Search Results (57)

Background/Objectives: Gingipains produced by P. gingivalis have been shown to be directly related to periodontal tissue degradation and are significant molecular targets in therapy of periodontitis. Blocking the activity of these enzymes should reduce survival of this pathogen and mitigate the effects of inflammation in periodontitis. Therefore, gingipains inhibitors and specific antibodies could be recommended in the treatment of periodontitis. Cysteine peptidase inhibitors can be obtained by chemical synthesis, or isolated from natural raw materials. This research has the following aims: 1. to analyze in vitro the inhibition of cysteine protease activity in the gingival crevicular fluid (GCF) and 2. to compare the toxicity of natural raw inhibitors (obtained from Fallopia japonica plant and egg white) with chlorhexidine (CHX) using an MTS viability test. Methods: Samples of GCF were collected from healthy (N = 17) individuals and (N = 65) periodontal patients. Cysteine peptidase activity was inhibited by adding a solution of cystatin from egg white (with 20% glycerol), or cystatin from knotweed, or low molecular weight inhibitors (MW < 3 kDa) from egg white and knotweed against Nα-Benzoyl-DL-arginine 4-nitroanilide hydrochloride. Results: There was a statistically significant difference between the inhibition means of cysteine protease activity for the five groups (p < 0.001). Means for the four groups of patients with periodontitis were not statistically significant different from each other (p = 0.320). The inhibition rates were higher in periodontitis patients. The toxicity of knotweed cystatin inhibitor was several times lower than the toxicity of E-64d, and of CHX. Conclusion: Cysteine protease inhibitors isolated from egg or plants were non-toxic, effectively inhibited the activity of cysteine proteases in GCF, and may be a promising alternative to more toxic standard antimicrobials (CHX) in preventing periodontal tissue breakdown. Full article

Chagas disease remains a significant public health concern, with limited treatment options and an urgent need for novel preventive strategies. Extracellular vesicles (EVs) from Trypanosoma cruzi have been shown to modulate host immune responses, often favoring parasite persistence. In this study, we characterized EVs derived from the non-pathogenic trypanosomatids Trypanosoma rangeli and Phytomonas serpens and evaluated their potential as immunogens capable of inducing cross-protection against T. cruzi infection. Isolated EVs were characterized by Nanoparticle Tracking Analysis (NTA) and electron microscopy. A comparative proteomic analysis of EVs was performed using Mass Spectrometry-Based Proteomic Analysis (LC-MS/MS). The effects of EVs on immunomodulation and T. cruzi infection were assessed through in vitro and in vivo assays, using peripheral blood mononuclear cells (PBMCs) and BALB/c mice. The proteomic analysis identified shared proteins between the EVs of T. rangeli, P. serpens, and T. cruzi, including immunogenic candidates such as calpain-like cysteine peptidase and elongation factor 2. In vitro, pre-stimulation with the T. rangeli EVs reduced infection rates of the host cells by T. cruzi. In vivo, immunization with the EVs from T. rangeli and P. serpens led to a significant reduction in parasitemia in the BALB/c mice challenged with T. cruzi, though this did not translate into improved survival compared to controls. Interestingly, the EVs from T. cruzi also reduced parasitemia but did not confer protection against mortality. These findings suggest that while non-pathogenic trypanosomatid EVs exhibit potential immunogenic properties and can reduce parasitic load, their efficacy in preventing disease progression remains limited. Further research is needed to explore the mechanisms underlying these effects and to optimize EV-based strategies for protective immunity against Chagas disease. Full article

Colletotrichum lindemuthianum is the most frequent pathogenic fungus of the common bean Phaseolus vulgaris. This filamentous fungus employs a hemibiotrophic nutrition/infection strategy, which is characteristic of many Colletotrichum species. Due to host–pathogen coevolution, C. lindemuthianum includes pathotypes with a diversity of virulence against differential common bean varieties. In this study, we performed comparative genomic analyses on three pathotypes with different virulence levels and a non-pathogenic pathotype, isolated from different geographical areas in Mexico. Our results revealed large genomes with high transposable element contents that have undergone expansions, generating intraspecific diversity. All the pathotypes exhibited a similar number of clusters of orthologous genes (COGs) and Gene Ontology (GO) terms. TFomes contain families that are typical in fungal genomes; however, they show different contents between pathotypes, mainly in transcription factors with the fungal-specific TF and Zn2Cys6 domains. Peptidase families mainly contain abundant serine peptidases, metallopeptidases, and cysteine peptidases. In the secretomes, the number of genes differed between the pathotypes, with a high percentage of candidate effectors. Both the virulence gene and CAZyme gene content for each pathotype was abundant and diverse, and the latter was enriched in hemicellulolytic enzymes. We provide new insights into the nature of intraspecific diversity among C. lindemuthianum pathotypes and the origin of their ability to rapidly adapt to genetic changes in its host and environmental conditions. Full article

An insect’s gut microbiome is an essential “organ” in their life cycle, playing a crucial role by aiding food digestion and nutrient absorption. This study employed both culture-independent and culture-dependent methods to explore the protease resources present in the gut of the omnivorous insect Gryllotalpa orientalis. The findings revealed that the gut extract of G. orientalis contained a diverse array of proteases, including cysteine proteases, pepsin, serine proteases, and trypsin, as well as some unidentified proteases. Furthermore, the protease gene htpX, derived from gut bacterium Priestia megaterium DX-3, has been cloned and recombinantly expressed. The recombinant DX-3-htpX protease exhibited a 61.9-fold increase in fermentation level compared to the DX-3 protease. This protease was characterized as a neutral, heat-resistant metalloprotease with an M48 peptidase domain, and it was observed that the binding of Ca²⁺ to the recombinant protease resulted in the formation of the largest active pocket. This study provides technical support for further development and utilization of functional protein resources in insect gut. Full article

To test the hypothesis that they serve as systemic biomarkers of chronic obstructive pulmonary disease (COPD), we profiled the mRNA expression of enzymes connected to systemic inflammation and GSH metabolism in peripheral blood mononuclear cells (PBMCs). These were taken from patients displaying acute exacerbation of COPD (AE-COPD) and stable COPD, and also from non-obstructive smokers and non-smokers. The expression of poly(ADP-ribose) polymerase-1 was increased, but that of histone deacetylase 2 was decreased in association with AE-COPD. The expression of modulatory subunit of glutamyl–cysteine ligase was higher and that of its catalytic subunit, together with the expression of dipeptidyl peptidase 4, was lower in COPD patients compared with non-obstructive smokers and non-smokers. Leukotriene A4 hydrolase saw increased expression in patients with COPD according to disease severity compared to non-obstructive individuals, whereas the expression of GSH peroxidase increased in non-obstructive smokers and COPD patients with the growing number of pack-years smoked. The results corroborate COPD and its acute exacerbation as a complex systemic disorder demonstrating distinct associations with the expression of enzymes linked to inflammation and the regulation of GSH metabolism. Full article

Aspergillus fungi constitute a pivotal element within ecosystems, serving as both contributors of biologically active compounds and harboring the potential to cause various diseases across living organisms. The organism’s proteolytic enzyme complex, termed the degradome, acts as an intermediary in its dynamic interaction with the surrounding environment. Using techniques such as genome and transcriptome sequencing, alongside protein prediction methodologies, we identified putative extracellular peptidases within Aspergillus ochraceus VKM-F4104D. Following manual annotation procedures, a total of 11 aspartic, 2 cysteine, 2 glutamic, 21 serine, 1 threonine, and 21 metallopeptidases were attributed to the extracellular degradome of A. ochraceus VKM-F4104D. Among them are enzymes with promising applications in biotechnology, potential targets and agents for antifungal therapy, and microbial antagonism factors. Thus, additional functionalities of the extracellular degradome, extending beyond mere protein substrate digestion for nutritional purposes, were demonstrated. Full article

The larvae of some lampyrid beetles are highly specialized predators of snails. They have been observed to climb on the shells of their prey and use this exposed position to bite and inject secretions potentially originating from the midgut. Besides serving the purpose of extra-oral digestion (EOD), injected compounds also seem to have a paralyzing effect. Up to now, the toxins causing this paralyzing activity have not been identified. In the current study, we provide a first compositional analysis of the midgut secretion from lampyrid larvae, with a focus on identifying putative neurotoxins causing the observed paralyzing effect. For this purpose, we utilized a combined proteo-transcriptomic approach to characterize the compounds present in the midgut secretion of larval stages of Lampyris noctiluca. In terms of the absolute numbers of identified compounds, the midgut secretion is dominated by hydrolyzing enzymes comprising peptidases, carboxylesterases, and glycosidases. However, when considering expression levels, a few rather short cysteine-rich peptides exceed all other compounds. Some of these compounds show moderate similarity to putative neurotoxins identified in the venom of other arthropods and could be responsible for paralyzing effects. In addition to these potential toxins, we provide a list of peptides typical of the midgut secretion of L. noctiluca, supplemented by the corresponding precursor sequences. Full article

Increased oxidative stress (OS) and systemic inflammation are key players in the pathophysiology of chronic obstructive pulmonary disease (COPD). We aimed to clarify the effects of synthetic glutathione (GSH) analogue peptides UPF1 and UPF17 on the mRNA levels of enzymes involved in systemic inflammation and GSH metabolism in peripheral blood mononuclear cells (PBMCs) from patients with acute exacerbation of COPD (AE-COPD) and stable COPD along with non-obstructive smokers and non-smokers. UPF1 and UPF17 increased the expression of enzymes involved in the formation of the antioxidant capacity: superoxide dismutase 1 (SOD1) and the catalytic subunit of glutamyl-cysteine ligase (GCLC) in patients with AE-COPD and stable COPD, but also in non-obstructive smokers and non-smokers. Similarly, both UPF1 and UPF17 increased the expression of inflammatory enzymes poly(ADP-ribose) polymerase-1 (PARP-1), dipeptidyl peptidase 4 (DPP4), and cyclooxygenase-2 (COX-2). Both UPF analogues acted in a gender-dependent manner by increasing the expression of certain anti-inflammatory (histone deacetylase 2 (HDAC2)) and GSH metabolism pathway (SOD1 and GSH reductase (GSR))-related enzymes in females and decreasing them in males. UPF1 and UPF17 are able to increase the expression of the enzymes involved in GSH metabolism and could serve as a lead for designing potential COPD therapies against excessive OS. Full article

Proteinaceous toxins are peptides or proteins that hold great biotechnological value, evidenced by their ecological role, whether as defense or predation mechanisms. Bioprospecting using bioinformatics and omics may render screening for novel bioactives more expeditious, especially considering the immense diversity of toxin-secreting marine organisms. Eulalia sp. (Annelida: Phyllodocidae), a toxin bearing marine annelid, was recently shown to secrete cysteine-rich protein (Crisp) toxins (hitherto referred to as ‘phyllotoxins’) that can immobilize its prey. By analyzing and validating transcriptomic data, we narrowed the list of isolated full coding sequences of transcripts of the most abundant toxins or accompanying bioactives secreted by the species (the phyllotoxin Crisp, hyaluronidase, serine protease, and peptidases M12A, M13, and M12B). Through homology matching with human proteins, the biotechnological potential of the marine annelid’s toxins and related proteins was tentatively associated with coagulative and anti-inflammatory responses for the peptidases PepM12A, SePr, PepM12B, and PepM13, and with the neurotoxic activity of Crisp, and finally, hyaluronidase was inferred to bear properties of an permeabilizing agent. The in silico analysis succeeded by validation by PCR and Sanger sequencing enabled us to retrieve cDNAs can may be used for the heterologous expression of these toxins. Full article

Trichomonas vaginalis is the causative agent of trichomoniasis, the most prevalent nonviral, neglected sexually transmitted disease worldwide. T. vaginalis has one of the largest degradomes among unicellular parasites. Cysteine peptidases (CPs) are the most abundant peptidases, constituting 50% of the degradome. Some CPs are virulence factors recognized by antibodies in trichomoniasis patient sera, and a few are found in vaginal secretions that show fluctuations in glucose concentrations during infection. The CPs of clan CD in T. vaginalis include 10 genes encoding legumain-like peptidases of the C13 family. TvLEGU-2 is one of them and has been identified in multiple proteomes, including the immunoproteome obtained with Tv (+) patient sera. Thus, our goals were to assess the effect of glucose on TvLEGU-2 expression, localization, and in vitro secretion and determine whether TvLEGU-2 is expressed during trichomonal infection. We performed qRT-PCR assays using parasites grown under different glucose conditions. We also generated a specific anti-TvLEGU-2 antibody against a synthetic peptide of the most divergent region of this CP and used it in Western blot (WB) and immunolocalization assays. Additionally, we cloned and expressed the tvlegu-2 gene (TVAG_385340), purified the recombinant TvLEGU-2 protein, and used it as an antigen for immunogenicity assays to test human sera from patients with vaginitis. Our results show that glucose does not affect tvlegu-2 expression but does affect localization in different parasite organelles, such as the plasma membrane, Golgi complex, hydrogenosomes, lysosomes, and secretion vesicles. TvLEGU-2 is secreted in vitro, is present in vaginal secretions, and is immunogenic in sera from Tv (+) patients, suggesting its relevance during trichomonal infection. Full article

A liver injury was recently reported for saxagliptin, which is a dipeptidyl peptidase-4 (DPP-4) inhibitor. However, the underlying mechanisms of saxagliptin-induced liver injury remain unknown. This study aimed to evaluate whether saxagliptin, a potent and selective DPP-4 inhibitor that is globally used for treating type 2 diabetes mellitus, binds to the nucleophiles in vitro. Four DPP-4 inhibitors, including vildagliptin, were evaluated for comparison. Only saxagliptin and vildagliptin, which both contain a cyanopyrrolidine group, quickly reacted with L-cysteine to enzyme-independently produce thiazolinic acid metabolites. This saxagliptin–cysteine adduct was also found in saxagliptin-administered male Sprague–Dawley rats. In addition, this study newly identified cysteinyl glycine conjugates of saxagliptin and 5-hydroxysaxagliptin. The observed metabolic pathways were hydroxylation and conjugation with cysteine, glutathione, sulfate, and glucuronide. In summary, we determined four new thiazoline-containing thiol metabolites (cysteine and cysteinylglycine conjugates of saxagliptin and 5-hydroxysaxagliptin) in saxagliptin-administered male rats. Our results reveal that saxagliptin can covalently bind to the thiol groups of cysteine residues of endogenous proteins in vivo, indicating the potential for saxagliptin to cause drug-induced liver injury. Full article

Bladder cancer (BLCA) is a prevalent malignancy of the urinary system, associated with a high recurrence rate and poor prognosis. FAM111B, which encodes a protein containing a trypsin-like cysteine/serine peptidase domain, has been implicated in the progression of various human cancers; however, its involvement in BLCA remains unclear. In this study, we investigated the expression of FAM111B gene in tumor tissues compared to para-tumor tissues using immunohistochemistry and observed a significantly higher FAM111B gene expression in tumor tissues. Furthermore, analysis of clinical characteristics indicated that the increased FAM111B gene expression correlated with lymphatic metastasis and reduced overall survival. To investigate its functional role, we employed FAM111B-knockdown BLCA cell models and performed cell proliferation, wound-healing, transwell, and flow cytometry assays. The results showed that decreased FAM111B gene expression inhibited proliferation and migration but induced apoptosis in BLCA cells. In vivo experiments further validated that FAM111B knockdown suppressed tumor growth. Overall, our findings suggest that FAM111B acts as an oncogene in BLCA, playing a critical role in tumorigenesis, progression, and metastasis of BLCA. In conclusion, we have demonstrated a strong correlation between the expression of FAM111B gene and the development, progression, and metastasis of bladder cancer (BLCA). Thus, FAM111B is an oncogene associated with BLCA and holds promise as a molecular target for future treatment of this cancer. Full article

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis. Full article

This review considers available data on the composition of the extracellular matrix (ECM) in echinoderms. The connective tissue in these animals has a rather complex organization. It includes a wide range of structural ECM proteins, as well as various proteases and their inhibitors. Members of almost all major groups of collagens, various glycoproteins, and proteoglycans have been found in echinoderms. There are enzymes for the synthesis of structural proteins and their modification by polysaccharides. However, the ECM of echinoderms substantially differs from that of vertebrates by the lack of elastin, fibronectins, tenascins, and some other glycoproteins and proteoglycans. Echinoderms have a wide variety of proteinases, with serine, cysteine, aspartic, and metal peptidases identified among them. Their active centers have a typical structure and can break down various ECM molecules. Echinoderms are also distinguished by a wide range of proteinase inhibitors. The complex ECM structure and the variety of intermolecular interactions evidently explain the complexity of the mechanisms responsible for variations in the mechanical properties of connective tissue in echinoderms. These mechanisms probably depend not only on the number of cross-links between the molecules, but also on the composition of ECM and the properties of its proteins. Full article

Peptidases of the papain family play a key role in protein degradation, regulated proteolysis, and the host–pathogen arms race. Although the papain family has been the subject of many studies, knowledge about its diversity, origin, and evolution in Eukaryota, Bacteria, and Archaea is limited; thus, we aimed to address these long-standing knowledge gaps. We traced the origin and expansion of the papain family with a phylogenomic analysis, using sequence data from numerous prokaryotic and eukaryotic proteomes, transcriptomes, and genomes. We identified the full complement of the papain family in all prokaryotic and eukaryotic lineages. Analysis of the papain family provided strong evidence for its early diversification in the ancestor of eukaryotes. We found that the papain family has undergone complex and dynamic evolution through numerous gene duplications, which produced eight eukaryotic ancestral paralogous C1A lineages during eukaryogenesis. Different evolutionary forces operated on C1A peptidases, including gene duplication, horizontal gene transfer, and gene loss. This study challenges the current understanding of the origin and evolution of the papain family and provides valuable insights into their early diversification. The findings of this comprehensive study provide guidelines for future structural and functional studies of the papain family. Full article