Monitoring Proteolytic Activity in Real Time: A New World of Opportunities for Biosensors.

Proteases play a pivotal role in several biological processes, from digestion, cell proliferation, and differentiation to fertility. Deregulation of protease metabolism can result in several pathological conditions (i.e., cancer, neurodegenerative disorders, and others). Therefore, monitoring proteolytic activity in real time could have a fundamental role in the early diagnosis of these diseases. Herein, the main approaches used to develop biosensors for monitoring proteolytic activity are reviewed. A comparison of the advantages and disadvantages of each approach is provided along with a discussion of their importance and promising opportunities for the early diagnosis of severe diseases. This new era of biosensors can be characterized by the ability to control and monitor biological processes, ultimately improving the potential of personalized medicine.

The proteolytic activity in inflammatory bowel disease: insight from gut microbiota.

Inflammatory bowel disease (IBD) is a chronic, recurrent inflammatory disease caused by the destruction of the intestinal mucosal epithelium that affects a growing number of people worldwide. Although the etiology of IBD is complex and still elucidated, the role of dysbiosis and dysregulated proteolysis is well recognized. Various studies observed altered composition and diversity of gut microbiota, as well as increased proteolytic activity (PA) in serum, plasma, colonic mucosa, and fecal supernatant of IBD compared to healthy individuals. The imbalance of intestinal microecology and intestinal protein hydrolysis were gradually considered to be closely related to IBD. Notably, the pivotal role of intestinal microbiota in maintaining proteolytic balance received increasing attention. In summary, we have speculated a mesmerizing story, regarding the hidden role of PA and microbiota-derived PA hidden in IBD. Most importantly, we provided the diagnosis and therapeutic targets for IBD as well as the formulation of new treatment strategies for other digestive diseases and protease-related diseases.

Phytaspase Does Not Require Proteolytic Activity for Its Stress-Induced Internalization.

Phytaspases differ from other members of the plant subtilisin-like protease family by having rare aspartate cleavage specificity and unusual localization dynamics. Phytaspases are secreted from healthy plant cells but are re-internalized upon perception of death-inducing stresses. Although proteolytic activity is required for the secretion of plant subtilases, its requirement for the retrograde transportation of phytaspases is currently unknown. To address this issue, we employed an approach to complement in trans the externalization of a prodomain-less form of Nicotiana tabacum phytaspase (NtPhyt) with the free prodomain in Nicotiana benthamiana leaf cells. Using this approach, the generation of the proteolytically active NtPhyt and its transport to the extracellular space at a level comparable to that of the native NtPhyt (synthesized as a canonical prodomain-containing precursor protein) were achieved. The application of this methodology to NtPhyt with a mutated catalytic Ser537 residue resulted in the secretion of the inactive, although processed (prodomain-free), protein as well. Notably, the externalized NtPhyt Ser537Ala mutant was still capable of retrograde transportation into plant cells upon the induction of oxidative stress. Our data thus indicate that the proteolytic activity of NtPhyt is dispensable for stress-induced retrograde transport of the enzyme.

SARS-CoV-2 Nucleocapsid Protein Is Not Responsible for Over-Activation of Complement Lectin Pathway.

The nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a viral structural protein that is abundant in the circulation of infected individuals. Previous published studies reported controversial data about the role of the N protein in the activation of the complement system. It was suggested that the N protein directly interacts with mannose-binding lectin-associated serine protease-2 (MASP-2) and stimulates lectin pathway overactivation/activity. In order to check these data and to reveal the mechanism of activation, we examined the effect of the N protein on lectin pathway activation. We found that the N protein does not bind to MASP-2 and MASP-1 and it does not stimulate lectin pathway activity in normal human serum. Furthermore, the N protein does not facilitate the activation of zymogen MASP-2, which is MASP-1 dependent. Moreover, the N protein does not boost the enzymatic activity of MASP-2 either on synthetic or on protein substrates. In some of our experiments, we observed that MASP-2 digests the N protein. However, it is questionable, whether this activity is biologically relevant. Although surface-bound N protein did not activate the lectin pathway, it did trigger the alternative pathway in 10% human serum. Additionally, we detected some classical pathway activation by the N protein. Nevertheless, we demonstrated that this activation was induced by the bound nucleic acid, rather than by the N protein itself.

Exploring the extraction, functional properties, and industrial applications of papain from Carica papaya.

Papain a protease enzyme naturally present in the Carica papaya has gained significant interest across several industries due to its unique properties and versatility. The unique structure of papain imparts the functionality that assists in elucidating how papain enzyme works and making it beneficial for a variety of purposes. This review highlights recent advancements in papain extraction techniques to enhance production efficiency to meet market demand. The extraction of papain from the Carica papaya plant offers various advantages such as cost-effectiveness, biodegradability, safety, and the ability to withstand a wide range of pH and temperature conditions. Key findings reveal that non-conventional papain extraction techniques offer significant advantages in terms of efficiency, product quality, and environmental sustainability. Furthermore, papain treatment enhances the value of final products due to its anti-bacterial, anti-oxidant, and anti-obesity properties. The ability of papain to hydrolyze a wide range of proteins across various conditions makes it a suitable protease enzyme. While the study emphasizes the advantages of papain, the study also acknowledges limitations such as the continuous research and development to optimize extraction processes which will help unlock papain's potential and meet the growing demand. © 2024 Society of Chemical Industry.

Phosphorylation of enteroviral 2Apro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis.

Enteroviral 2A proteinase (2Apro ), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential for enterovirus A71 (EV-A71) and other viruses replication. We recently showed that ERK1/2 is required for the proteolytic activity of viral 2Apro ; however, the mechanism underlying the regulation of 2Apro remains unknown. Here, we demonstrated that the 125th residue Ser125 of EV-A71 2Apro or Thr125 of coxsackievirus B3 2Apro , which is highly conserved in the Enterovirus, was phosphorylated by ERK1/2. Importantly, 2Apro with phosphor-Ser/Thr125 had much stronger proteolytic activity toward eukaryotic initiation factor 4GI and rendered the virus more efficient for multiplication and pathogenesis in hSCARB2 knock-in mice than that in nonphospho-Ser/Thr125A (S/T125A) mutants. Notably, phosphorylation-mimic mutations caused deleterious changes in 2Apro catalytic function (S/T125D/E) and in viral propagation (S125D). Crystal structure simulation analysis showed that Ser125 phosphorylation in EV-A71 2Apro enabled catalytic Cys to adopt an optimal conformation in the catalytic triad His-Asp-Cys, which enhances 2Apro proteolysis. Therefore, we are the first to report Ser/Thr125 phosphorylation of 2Apro increases enteroviral adaptation to the host to ensure enteroviral multiplication, causing pathogenicity. Additionally, weakened viruses containing a S/T125A mutation could be a general strategy to develop attenuated Enterovirus vaccines.

Initial study of the detection of ADAM 10 in the urine of type-2 diabetic patients.

Diabetes mellitus (DM) is a disease of civilization. If left untreated, it can cause serious complications and significantly shortens the life time. DM is one of the leading causes of end-stage renal disease (uremia) worldwide. Early diagnosis is a prerequisite for successful treatment, preferably before the first symptoms appear. In this paper, we describe the optimization and synthesis of the internally quenched fluorescent substrate disintegrin and metalloproteinase 10 (ADAM10). Using combinatorial chemistry methods with iterative deconvolution, the substrate specificity of the enzyme in non-primed and primed positions was determined. We used the ABZ-Lys-Ile-Ile-Asn-Leu-Lys-Arg-Tyr(3-NO2)-NH2 peptide to study ADAM10 activity in urine samples collected from patients diagnosed with type 2 diabetes, compared to urine samples from healthy volunteers. The proteolytically active enzyme was present in diabetes samples, while in the case of healthy people we did not observe any activity. In conclusion, our study provides a possible basis for further research into the potential role of ADAM10 in the diagnosis of type 2 diabetes.

Analysis of Some Biochemical Properties of Recombinant Siberian Roe Deer (Capreolus pygargus) Chymosin Obtained in the Mammalian Cell Culture (CHO-K1).

Structure of the chymosin gene of Siberian roe deer (Capreolus pygargus) was established for the first time and its exon/intron organization was determined. Coding part of the chymosin gene of C. pygargus was reconstructed by the Golden Gate method and obtained as a DNA clone. Comparative sequence analysis of the roe deer, cow, and one-humped camel prochymosins revealed a number of amino acid substitutions at the sites forming the substrate-binding cavity of the enzyme and affecting the S4 and S1' + S3' specificity subsites. Integration vector pIP1 was used to construct a plasmid pIP1-Cap in order to express recombinant roe deer prochymosin gene in CHO-K1 cells. CHO-K1-CYM-Cap pool cells were obtained, allowing synthesis and secretion of recombinant prochymosin into the culture fluid. As a result of zymogen activation, a recombinant roe deer chymosin was obtained and its total milk-clotting activity was estimated to be 468.4 ± 11.1 IMCU/ml. Yield of the recombinant roe deer chymosin was 500 mg/liter or ≈468,000 IMCU/liter, which exceeds the yields of genetically engineered chymosins in most of the expression systems used. Basic biochemical properties of the obtained enzyme were compared with the commercial preparations of recombinant chymosins from one-humped camel (Camelus dromedarius) and cow (Bos taurus). Specific milk-clotting activity of the recombinant chymosin of C. pygargus was 938 ± 22 IMCU/mg, which was comparable to that of the reference enzymes. Non-specific proteolytic activity of the recombinant roe deer chymosin was 1.4-4.5 times higher than that of the cow and camel enzymes. In terms of coagulation specificity, recombinant chymosin of C. pygargus occupied an intermediate position between the genetically engineered analogs of B. taurus and C. dromedarius chymosins. Thermostability threshold of the recombinant roe deer chymosin was 55°C. At 60°C, the enzyme retained <1% of its initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C.

Multifunctional potentials of lactic acid bacterial isolates from Turkish traditional fermented foods.

This study aimed to isolate LAB strains with antimicrobial activity that can be used as bio-protective agents, from yogurt, pickle, and cheese samples, which are Turkish traditional fermented foods, and to reveal the potential probiotic properties of these isolates with antimicrobial activity. In addition, it was aimed for the first time to examine the potential and uniqueness of these domestic and local traditional fermented foods. In the study, a total of 682 LAB isolates from Turkish traditional fermented samples were isolated. Thirteen isolates with both high and antifungal activities were selected; four Lactiplantibacillus pentosus strains, six Lactiplantibacillus plantarum strains, and one each Enterococcus lactis, Enterococcus durans, and Enterococcus faecalis strains. Ten isolates of them were suggested as potential probiotics (excluding 1Y2-1, 1Y7-3, and 3Y12-4). It was thought that the L. pentosus 2Y7-1, which was slow to produce acid, could be used as a co-culture in fermented products with a long ripening stage, supported by enriching the product of its milk coagulation, ß-galactosidase, proteolytic, and cholesterol assimilation abilities in terms of bioactive peptide and prebiotics. Another strain, E. faecalis 3Y4-3, which was high proteolytic activity, was suggested as a co-culture in fermented products to improve both organoleptic properties and increase bioactive peptides. On the other hand, among non-probiotics, L. plantarum 3Y12-4 can be used as a starter culture in fermented milk products in order to evaluate its milk coagulation, cholesterol assimilation, and proteolytic abilities. As a result, these three strains can be used in many areas, such as fermented and/or functional food production and enzyme production. In addition, this has been the first study to examine the potential and uniqueness of Turkish domestic and local traditional fermented foods in Samsun, Turkey, together, in terms of both antimicrobial and probiotic LAB isolate (multifunctional) potential and diversity.

Volatilome of brine-related microorganisms in a curd-based medium.

The possible contribution of brine-derived microflora to the sensory attributes of cheese is still a rather unexplored field. In this study, 365 bacteria and 105 yeast strains isolated from 11 cheese brines were qualitatively tested for proteolytic and lipolytic activities, and positive strains were identified by sequencing. Among bacteria, Staphylococcus equorum was the most frequent, followed by Macrococcus caseolyticus and Corynebacterium flavescens. As for yeasts, Debaryomyces hansenii, Clavispora lusitaniae, and Torulaspora delbrueckii were most frequently identified. A total of 38% of bacteria and 59% of yeasts showed at least 1 of the metabolic activities tested, with lipolytic activity being the most widespread (81% of bacteria and 95% of yeasts). Subsequently 15 strains of bacteria and 10 yeasts were inoculated in a curd-based medium and assessed via headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry to determine their volatilome. After a 30-d incubation at 12°C, most strains showed a viability increase of about 2 log cfu/mL, suggesting good adaptability to the cheese environment. A total of 26 compounds were detected in the headspace, carbonyl compounds and alcohols being the major contributors to the volatile profile of the curd-based medium. Multivariate analysis was carried out to elucidate the overall differences in volatiles produced by selected strains. Principal component analysis and hierarchical clustering analysis demonstrated that the brine-related microorganisms were separated into 3 different groups, suggesting their different abilities to produce volatile compounds. Some of the selected strains have been shown to have interesting aromatic potential and to possibly contribute to the sensory properties of cheese.

In vitro assessment of antimicrobial potential of low molecular weight chitosan and its ability to mechanically reinforce and control endogenous proteolytic activity of dentine.

AIMS: Chitosan-based biomaterials exhibit several properties of biological interest for endodontic treatment. Herein, a low molecular weight chitosan (CH) solution was tested for its antimicrobial activity against Enterococcus faecalis (E. faecalis) and effects on dentine structure. METHODOLOGY: The root canal of 27 extracted uniradicular teeth were biomechanically prepared, inoculated with a suspension of E. faecalis and randomly assigned to be irrigated with either 5.25% sodium hypochlorite (NaClO), 0.2% CH or sterile ultrapure water (W). Bacteriologic samples were collected from root canals and quantified for of E. faecalis colony-forming units (CFUs). The effectiveness of CH over E. faecalis biofilms was further measured using the MBEC Assay®. Additionally, dentine beams and dentine powder were obtained, respectively, from crowns and roots of 20 extracted third molars. Dentine samples were treated or not with 17% EDTA and immersed in either CH or W for 1 min. The effects of CH on dentine structure were evaluated by assessment of the modulus of elasticity, endogenous proteolytic activity and biochemical modifications. RESULTS: The number of E. faecalis CFUs was significantly lower for samples irrigated with CH and NaClO. No significant differences were found between CH and NaClO treatments. Higher modulus of elasticity and lower proteolytic activity were reported for dentine CH-treated specimens. Chemical interaction between CH and dentine was observed for samples treated or not with EDTA. CONCLUSIONS: Present findings suggest that CH could be used as an irrigant during root canal treatment with the triple benefit of reducing bacterial activity, mechanically reinforcing dentine and inhibiting dentine proteolytic activity.

Complexation of Bromelain, Ficin, and Papain with the Graft Copolymer of Carboxymethyl Cellulose Sodium Salt and N-Vinylimidazole Enhances Enzyme Proteolytic Activity.

This study investigates the features of interactions between cysteine proteases (bromelain, ficin, and papain) and a graft copolymer of carboxymethyl cellulose sodium salt with N-vinylimidazole. The objective is to understand the influence of this interactions on the proteolytic activity and stability of the enzymes. The enzymes were immobilized through complexation with the carrier. The interaction mechanism was examined using Fourier-transform infrared spectroscopy and flexible molecular docking simulations. The findings reveal that the enzymes interact with the functional groups of the carrier via amino acid residues, resulting in the formation of secondary structure elements and enzyme's active sites. These interactions induce modulation of active site of the enzymes, leading to an enhancement in their proteolytic activity. Furthermore, the immobilized enzymes demonstrate superior stability compared to their native counterparts. Notably, during a 21-day incubation period, no protein release from the conjugates was observed. These results suggest that the complexation of the enzymes with the graft copolymer has the potential to improve their performance as biocatalysts, with applications in various fields such as biomedicine, pharmaceutics, and biotechnology.

Calpain-3 Is Not a Sodium Dependent Protease and Simply Requires Calcium for Activation.

Calpain-3 (CAPN3) is a muscle-specific member of the calpain family of Ca2+-dependent proteases. It has been reported that CAPN3 can also be autolytically activated by Na+ ions in the absence of Ca2+, although this was only shown under non-physiological ionic conditions. Here we confirm that CAPN3 does undergo autolysis in the presence of high [Na+], but this only occurred if all K+ normally present in a muscle cell was absent, and it did not occur even in 36 mM Na+, higher than what would ever be reached in exercising muscle if normal [K+] was present. CAPN3 in human muscle homogenates was autolytically activated by Ca2+, with ~50% CAPN3 autolysing in 60 min in the presence of 2 µM Ca2+. In comparison, autolytic activation of CAPN1 required about 5-fold higher [Ca2+] in the same conditions and tissue. After it was autolysed, CAPN3 unbound from its tight binding on titin and became diffusible, but only if the autolysis led to complete removal of the IS1 inhibitory peptide within CAPN3, reducing the C-terminal fragment to 55 kDa. Contrary to a previous report, activation of CAPN3, either by raised [Ca2+] or Na+ treatment, did not cause proteolysis of the skeletal muscle Ca2+ release channel-ryanodine receptor, RyR1, in physiological ionic conditions. Treatment of human muscle homogenates with high [Ca2+] caused autolytic activation of CAPN1, accompanied by proteolysis of some titin and complete proteolysis of junctophilin (JP1, full length ~95 kDa), generating an equimolar amount of a diffusible ~75 kDa N-terminal JP1 fragment, but without any proteolysis of RyR1.

Novel Fecal Biomarkers That Precede Clinical Diagnosis of Ulcerative Colitis.

BACKGROUND & AIMS: Altered gut microbiota composition and function have been associated with inflammatory bowel diseases, including ulcerative colitis (UC), but the causality and mechanisms remain unknown. METHODS: We applied 16S ribosomal RNA gene sequencing, shotgun metagenomic sequencing, in vitro functional assays, and gnotobiotic colonizations to define the microbial composition and function in fecal samples obtained from a cohort of healthy individuals at risk for inflammatory bowel diseases (pre-UC) who later developed UC (post-UC) and matched healthy control individuals (HCs). RESULTS: Microbiota composition of post-UC samples was different from HC and pre-UC samples; however, functional analysis showed increased fecal proteolytic and elastase activity before UC onset. Metagenomics identified more than 22,000 gene families that were significantly different between HC, pre-UC, and post-UC samples. Of these, 237 related to proteases and peptidases, suggesting a bacterial component to the pre-UC proteolytic signature. Elastase activity inversely correlated with the relative abundance of Adlercreutzia and other potentially beneficial taxa and directly correlated with known proteolytic taxa, such as Bacteroides vulgatus. High elastase activity was confirmed in Bacteroides isolates from fecal samples. The bacterial contribution and functional significance of the proteolytic signature were investigated in germ-free adult mice and in dams colonized with HC, pre-UC, or post-UC microbiota. Mice colonized with or born from pre-UC-colonized dams developed higher fecal proteolytic activity and an inflammatory immune tone compared with HC-colonized mice. CONCLUSIONS: We have identified increased fecal proteolytic activity that precedes the clinical diagnosis of UC and associates with gut microbiota changes. This proteolytic signature may constitute a noninvasive biomarker of inflammation to monitor at-risk populations that can be targeted therapeutically with antiproteases.

Detection of ADAM15 in urine from patients with bladder cancer.

Cancer is one of the leading causes of death in the United States and Europe. Of the cancers, bladder cancer is the 10th most frequently diagnosed cancer and the 13th most frequently diagnosed cancer in men. There are many studies showing that proteolytic enzymes, e.g. A Disintegrin and Metalloproteinases (ADAMs), play a key role in the development and progression of neoplasms. In this paper, we present the use of chromogenic substrate of ADAM15 for the qualitative determination of specific activity of enzyme in urine of patients with confirmed bladder cancer. In the first step, we optimized the substrate molecule in non-primed positions using combinatorial chemistry. By means of the obtained ABZ-His-Ala-Arg-Gly-ANB-NH2 peptide, we detected ADAM15 activity in urine samples collected from patients diagnosed with bladder cancer. In contrast, we did not observe such activity in urine obtained from healthy volunteers.

Enhancing proteolytic activity of Lysobacter enzymogenes using cold atmospheric plasma.

Cold atmospheric plasma (CAP) is being used recently as a modern technique for microbial random mutagenesis. In the present study, CAP was used to induce mutagenesis in L. enzymogenes which is the bacteria known for producing proteolytic enzymes especially lysyl endopeptidase (Lys C). Enhanced proteolytic activity was the main criteria to select mutant strains. Therefore, the cell suspension of L. enzymogenes strain (ATCC 29487), was exposed to CAP for 30, 45, 90, and 150 s. The proteolytic activity of mutant strains was screened initially by radial caseinolytic assay and then by Ansons method in different phases of bacterial growth in the selected mutants. The purification process of Lysyl endopeptidase as the target enzyme was optimized and for enlightening molecular aspect of CAP mutagenesis, the sequences of the upstream and coding regions of lys C gene from 10 selected mutant strains were determined. The bacterial survival assessment showed that the more CAP treatment time, the less survival rate, however, in all exposure times, a number of survived mutants showed enhanced proteolytic activity. Among 38 out of 100 examined mutants which showed higher proteolytic activity than that of wild type, the M1-30 s mutant exhibited the highest increment to 1.94 fold. The SDS-PAGE analysis showed expected size of purified Lys C from M1-30 s. The Lys C gene from M14-150 s mutant strain (1.4-fold increment) harbored three point mutations which can be effective in enhancing protease activity. In conclusion, the results highlighted the role of CAP for strain improvement process to obtain industrial strains.

New sources of lactic acid bacteria with potential antibacterial properties.

In the face of the growing demand for functional food, the search for new sources of lactic acid bacteria (LAB) becomes a priority. In our research, we used multiplied culture conditions followed by identification via the matrix-assisted laser desorption ionization-time of flight mass spectrometry for seeking LAB strains in plant- and animal-derived sources. Furthermore, the selected LAB isolates were examined for their proteolytic activity as well as antimicrobial action against different bacterial pathogens. The applied method appeared to be useful tool for searching LAB strains within different types of the biological matrices. The best source of the LABs was from calf. Comparing properties of the two selected LABs, those isolated from calf demonstrated the greatest proteolytic and antibacterial properties suggesting that gastrointestinal microbiota are the most valuable LAB source. Nevertheless, second selected strain derived from pickled cucumber juice may be also treated as a promising source of potential probiotic strains.

Simultaneous targeting of CD44 and MMP9 catalytic and hemopexin domains as a therapeutic strategy.

Crosstalk of the oncogenic matrix metalloproteinase-9 (MMP9) and one of its ligands, CD44, involves cleavage of CD44 by the MMP9 catalytic domain, with the CD44-MMP9 interaction on the cell surface taking place through the MMP9 hemopexin domain (PEX). This interaction promotes cancer cell migration and invasiveness. In concert, MMP9-processed CD44 induces the expression of MMP9, which degrades ECM components and facilitates growth factor release and activation, cancer cell invasiveness, and metastasis. Since both MMP9 and CD44 contribute to cancer progression, we have developed a new strategy to fully block this neoplastic process by engineering a multi-specific inhibitor that simultaneously targets CD44 and both the catalytic and PEX domains of MMP9. Using a yeast surface display technology, we first obtained a high-affinity inhibitor for the MMP9 catalytic domain, which we termed C9, by modifying a natural non-specific MMP inhibitor, N-TIMP2. We then conjugated C9 via a flexible linker to PEX, thereby creating a multi-specific inhibitor (C9-PEX) that simultaneously targets the MMP9 catalytic and PEX domains and CD44. It is likely that, via its co-localization with CD44, C9-PEX may compete with MMP9 localization on the cell surface, thereby inhibiting MMP9 catalytic activity, reducing MMP9 cellular levels, interfering with MMP9 homodimerization, and reducing the activation of downstream MAPK/ERK pathway signaling. The developed platform could be extended to other oncogenic MMPs as well as to other important target proteins, thereby offering great promise for creating novel multi-specific therapeutics for cancer and other diseases.

Differences in Charge Distribution in Leishmania tarentolae Leishmanolysin Result in a Reduced Enzymatic Activity.

Leishmania tarentolae is a non-pathogenic trypanosomatid isolated from lizards widely used for heterologous protein expression and extensively studied to understand the pathogenic mechanisms of leishmaniasis. The repertoire of leishmanolysin genes was reported to be expanded in L. tarentolae genome, but no proteolytic activity was detected. Here, we analyzed L. tarentolae leishmanolysin proteins from the genome to the structural levels and evaluated the enzymatic activity of the wild-type and overexpressing mutants of leishmanolysin. A total of 61 leishmanolysin sequences were retrieved from the L. tarentolae genome. Five of them were selected for phylogenetic analysis, and for three of them, we built 3D models based on the crystallographic structure of L. major ortholog. Molecular dynamics simulations of these models disclosed a less negative electrostatic potential compared to the template. Subsequently, L. major LmjF.10.0460 and L. tarentolae LtaP10.0650 leishmanolysins were cloned in a pLEXSY expression system into L. tarentolae. Proteins from the wild-type and the overexpressing parasites were submitted to enzymatic analysis. Our results revealed that L. tarentolae leishmanolysins harbor a weak enzymatic activity about three times less abundant than L. major leishmanolysin. Our findings strongly suggest that the less negative electrostatic potential of L. tarentolae leishmanolysin can be the reason for the reduced proteolytic activity detected in this parasite.

Influence of Calcium Resonance-Tuned Low-Frequency Magnetic Fields on Daphnia magna.

A biophysical model for calculating the effective parameters of low-frequency magnetic fields was developed by Lednev based on summarized empirical data. According to this model, calcium ions as enzyme cofactors can be the primary target of low-frequency magnetic fields with different parameters tuned to calcium resonance. However, the effects of calcium-resonant combinations of static and alternating magnetic fields that correspond to Lednev's model and differ by order in frequency and intensity were not studied. It does not allow for confidently discussing the primary targets of low-frequency magnetic fields in terms of the magnetic influence on ions-enzyme cofactors. To clarify this issue, we examined the response of freshwater crustaceans Daphnia magna to the impact of combinations of magnetic fields targeted to calcium ions in enzymes according to Lednev's model that differ in order of magnitude. Life-history traits and biochemical parameters were evaluated. Exposure of daphnids to both combinations of magnetic fields led to a long-term delay of the first brood release, an increase in the brood size, a decrease in the number of broods, and the period between broods. The amylolytic activity, proteolytic activity, and sucrase activity significantly decreased in whole-body homogenates of crustaceans in response to both combinations of magnetic fields. The similarity in the sets of revealed effects assumes that different magnetic fields tuned to calcium ions in biomolecules can affect the same primary molecular target. The results suggest that the low-frequency magnetic fields with parameters corresponding to Lednev's model of interaction between biological molecules and ions can remain effective with a significant decrease in the static magnetic background.