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Serine peptidases (SPs) of the chymotrypsin S1A subfamily are an extensive group of enzymes found in all animal organisms, including insects. Here, we provide analysis of SPs in the yellow mealworm Tenebrio molitor transcriptomes and genomes datasets and profile their expression patterns at various stages of ontogeny. A total of 269 SPs were identified, including 137 with conserved catalytic triad residues, while 125 others lacking conservation were proposed as non-active serine peptidase homologs (SPHs). Seven deduced sequences exhibit a complex domain organization with two or three peptidase units (domains), predicted both as active or non-active. The largest group of 84 SPs and 102 SPHs had no regulatory domains in the propeptide, and the majority of them were expressed only in the feeding life stages, larvae and adults, presumably playing an important role in digestion. The remaining 53 SPs and 23 SPHs had different regulatory domains, showed constitutive or upregulated expression at eggs or/and pupae stages, participating in regulation of various physiological processes. The majority of polypeptidases were mainly expressed at the pupal and adult stages. The data obtained expand our knowledge on SPs/SPHs and provide the basis for further studies of the functions of proteins from the S1A subfamily in T. molitor. [ABSTRACT FROM AUTHOR]

We characterized an alkaline chymotrypsin-like serine peptidase from the yellow mealworm Te-nebrio molitor with a non-canonical substrate-binding subsite for its possible application as a component (an additive) in various biological products. The enzyme was obtained as a recombinant preparation. Purification was carried out using affinity chromatography on Ni2+-NTA agarose. The specificity constants (kcat/KM) for the chymotrypsin substrates, Glp-AAF-pNA, Suc-AAPF-pNA, and Ac-Y-pNA, were 7, 4.2, and 0.9 (µM min)–1, respectively. The optimum of the proteolytic activity was observed at pH 9.0. The enzyme was stable in the alkaline pH range, and in the presence of BSA, also in the acidic region. Peptidase was inhi-bited by synthetic inhibitors such as PMSF, TPCK, and chymostatin, while EDTA, E-64, and pepstatin had no effect on the enzyme activity. The purified enzyme showed high stability over time in the presence of BSA. The short life cycle of the insect and the production of a large number of peptidases in the midgut with high catalytic activity and stability can make T. molitor an excellent alternative source of industrially important enzymes for application as components (additives) in various biological products (e.g., stain removers, detergents, etc.). [ABSTRACT FROM AUTHOR]

Background: The invasive gastropod Pomacea canaliculata has received great attention in the last decades as a result of its negative impact on crops agriculture, yet knowledge of their digestive physiology remains incomplete, particularly the enzymatic breakdown of macromolecules such as proteins and lipids. Results: Discovery proteomics revealed aspartic peptidases, cysteine peptidases, serine peptidases, metallopeptidases and threonine peptidases, as well as acid and neutral lipases and phospholipases along the digestive tract of P. canaliculata. Peptides specific to peptidases (139) and lipases (14) were quantified by targeted mass spectrometry. Digestion begins in the mouth via diverse salivary peptidases (nine serine peptidases; seven cysteine peptidases, one aspartic peptidase and 22 metallopeptidases) and then continues in the oesophagus (crop) via three luminal metallopeptidases (Family M12) and six serine peptidases (Family S1). Downstream, the digestive gland provides a battery of enzymes composed of aspartic peptidase (one), cysteine peptidases (nine), serine peptidases (12) and metallopeptidases (24), including aminopeptidases, carboxypeptidases and dipeptidases). The coiled gut has M1 metallopeptidases that complete the digestion of small peptides. Lipid extracellular digestion is completed by triglyceride lipases. Conclusion: From an integrative physiological and anatomical perspective, P. canaliculata shows an unexpected abundance and diversity of peptidases, which participate mainly in extracellular digestion. Moreover, the previously unknown occurrence of luminal lipases from the digestive gland is reported for the first time. Salivary and digestive glands were the main tissues involved in the synthesis and secretion of these enzymes, but plausibly the few luminally exclusive peptidases are secreted by ventrolateral pouches or epithelial unicellular glands. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Thraustochytrids have attracted attention due to the high contents of useful lipids and growth rate. Genus Schizochytrium is commonly used for docosahexaenoic acid (DHA) production, while a strain, which produces a high amount of squalene, has been reported in the genus Aurantiochytrium. These organisms are heterotrophic, and Schizochytrium degrades the extracellular macromolecules, e.g., proteins and polysaccharides, as the nutrients. However, the extracellular lytic enzymes are not well-studied yet. Here, we investigated the induction of extracellular proteases of Schizochytrium aggregatum ATCC 28209. A casein-hydrolytic activity was induced in the nitrogen-limited conditions, and that was also detected by zymography after fractionation by non-heat denatured SDS-PAGE. The proteinous band corresponding to the protease activity was analyzed by MALDI-TOF mass spectrometry after digestion with trypsin. The molecular mass data of the protein fragments were compared to the protein database of S. aggregatum ATCC 28209 in the Joint Genome Institute, and we found that the molecular masses of the six peptides were matched with the prediction from the sequence of a protein, ID 63992, which was annotated as a peptidase S8 family protein. Interestingly, we found that a paralogous protein, ID 99856, was encoded by a gene flanking at the downstream of the gene for ID 63992, and the expression of both genes was similarly induced under the nitrogen-limited conditions. These findings may provide us a key to disclose the induction mechanisms of the extracellular lytic enzymes and the function of the proteolytic enzyme for the nutrition acquisition in thraustochytrids. [ABSTRACT FROM AUTHOR]

As one of the most abundant bacteria in the human oral cavity, Fusobacterium nucleatum is closely involved in various oral diseases and is also a risk factor for other diseases. The peptidases of F. nucleatum can digest exogenous peptides into amino acids to satisfy its nutrient requirements. Here, a putative F. nucleatum peptidase, termed S9Cfn, which belongs to the S9C peptidase family was identified. Enzymatic activity assays combined with mass‐spectrometric analysis revealed that S9Cfn is a carboxypeptidase, but not an aminopeptidase as previously annotated. The crystal structure of the S9Cfn tetramer was solved at 2.6 Å resolution and was found to contain a pair of oligomeric pores in the center. Structural analysis, together with site‐directed mutagenesis and enzymatic activity assays, revealed a substrate‐entrance tunnel that extends from each oligomeric pore to the catalytic triad, adjacent to which three conserved arginine residues are responsible for substrate binding. Moreover, comparison with other S9 peptidase structures indicated drastic conformational changes of the oligomeric pores during the catalytic cycle. Together, these findings increase the knowledge of this unique type of tetrameric carboxypeptidase and provide insight into the homeostatic control of microbiota in the human oral cavity. [ABSTRACT FROM AUTHOR]

Tenebrio molitor is an important coleopteran model insect and agricultural pest from the Tenebrionidae family. We used RNA-Seq transcriptome data from T. molitor to annotate trypsin-like sequences from the chymotrypsin S1 family of serine peptidases, including sequences of active serine peptidases (SerP) and their inactive homologs (SerPH) in T. molitor transcriptomes. A total of 63 S1 family tryspin-like serine peptidase sequences were de novo assembled. Among the sequences, 58 were predicted to be active trypsins and five inactive SerPH. The length of preproenzyme and mature form of the predicted enzyme, position of signal peptide and proenzyme cleavage sites, molecular mass, active site and S1 substrate binding subsite residues, and transmembrane and regulatory domains were analyzed using bioinformatic tools. The data can be used for further physiological, biochemical, and phylogenetic study of tenebrionid pests and other animal systems.

In this work, we report on the halophilic bacterium Halobacillus blutaparonensis strain M9, which was isolated from a preserved Brazilian restinga, as a producer of alkaline serine peptidases with potential application as additives for biotechnological purposes. Gelatin-zymography assay revealed the presence of the major peptidase of 70 kDa and two minor ones (50 and 40 kDa) secreted by the living bacterial cells. When a specific chromogenic substrate for chymotrypsin-type serine peptidases was employed by means of an in-solution assay, the bacterial peptidases were completely inhibited by phenylmethylsulphonyl fluoride (PMSF) and slightly inhibited by benzamidine, N-tosyl-L-lysine chloromethyl ketone (TLCK) and 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), which are classical serine peptidase inhibitors. The proteolytic activity remained extremely stable within a broad pH range (from 5.0 to 9.0) and also displayed moderate thermostability. In addition, the effects of osmolytes/polyols, divalent cations, denaturing agents, and detergents on the enzymatic activity and stability of these serine peptidases were tested. The proteolytic activity increased in the presence of different osmolytes/polyols (e.g., glycerol, glutamate, and mannitol). The enzymatic activity increased to 110% when incubated in the presence of 10 mM Ca2+, while Mg2+ and Mn2+ (both at 10 mM) decreased the proteolytic activity to 95 and 85%, respectively. Interestingly, urea at 1% induced a significant increase (around 160%) in the proteolytic activity. The peptidases showed high stability when incubated with various detergent agents and commercial detergent powders. Collectively, all these properties indicate H. blutaparonensis as a producer of extracellular serine peptidases with possible industrial applications, such as in detergent formulations. [ABSTRACT FROM AUTHOR]

Candida parapsilosis sensu stricto (C. parapsilosis) has emerged as the second/third commonest Candida species isolated from hospitals worldwide. Candida spp. possess numerous virulence attributes, including peptidases that play multiple roles in both physiological and pathological events. So, fungal peptidases are valid targets for new drugs development. With this premise in mind, we have evaluated the effect of serine peptidase inhibitors (SPIs) on both cell biology and virulence aspects of C. parapsilosis. First, five different SPIs, phenylmethylsulfonyl fluoride, benzamidine, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, N -α-tosyl-L-lysine chloromethyl ketone hydrochloride, and N -tosyl-L-phenylalanine chloromethyl ketone (TPCK) were tested, and TPCK showed the best efficacy to arrest fungal growth. Subsequently, the ability of TPCK to modulate physiopathological processes was investigated. Overall, TPCK was able to (i) inhibit the cell-associated serine peptidase activities, (ii) promote morphometric and ultrastructural alterations, (iii) induce an increase in the intracellular oxidation level, which culminates in a vigorous lipid peroxidation and accumulation of neutral lipids in cytoplasmic inclusions, (iv) modulate the expression/exposition of surface structures, such as mannose/glucose-rich glycoconjugates, N -acetylglucosamine-containing molecules, chitin, polypeptides and surface aspartic peptidases, (v) reduce the adhesion to either polystyrene or glass surfaces as well as to partially disarticulate the mature biofilm, (vi) block the fungal interaction with macrophages, and (vii) protect Galleria mellonella from fungal infection, enhancing larvae survivability. Altogether, these results demonstrated that TPCK induced several changes over fungal biology besides the interference with aspects associated to C. parapsilosis virulence and pathogenesis, which indicates that SPIs could be novel promising therapeutic agents in dealing with candidiasis. [ABSTRACT FROM AUTHOR]

In Brazil, envenomation by Bothrops pitvipers is responsible for over 73% of snakebites, and their venom is a rich source of proteolytic enzymes. Most studies have demonstrated that Bothrops jararaca venom acts on macromolecular substrates, causing an imbalance in the victim's hemostatic system. In contrast, fewer studies have examined the proteolytic activity on small molecules such as peptides. In this study, we used a set of bioactive peptides (insulin B chain, Met-enkephalin, Leu-enkephalin, neuropeptide Y, peptide YY, pancreatic polypeptide, substance P and somatostatin) to identify new peptide substrates for the metallopeptidases and serine peptidases from the B. jararaca venom. The majority of these peptides were substrates for the venom, but neuropeptide Y and pancreatic polypeptide presented higher hydrolyses rates. Although most of the peptides were simultaneously substrates for both classes of proteases, serine peptidases were the most active. Substance P was an exclusive substrate for metallopeptidases, while somatostatin was a selective substrate for serine peptidases. The neutralizing efficacy of the bothropic antivenom produced by the Butantan Institute was also assessed and found to totally prevent substance P hydrolysis, whereas somatostatin cleavage was not inhibited. Thus, the antivenom effectively inhibited metallopeptidase activity, but did not neutralize some of the serine peptidases. These results indicate that, in addition to cleaving proteins, the proteolytic enzymes from this venom also hydrolyze bioactive peptides, and this peptidase activity could effectively contribute to some of the many dire manifestations of envenomation. [ABSTRACT FROM AUTHOR]

Vip3 proteins have been described to be secreted by Bacillus thuringiensis during the vegetative growth phase and to display a broad insecticidal spectrum against lepidopteran larvae. Vip3Aa protoxin has been reported to be significantly more toxic to Spodoptera frugiperda than to Spodoptera exigua and differences in the midgut processing have been proposed to be responsible. In contrast, we have found that Vip3Ae is essentially equally toxic against these two species. Proteolysis experiments were performed to study the stability of Vip3A proteins to peptidase digestion and to see whether the differences found could explain differences in toxicity against these two Spodoptera species. It was found that activation of the protoxin form and degradation of the 62kDa band took place at lower concentrations of trypsin when using Vip3Aa than when using Vip3Ae. The opposite effect was observed for chymotrypsin. Vip3Aa and Vip3Ae protoxins were effectively processed by midgut content extracts from the two Spodoptera species and the proteolytic activation did not produce a peptidase resistant core under these in vitro conditions. Digestion experiments performed with S. frugiperda chromatography-purified digestive serine peptidases showed that the degradation of the Vip3A toxins active core is mainly due to the action of cationic chymotrypsin-like peptidase. Although the digestion patterns of Vip3A proteins do not always correlate with toxicity, the peptidase stability of the 62kDa core is in agreement with intraspecific differences of toxicity of the Vip3Aa protein. [ABSTRACT FROM AUTHOR]

Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5), also known as CD26, is a serine aminopeptidase that preferentially cleaves Xaa-Pro or Xaa-Ala dipeptides from the N-terminus of oligopeptides and processes regulatory peptides in vivo, leading to their biological activation or inactivation. The ezyme is a homodimer and each subunit is formed by a aß-hydrolase domain and a ß-propeller domain, involved in the enzymatic activity and its interaction with other proteins. It has an important role in multiple physiological functions, including the regulation of glucose metabolism being one of the current targets for the treatment of type II diabetes mellitus. This enzyme also regulates immune system responses mediated by CD4+ T lymphocytes, and recently has been identified a high/low DPP-IV activity regarding physiological levels, in pathologies like thyroid, ovarian, lung, skin, prostate cancers and central nervous system tumors. For these reasons this enzyme evolves as a new target of attention for the development of more efficient diagnostics being considering as molecular markers for some pathologies and target for the development of new therapeutic assessments in cancer. Current research interests are focused in depth in the structure-function relation for this enzyme, as a key point for the development of new therapies in pathologies involving DPP-IV activity or its interaction with other proteins. [ABSTRACT FROM AUTHOR]

Abstract: pks genomic island of Escherichia coli is involved in the synthesis of the non-ribosomal peptide-type genotoxin colibactin, which has been suggesting as affecting the host immune response and having an impact on cancer development. The pks-encoded enzyme ClbP is an atypical peptidase that contributes to the synthesis of colibactin. In this work, we identified key features of ClbP. Bacterial fractionation and Western-blot analysis revealed the docking of ClbP to the bacterial inner membrane via a C-terminal domain harboring three predicted transmembrane helices. Whereas only one helix was necessary for the location in the inner membrane, the complete sequence of the C-terminal domain was necessary for ClbP bioactivity. In addition, the N-terminal sequence of ClbP allowed the SRP/Sec/YidC- and MreB-dependent translocation of the enzymatic domain in the periplasmic compartment, a feature also essential for ClbP bioactivity. Finally, the comparison of ClbP structure with that of the paralogs FmtA-like and AmpC revealed at an extremity of the catalytic groove a negative electrostatic potential surface characteristic of ClbP. Site-directed mutagenesis experiments identified in this zone two aspartic residues that were important for ClbP bioactivity. Overall, these results suggest a model for precolibactin activation by ClbP and pave a way for the design of inhibitors targeting colibactin production. [Copyright &y& Elsevier]

Abstract: Snakebite envenomation is considered a highly relevant public health hazard in South America, having an impact in terms of mortality and morbidity. In Brazil, Bothrops (sensu latu) poisoning is responsible for 90% of the snakebites and in patients treated at the Vital Brazil Hospital (Butantan Institute) this index reaches 97.5%. The objective of the present study was to analyze more specifically the ability of the antibothropic antivenom, produced by the Butantan Institute, São Paulo, Brazil, to neutralize metallo-and serine peptidases, known as the major toxins present in Bothrops jararaca venom. A set of Fret peptides (Free Ressonance Energy Transfer) was studied using the BjV (B. jararaca venom) and site-directed inhibitors PMSF, EDTA and 1,10-phenanthroline. Two substrates were reached to be used as specific tools for studies with metallo peptidases, Abz-FASSAQ-EDDnp, and the serine peptidases, Abz-RPPGFSPFRQ-EDDnp. In disagreement with the literature, the use of both substrates and the antibothropic serum showed a weak neutralization of the serine peptidases present in this venom and a strong neutralization of the metallo peptidases. In order to investigate possible mechanisms of action that have not yet been described for the serine peptidases from the BjV, the present study shows for the first time a new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine peptidase activity, that was partially blocked by the antibothropic serum. In conclusion, the antivenom presented a good neutralization of metallo peptidases but not of serine peptidases, indicating that further studies about serine peptidases immunogenicity are necessary to improve the antibothropic serum. [Copyright &y& Elsevier]

Abstract: Chymotrypsin-like peptidases (CTLPs) of insects are primarily secreted into the gut lumen where they act as digestive enzymes. We studied the gene family encoding CTLPs in the genome of the red flour beetle, Tribolium castaneum. Using an extended search pattern, we identified 14 TcCTLP genes that encode peptidases with S1 specificity pocket residues typically found in chymotrypsin-like enzymes. We further analyzed the expression patterns of seven TcCTLP genes at various developmental stages. While some TcCTLP genes were exclusively expressed in feeding larval and adult stages (TcCTLP-5A/B, TcCTLP-6A), others were also detected in non-feeding embryonic (TcCTLP-5C, TcCTLP-6D) and pupal stages (TcCTLP-5C, TcCTLP-6C/D/E). TcCTLP genes were expressed predominantly in the midgut, where they presumably function in digestion. However, TcCTLP-6C and TcCTLP-5C also showed considerable expression in the carcass. The latter two genes might therefore encode peptidases that act as molting fluid enzymes. To test this hypothesis, we performed western blots using protein extracts from larval exuviae. The extracts reacted with antibodies to TcCTLP-5C and TcCTLP-6E suggesting that the corresponding peptidases are secreted into the molting fluid. Finally, we performed systemic RNAi experiments. While injections of five TcCTLP-dsRNAs into penultimate larvae did not affect growth or development, injection of dsRNA for TcCTLP-5C and TcCTLP-6C resulted in severe molting defects. [Copyright &y& Elsevier]

Abstract: Acanthamoeba spp., known to cause keratitis and granulomatous encephalitis in humans, are frequently isolated from a variety of water sources. Here we report for the first time the characterization of an Acanthamoeba sp. (ACC01) isolated from tap water in Brazil. This organism is currently being maintained in an axenic growth medium. Phylogenetic analysis based on SSU rRNA gene sequences positioned the new isolate in genotype T4, closest to the keratitis-causing isolate, A. polyphaga ATCC 30461 (∼99% similarity). Acanthamoeba ACC01 and A. polyphaga 30461 both grew at 37°C and were osmotically resistant, multiplying in hyperosmolar medium. Both isolates secreted comparable amounts of proteolytic enzymes, including serine peptidases that were optimally active at a near neutral/alkaline pH and resolved identically in gelatin gels. Incubation of gels at pH 4.0 with 2mM DTT also indicated the secretion of similar cysteine peptidases. Altogether, the results point to the pathogenic potential of Acanthamoeba ACC01. [Copyright &y& Elsevier]

Abstract: Acanthamoeba species are frequently isolated from soil and water collections. In the environment, the organisms multiply as phagotrophic trophozoites and encyst under adverse conditions. Several species are known to infect man, causing keratitis and opportunistic diseases. The mechanisms underlying tissue damage and invasion by the amoebae are being elucidated and the involvement of secreted peptidases, particularly serine peptidases, has been demonstrated. Here, elastase activity was examined in Acanthamoeba-conditioned medium (ACM), making use of elastin-Congo red (ECR) and synthetic peptide p-nitroanilide substrates. ACM hydrolysed ECR over a broad pH range and optimally at a pH of 7.5 and above. Indicating the activity of serine and metallopeptidases, Congo red release was potently inhibited by PMSF, antipain, chymostatin and 1,10-phenanthroline, partially reduced by elastatinal and EDTA, and unaffected by 1,7-phenanthroline and E-64. Screening with synthetic substrates mainly showed the activity of serine peptidases. ACM efficiently hydrolysed Suc-Ala2-Pro-Leu-pNA and Suc-Ala2-Pro-Phe-pNA over a broad pH range (7.0–9.5) and was weakly active against Suc-Ala3-pNA, a substrate found to be optimally hydrolysed at a pH around 7.0. Following ammonium sulfate precipitation of ACM proteins and FPLC analysis, the majority of the ECR-splitting activity, characterised as serine peptidases, bound to CM-sepharose and co-eluted with part of the Suc-Ala2-Pro-Phe-pNA-hydrolysing activity in a gradient of 0–0.6M NaCl. In the corresponding FPLC fractions, serine peptidases resolving in the region of 70–130kDa were detected in gelatin gels. Overall, the results demonstrate that trophozoites secrete elastases, and additionally suggest the high molecular weight serine peptidases as possible elastase candidates. [Copyright &y& Elsevier]

Candida albicans expresses a vast number of hydrolytic enzymes, playing roles in several phases of yeast–host interactions. Here, we identified two novel extracellular peptidase classes in C. albicans. Using gelatin–sodium dodecyl sulfate polyacrylamide gel electrophoresis two gelatinolytic activities were detected at physiological pH: a 60-kDa metallopeptidase, completely blocked by 1,10-phenanthroline, and a 50-kDa serine peptidase inhibited by phenylmethylsulfonyl fluoride. In an effort to establish a probable functional implication for these novel peptidase classes, we demonstrated that the 50-kDa secretory serine peptidase was active over a broad pH range (5.0–7.2) and was capable to hydrolyze some soluble human serum proteins and extracellular matrix components. Conversely, when this isolate was grown in yeast carbon base supplemented with bovine serum albumin, a secretory aspartyl peptidase activity was measured, instead of metallo- and serine peptidases, suggesting that distinct medium composition induces different expression of released peptidases in C. albicans. Additionally, we showed by quantitative proteolytic measurement, flow cytometry and immunoblotting assays that the brain heart infusion medium might repress the Sap1–3 production. Collectively, our results showed for the first time the capability of an extracellular proteolytic enzyme other than aspartic-type peptidases to cleave a broad spectrum of relevant host proteinaceous substrates by the human pathogen C. albicans. [ABSTRACT FROM AUTHOR]

A group of serine peptidases, the prolyl oligopeptidase family, cannot hydrolyze peptides containing more than about 30 residues. This group is unrelated to the classical trypsin and subtilisin families, and includes dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B, in addition to the prototype prolyl oligopeptidase. The recent crystal structure determination of prolyl oligopeptidase (80 kDa) has shown that the enzyme contains a peptidase domain with an α/β hydrolase fold, and its catalytic triad is covered by the central tunnel of an unusual seven-bladed β-propeller. This domain operates as a gating filter, excluding large, structured peptides from the active site. The binding mode of substrates and the catalytic mechanism differ from that of the classical serine peptidases in several features. The members of the family are important targets of drug design. Prolyl oligopeptidase is involved in amnesia, depression and blood pressure control, dipeptidyl peptidase IV in type 2 diabetes and oligopeptidase B in trypanosomiasis. [ABSTRACT FROM AUTHOR]

There have been only a few studies of how allergens cross the airway epithelium to cause allergic sensitization. House dust mite fecal pellets (HDMFP) contain several proteolytic enzymes. Group 1 allergens are cysteine peptidases, whilst those of groups 3, 6 and 9 have catalytic sites indicative of enzymes that mechanistically behave as serine peptidases. We have previously shown that the group 1 allergen Der p 1 leads to cleavage of tight junctions (TJs), allowing allergen delivery to antigen presenting cells. In this study we determined whether HDMFP serine peptidases similarly compromise the airway epithelium by attacking TJs, desmosomes and adherens junctions. Experiments were performed in monolayers of MDCK, Calu-3 or 16HBE14o-epithelial cells. Cell junction morphology was examined by 2-photon molecular excitation microscopy and digital image analysis. Barrier function was measured as mannitol permeability. Cleavage of cell adhesion proteins was studied by immunoblotting and mass spectrometry. HDMFP serine peptidases led to a progressive cleavage of TJs and increased epithelial permeability. Desmosomal puncta became more concentrated. Cleavage of TJs involved proteolysis of the TJ proteins, occludin and ZO-1. This was associated with activation of intracellular proteolysis of ZO-1. In contrast to occludin, E-cadherin of adherens junctions was cleaved less extensively. Although Calu-3 and 16HBE14o-cells expressed tethered ligand receptors for serine peptidases, these were not responsible for transducing the changes in TJs. HDMFP serine peptidases cause cleavage of TJs. This study identifies a second general class of HDM peptidase capable of increasing epithelial permeability and thereby creating conditions that would favour transepithelial delivery of allergens. [ABSTRACT FROM AUTHOR]

Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5), also known as CD26, is a serine aminopeptidase that preferentially cleaves Xaa-Pro or Xaa-Ala dipeptides from the N-terminus of oligopeptides and processes regulatory peptides in vivo, leading to their biological activation or inactivation. The enzyme is a homodimer and each subunit is formed by a aß-hydrolase domain and a ß-propeller domain, involved in the enzymatic activity and its interaction with other proteins. It has an important role in multiple physiological functions, including the regulation of glucose metabolism being one of the current targets for the treatment of type II diabetes mellitus. This enzyme also regulates immune system responses mediated by CD4+ T lymphocytes, and recently has been identified a high/low DPP-IV activity regarding physiological levels, in pathologies like thyroid, ovarian, lung, skin, prostate cancers and central nervous system tumors. For these reasons, this enzyme evolves as a new target of attention for the development of more efficient diagnostics being considering as molecular markers for some pathologies and a target for the development of new therapeutic assessments in cancer. Current research interests are focused on depth in the structure-function relation for this enzyme, as a key point for the development of new therapies in pathologies involving DPP-IV activity or its interaction with other proteins.

Abstract: Candida haemulonii complex (C. haemulonii, C. haemulonii var. vulnera and C. duobushaemulonii) consists of emergent multidrug-resistant pathogens that cause bloodstream and deep-seated infections. However, little is known about their virulence factors. Herein, we evaluated the presence of extracellular serine peptidases in this fungal complex. Serine peptidase activity was measured by spectrophotometry using chromogenic peptide substrates to the S1 family. Chymotrypsin-, trypsin- and elastase-like activities were detected in all fungal isolates. Since higher chymotrypsin- and trypsin-like activities were observed from the cleavage of N-succinyl-Ala-Ala-Pro-Phe-pNa and N-benzoyl-Phe-Val-Arg-pNa, respectively, these substrates were selected for further experiments. Overall, pHs 7.0 and 9.0 were those in which higher chymotrypsin- and trypsin-like activities were observed, respectively, displaying higher hydrolytic activities at 37-45°C. Additionally, the serine peptidases produced by C. haemulonii complex were inhibited by PMSF and AEBSF in a typically concentration-dependent manner. Although the Michaelis constant (Km) values obtained for chymotrypsin-like peptidases were similar, greater differences were observed for trypsin-like enzymes secreted by the different fungal isolates. This is the first time that peptidases belonging to the S1 family are described in the C. haemulonii species complex. Thus, these data open the doors for more detailed studies into potential roles of these peptidases in fungal virulence.