities, inflammatory response and intestinal barrier-related gene expression, and microbiota dysbiosis in L. maculatus. The suitable supplemental level of Rhodiola rosea L. in high-carbohydrate diets of L. maculatus is 60 mg/kg.

Measurement of enzyme activity of insoluble substrates based on ordered porous layer interferometry and the application in evaluation of thrombolytic drugs.

The development of innovative methods for real-time surveillance of enzymatic activity determination processes is essential, particularly for insoluble substrate enzymatic assessments. In this work, a novel method for enzymatic activity determination was devised by assembling a 190 nm silica colloidal crystal (SCC) film onto a glass slide, coupled with Ordered Porous Layer Interferometry (OPLI) technology. By fixing the substrate of the enzyme on the surface of the silica sphere, a solid-liquid interface can be formed for monitoring enzymatic activity. The enzymatic activity is gauged by the change in the SCC film's thickness caused by the digestion of the loaded substrate. The procedure of chymotrypsin-mediated casein digestion was documented in real time, facilitating the examination of chymotrypsin's activity and kinetics. The newly-developed enzymatic activity determination method demonstrated exceptional sensitivity towards chymotrypsin activity, with a linear range spanning 0.0505-2.02 units per mg. Additionally, the method was extended to the assessment of fibrinolysis enzyme activity and kinetic analysis, yielding promising results. Therefore, this technique can serve as a real-time, user-friendly, cost-effective novel approach for enzymatic activity determination, providing fresh perspectives for enzymatic activity determination studies.

Evaluation of pancreatic chymotrypsin activity for on-site prediction of clinically relevant postoperative pancreatic fistula.

OBJECTIVES: Although the risk of complications due to postoperative pancreatic fistula (POPF) have been evaluated based on the amylase level in drained ascitic fluid, this method has much room for improvement regarding diagnostic accuracy and facility of the measurement. This study aimed to investigate the clinical value of measuring pancreatic chymotrypsin activity for rapid and accurate prediction of POPF after pancreaticoduodenectomy. METHODS: In 52 consecutive patients undergoing pancreaticoduodenectomy, the chymotrypsin activity in pancreatic juice was measured by calculating the increase in fluorescence intensity during the first 5 min after activation with an enzyme-activatable fluorophore. The predictive value for clinically relevant POPF (CR-POPF) was compared between this technique and the conventional method based on the amylase level. RESULTS: According to receiver operating characteristic analyses, pancreatic chymotrypsin activity on postoperative day (POD) 3 measured with a multiplate reader had the highest predictive value for CR-POPF (area under the curve [AUC], 0.752; P < 0.001), yielding 77.8 % sensitivity and 68.8 % specificity. The AUC and sensitivity/specificity of the amylase level in ascitic fluid on POD 3 were 0.695 (P = 0.053) and 77.8 %/41.2 %, respectively. Multivariable analysis identified high pancreatic chymotrypsin activity on POD 3 as an independent risk factor for CR-POPF. Measurement of pancreatic chymotrypsin activity with a prototype portable fluorescence photometer could significantly predict CR-POPF (AUC, 0.731; P = 0.010). CONCLUSION: Measurement of pancreatic chymotrypsin activity enabled accurate and rapid prediction of CR-POPF after pancreaticoduodenectomy. This can help surgeons to implement appropriate drain management at the patient's bedside without delay.

Native top-down mass spectrometry for monitoring the rapid chymotrypsin catalyzed hydrolysis reaction.

Enzymes play crucial roles in life sciences, pharmaceuticals and industries as biological catalysts that speed up biochemical reactions in living organisms. New catalytic reactions are continuously developed by enzymatic engineering to meet industrial needs, which thereby drives the development of analytical approaches for real-time reaction monitoring to reveal catalytic processes. Here, taking the hydrolase- chymotrypsin as a model system, we proposed a convenient method for monitoring catalytic processes through native top-down mass spectrometry (native TDMS). The chymotrypsin sample heterogeneity was first explored. By altering sample introduction modes and pHs, covalent and noncovalent enzymatic complexes, substrates and products can be monitored during the catalysis and further confirmed by tandem MS. Our results demonstrated that native TDMS based catalysis monitoring has distinctive strength on real-time inspection and continuous observation, making it a promising tool for characterizing more biocatalysts.

Thrombin has dual trypsin-like and chymotrypsin-like specificity.

BACKGROUND: The residue at the site of activation of protein C is Arg in all species except the ray-finned fish, where it is Trp. This feature raises the question of whether thrombin is the physiological activator of protein C across vertebrates. OBJECTIVES: To establish if thrombin can cleave at Trp residues. METHODS: The activity of wild-type thrombin and mutant D189S was tested with a library of chromogenic substrates and toward wild-type protein C and mutants carrying substitutions at the site of cleavage. RESULTS: Thrombin has trypsin-like and chymotrypsin-like specificity and cleaves substrates at Arg or Trp residues. Cleavage at Arg is preferred, but cleavage at Trp is significant and comparable with that of chymotrypsin. The D189S mutant of thrombin has broad specificity and cleaves at basic and aromatic residues without significant preference. Thrombin also cleaves natural substrates at Arg or Trp residues, showing activity toward protein C across vertebrates, including the ray-finned fish. The rate of activation of protein C in the ray-finned fish is affected by the sequence preceding Trp at the scissile bond. CONCLUSION: The results provide a possible solution for the paradoxical presence of a Trp residue at the site of cleavage of protein C in ray-finned fish and support thrombin as the physiological activator of protein C in all vertebrates. The dual trypsin-like and chymotrypsin-like specificity of thrombin suggests that the spectrum of physiological substrates of this enzyme is broader currently assumed.

Antagonizing pathological α-synuclein-mediated neurodegeneration by J24335 via the activation of immunoproteasome.

The aggregation of misfolded proteins, such as α-synuclein in Parkinson's disease (PD), occurs intracellularly or extracellularly in the majority of neurodegenerative diseases. The immunoproteasome has more potent chymotrypsin-like activity than normal proteasome. Thus, degradation of α-synuclein aggregation via immunoproteasome is an attractive approach for PD drug development. Herein, we aimed to determine if novel compound, 11-Hydroxy-1-(8-methoxy-5-(trifluoromethyl)quinolin-2-yl)undecan-1-one oxime (named as J24335), is a promising candidate for disease-modifying therapy to prevent the pathological progression of neurodegenerative diseases, such as PD. The effects of J24335 on inducible PC12/A53T-α-syn cell viability and cytotoxicity were evaluated by MTT assay and LDH assay, respectively. Evaluation of various proteasome activities was done by measuring the luminescence of enzymatic activity after the addition of different amounts of aminoluciferin. Immunoblotting and real-time PCR were employed to detect the expression of various proteins and genes, respectively. We also used a transgenic mouse model for behavioral testing and immunochemical analysis, to assess the neuroprotective effects of J24335. J24335 inhibited wild-type and mutant α-synuclein aggregation without affecting the growth or death of neuronal cells. The inhibition of α-synuclein aggregation by J24335 was caused by activation of immunoproteasome, as mediated by upregulation of LMP7, and increased cellular chymotrypsin-like activity in 20S proteasome. J24335-enhanced immunoproteasome activity was mediated by PKA/Akt/mTOR pathway activation. Moreover, animal studies revealed that J24335 treatment markedly mitigated both the loss of tyrosine hydroxylase-positive (TH-) neurons and impaired motor skill development. This is the first report to use J24335 as an immunoproteasome enhancing agent to antagonize pathological α-synuclein-mediated neurodegeneration.

Quantification of human serum albumin by combining chymotrypsin/trypsin digestion coupled with LC-MS/MS technique.

The quantification of albumin is important in clinical medicine because the concentration of albumin in biological fluids is closely related to human health. In this study, we developed a highly selective and robust assay to determine human serum albumin (HSA) in human plasma by combining chymotrypsin/trypsin digestion coupled with targeted LC-MS/MS technique. Human plasma samples were denatured, reduced, alkylated, and digested with both chymotrypsin and trypsin to generate surrogate peptides. A unique chymotryptic peptide (NAETF) arising from human serum albumin was finally selected for targeted LC-MS/MS detection and quantification. Numerous parameters related to the targeted LC-MS/MS assay were evaluated, including lower limit of quantitation (LLOQ), linearity range, enzyme digestion efficiency, accuracy and precision. The LC-MS/MS assay was linear in the concentration range 0.05-1 mg/mL with intra-day and inter-day precision <10.2% and accuracy ranging from -3.94% to 4.89%. The assay was successfully applied to determine HSA in 148 human plasma samples.

Structural dynamics influences the antibacterial activity of a cell-penetrating peptide (KFF)3K.

Given the widespread demand for novel antibacterial agents, we modified a cell-penetrating peptide (KFF)3K to transform it into an antibacterial peptide. Namely, we inserted a hydrocarbon staple into the (KFF)3K sequence to induce and stabilize its membrane-active secondary structure. The staples were introduced at two positions, (KFF)3K[5-9] and (KFF)3K[2-6], to retain the initial amphipathic character of the unstapled peptide. The stapled analogues are protease resistant contrary to (KFF)3K; 90% of the stapled (KFF)3K[5-9] peptide remained undigested after incubation in chymotrypsin solution. The stapled peptides showed antibacterial activity (with minimal inhibitory concentrations in the range of 2-16 µM) against various Gram-positive and Gram-negative strains, contrary to unmodified (KFF)3K, which had no antibacterial effect against any strain at concentrations up to 32 µM. Also, both stapled peptides adopted an α-helical structure in the buffer and micellar environment, contrary to a mostly undefined structure of the unstapled (KFF)3K in the buffer. We found that the antibacterial activity of (KFF)3K analogues is related to their disruptive effect on cell membranes and we showed that by stapling this cell-penetrating peptide, we can induce its antibacterial character.

[Dynamic Changes in the Activities and Contents of Particular Proteasome Forms in the Cerebral Cortex of C57BL/6 Mice during Aging].

Proteasomes are key components of the ubiquitin-proteasome system. Various forms of proteasomes are known. During aging, disturbances in the functioning of proteasomes have been revealed, as well as increased expression of their particular forms. Considering these data, we studied the expression of genes encoding the constitutive and immune subunits of proteasomes in cerebral cortex samples from C57BL/6 mice at the ages of 60, 190, 380, and 720 days. In addition, the contents of constitutive and immune proteasome subunits, chymotrypsin-like and caspase-like activities of proteasome pools, as well as the activity of the ß5i immune subunit were studied in tissue homogenates. The chymotrypsin-like activity and the activity of the ß5i subunit of different forms of proteasomes separated by electrophoresis in native gel were characterized. Compared with samples from young animals, in the cerebral cortex of animals at an age of 720 days the following changes in the expression patterns of proteasome genes were revealed: a decreased expression of the PSMB5 gene encoding constitutive proteasome subunit ß5; increased expression of genes encoding immune proteasome subunits ß5i and ß1i. In tissue homogenates of aged mice, an increase in the content of immune subunits ß1i and ß2i was shown. In samples from old animals, chymotrypsin-like activity was decreased and a tendency to a decrease in caspase-like activity of proteasomes as well as the ß5i subunit activity was revealed. Analysis of the activity of native complexes in tissues obtained from old animals revealed decreased chymotrypsin-like activity of 26S and 20S proteasomes containing the ß5i subunit. Based on the obtained data, it can be assumed that changes in the pool of nonconstitutive proteasomes reflect aging-associated adaptive processes in the mouse brain.

Changes in digestive enzyme activities during the early ontogeny of milkfish, Chanos chanos larvae.

Knowledge of the developmental ontogeny of the digestive system and nutritional requirements of marine fish larvae is a primary requisite for their successful rearing under an optimal feeding regime. In this context, we assessed the activity profile of key digestive enzymes viz., trypsin, chymotrypsin, leucine aminopeptidase, lipase, amylase, and alkaline phosphatase during the early ontogeny of milkfish, Chanos chanos (0 day, 3 days, 6 days, 9 days, 12 days, 15 days, 18 days, 21 days, 25 days, and 30 days post-hatch). Larvae for this study were obtained from the successful breeding of milkfish at ICAR-Central Institute of Brackishwater Aquaculture, India. Growth curves (length and weight) of the larvae indicated a positive morphological development under a standardized feeding regime that comprised Chlorella salina, Brachionus plicatilis, Artemia salina nauplii, and commercial weaning feed for different larval stages. With respect to protein digestion, the specific activity of pancreatic enzymes trypsin and chymotrypsin and intestinal brush border leucine aminopeptidase showed two peaks at 3 dph and 15 dph, following the introduction of rotifer and Artemia nauplii. Similar bimodal peaks were observed for alkaline phosphatase and amylase activities, with the first peak at 3 dph and the second peak at 18 dph and 21 dph, respectively. Whereas in the case of lipase, high activity levels were observed at 0 dph, 3 dph, and 18 dph, with subsequent decreases and fluctuations. Overall, as most of the enzymes were found to have peak activities at 15 to 21 dph, this period can be potentially considered as the developmental window for weaning larvae from live to formulated feeds in milkfish hatcheries.

Recent increase in low complexity polygenomic infections and sialic acid-independent invasion pathways in Plasmodium falciparum from Western Gambia.

BACKGROUND: The malaria parasite Plasmodium falciparum utilizes multiple alternative receptor-ligand interactions for the invasion of human erythrocytes. While some P. falciparum clones make use of sialic acid (SA) residues on the surface of the human glycophorin receptors to invade the erythrocyte, others use alternative receptors independent of sialic acid residues. We hypothesized that over the years, intensified malaria control interventions and declining prevalence in The Gambia have resulted in a selection of parasites with a dominant invasion pathways and ligand expression profiles. METHODS: Blood samples were collected from 65 malaria-infected participants with uncomplicated malaria across 3 years (2015, 2016, and 2021). Genetic diversity was determined by genotyping the merozoite surface protein 2 (msp2) polymorphic gene of P. falciparum. Erythrocyte invasion phenotypes were determined using neuraminidase, trypsin, and chymotrypsin enzymes, known to cleave different receptors from the surface of the erythrocyte. Schizont-stage transcript levels were obtained for a panel of 6 P. falciparum invasion ligand genes (eba175, eba181, Rh2b, Rh4, Rh5, and clag2) using 48 successfully cultured isolates. RESULTS: Though the allelic heterozygosity of msp2 repeat region decreased as expected with reduced transmission, there was an increase in infections with more than a single msp2 allelotype from 2015 to 2021. The invasion phenotypes of these isolates were mostly SA independent with a continuous increase from 2015 to 2021. Isolates from 2021 were highly inhibited by chymotrypsin treatment compared to isolates from 2015 and 2016. Higher invasion inhibition for 2021 isolates was further obtained following erythrocyte treatment with a combination of chymotrypsin and trypsin. The transcript levels of invasion ligand genes varied across years. However, levels of clag2, a rhoptry-associated protein, were higher in 2015 and 2016 isolates than in 2021 isolates, while Rh5 levels were higher in 2021 compared to other years. CONCLUSIONS: Overall, these findings suggest increasing mixed infections with an increase in the use of sialic-acid independent invasion pathways by P. falciparum clinical isolates in the Western part of Gambia.

Cyclotheonellazoles D-I, Potent Elastase Inhibitory Thiazole-Containing Cyclic Peptides from Theonella sp. (2131).

Six new thiazole-containing cyclic peptides, the cyclotheonellazoles D-I (1-6), were isolated from the Australian marine sponge Theonella sp. (2131) with their structures assigned by comprehensive 1D and 2D NMR spectroscopic and MS spectrometric analyses, Marfey's derivatization studies, and comparison with time-dependent density functional theory (TDDFT) calculated ECD data. The Type 2 azole-homologated peptides herein comprise up to five nonproteinogenic amino acids, including the protease transition state mimic α-keto-ß-amino acid residue 3-amino-4-methyl-2-oxohexanoic acid (Amoha), while 1-3 also contain a terminal hydantoin residue not previously found in cyclotheonellazoles. The keramamides A (7) and L (8) were reisolated affording expanded exploration of their biological activities. The peptides were examined for protease inhibitory activities against two mammalian serine proteases (elastase and chymotrypsin) and SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), a validated antiviral therapeutic target for COVID-19. Peptides 1-6 and keramamide A (7) displayed potent nanomolar inhibition of elastase (IC50 16.0 to 61.8 nM), while 7 also contained modest inhibition of chymotrypsin and SARS-CoV-2 3CLpro (IC50 0.73 and 1.1 µM, respectively). The cyclotheonellazoles D-E (1-3) do not affect the viability of human breast, ovarian, and colon cancer cells (>100 µM), with the cytotoxicity previously reported for keramamide L (8) not replicated (inactive >20 µM).

Substrate specificity of human chymotrypsin-like protease (CTRL) characterized by phage display-selected small-protein inhibitors.

Chymotrypsin-like protease (CTRL) is one of the four chymotrypsin isoforms expressed in the human exocrine pancreas. Human genetic and experimental evidence indicate that chymotrypsins B1, B2, and C (CTRB1, CTRB2 and CTRC) are important not only for protein digestion but also for protecting the pancreas against pancreatitis by degrading potentially harmful trypsinogen. CTRL has not been reported to play a similar role, possibly due to its low abundance and/or different substrate specificity. To address this problem, we investigated the specificity of the substrate-binding groove of CTRL by evolving the substrate-like canonical loop of the Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), a small-protein reversible chymotrypsin inhibitor to bind CTRL. We found that phage-associated SGPI-2 variants with strong affinity to CTRL were similar to those evolved previously against CTRB1, CTRB2 or bovine chymotrypsin A (bCTRA), indicating comparable substrate specificity. When tested as recombinant proteins, SGPI-2 variants inhibited CTRL with similar or slightly weaker affinity than bCTRA, confirming that CTRL is a typical chymotrypsin. Interestingly, an SGPI-2 variant selected with a Thr29His mutation in its reactive loop was found to inhibit CTRL strongly, but it was digested rapidly by bCTRA. Finally, CTRL was shown to degrade human anionic trypsinogen, however, at a much slower rate than CTRB2, suggesting that CTRL may not have a significant role in the pancreatic defense mechanisms against inappropriate trypsinogen activation and pancreatitis.

Heliothis virescens chymotrypsin is translationally controlled by AeaTMOF binding ABC putative receptor.

Heliothis virescens larval chymotrypsin (GenBank accession number AF43709) was cloned, sequenced and its three dimensional (3D) conformation modeled. The enzyme's transcript was first detected 6 days after larval emergence and the transcript level was shown to fall between larval ecdysis periods. Comparisons between the activities of larval gut chymotrypsin and trypsin shows that chymotrypsin activity is only 16% of the total trypsin activity and the pH optimum of the larval chymotrypsin is between pH 9-10, however the enzyme also exhibited a broad activity between pH 4-6. Injections of AeaTMOF and several shorter analogues into 3rd instar larvae followed by Northern blot analyses showed that although the chymotrypsins activities were inhibited by 60%-80% the transcript level of the sequenced chymotrypsin was not reduced and was similar to controls in which the chymotrypsin activity was not inhibited, indicating that AeaTMOF and its analogues exert a translational control. Based on these observations a putative AeaTMOF receptor (ABCC4) homologous to the Ae. aegypti ABC receptor sequence was found in the H. virescens genome. 3D molecular modeling and docking of the AeaTMOF and several of its analogues to the ABCC4 receptor showed that it can bind AeaTMOF and its analogues as was shown before for the Ae. aegypti receptor.

First insights into the activity of major digestive enzymes in the intestine of the European catfish Silurus glanis and protective anti-enzymatic potential of its gut parasite Silurotaenia siluri.

The European catfish Silurus glanis is attracting growing interest as an object of fisheries and aquaculture, which is reinforced by the expansion of its natural range under climate change. Shaping the effective exploitation strategy for this valuable species requires detailed knowledge of its biology, including feeding and digestion processes, especially near the natural limits of the species range. Meanwhile, the digestion physiology of the European catfish remains poorly explored, including the activity of major digestive enzymes and the possible effect of intestinal parasites on this activity. In this regard, the activity of proteinases and α-amylase in the intestinal mucosa of the catfish was studied. Adult catfish were collected in the Rybinsk reservoir (Upper Volga) located close to the northern limit of the species range. It was shown that all subclasses of intestinal digestive proteinases, including serine proteinases, metalloproteases and cysteine (thiol) proteinases, function in the gut mucosa of the catfish. The mucosal levels of total proteolytic activity depended on fish size, in contrast to those of trypsin, chymotrypsin and α-amylase. The level of chymotrypsin activity was significantly higher than that of trypsin activity. It was also found that the incubation medium and extract of the cestodes Silurotaenia siluri parasitizing the catfish gut had a significant inhibitory effect on the activity of serine proteases (trypsin and chymotrypsin) operating in the intestines of the host fish.