This study aims to identify, purify, and characterize an endogenous serine protease inhibitor from an Indian strain of Leishmania donovani, which causes the fatal visceral leishmaniasis.
(i) Reverse ...zymography was used to identify the serine protease inhibitor by inhibiting the gelatinolytic activity of serine protease. (ii) Purification was performed by combining heat treatment, ultracentrifugation, and affinity and gel permeation chromatography. (iii) Spectrophotometric assays were conducted to quantify and compare the inhibitory activity of the L. donovani serine protease inhibitor (LdISP). (iv) Further, the protein was identified by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF) mass spectrometry (MS).
An endogenous inhibitor with an apparent molecular weight of 21.8kDa, which is acidic in nature, having a pI of 5.9 was identified. The Ki value of the inhibitor for trypsin was determined to be in the nanomolar range. The protein has the following features: (i) ecotin-like nature, (ii) cross-organism functionality, that is, an inhibitory effect on the serine proteases of higher organisms other than its own, and (iii) homology with other such proteins from a different species of Leishmania on conducting protein mass fingerprinting after MALDI ToF MS.
The inhibitor shows varying and entirely contrasting efficacies toward serine proteases of its own as well as of higher organisms. This indicates that it accelerates disease progression and drives parasite survival as it inhibits the activities of the host serine proteases.
Serine proteases are among the important groups of enzymes having significant roles in cell biology. Trypsin is a representative member of the serine superfamily of enzymes, produced by acinar cells ...of pancreas. It is a validated drug target for various ailments including pancreatitis and colorectal cancer. Premature activation of trypsin is involved in the lysis of pancreatic tissues, which causes pancreatitis. It is also reported to be involved in colorectal carcinoma by activating other proteases, such as matrix metalloproteinase (MMPs). The development of novel trypsin inhibitors with good pharmacokinetic properties could play important roles in pharmaceutical sciences. This study reports the crystal structures of bovine pancreatic trypsin with four molecules; cimetidine, famotidine, pimagedine, and guanidine. These compounds possess binding affinity towards the active site (S1) of trypsin. The structures of all four complexes provided insight of the binding of four different ligands, as well as the dynamics of the active site towards the bind with different size ligands. This study might be helpful in designing of new potent inhibitors of trypsin and trypsin like serine proteases.
•Trypsin is serine protease which involved in pancreatitis.•Trypsin is crystallized with cimetidine, famotidine, pimagedine, and guanidine.•All ligands showed their binding with active site of trypsin.•The binding of different ligands showed different effect on the dynamical properties of active side residues.
Summary
Mannose‐binding lectin (MBL)‐associated serine proteases (MASPs) are the enzymatic constituents of the lectin pathway of the complement system. They are complexed with large pattern ...recognition molecules (PRMs) such as MBL, other collectins, and ficolins. The main function of two of the three MASPs has crystallized lately: MASP‐1 autoactivates first, then it activates MASP‐2, and finally both participate in the formation of the C4b2a convertase. In addition to this, both enzymes are involved in several other processes which are subject to intense research nowadays. Notably, MASP‐1, as a promiscuous enzyme, has been implicated in the coagulation cascade, in the kinin generating contact system, and in cellular activation through protease‐activated receptor (PAR) cleavage on endothelial cells. The third protease MASP‐3 has emerged recently as the protease responsible for pro‐factor D activation in resting blood, providing a fundamental link between two complement pathways. At present all three MASPs have at least one well‐defined role and several other possible functions were implicated. Defect or more likely over‐activation of MASPs may culminate into diseases such as ischemia–reperfusion injury (IRI); hence, MASPs are all potential targets of drug development.
The serine protease gene was heterologously expressed in Escherichia coli BL21 (DE3) using the PET 28a vector. The purified enzyme was immobilized on a nanohybrid of amino graphene and chitosan. The ...characterization of synthesized nanohybrids and immobilized enzymes was confirmed by Fourier transform infrared (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and field emission scanning electron microscopy (FE-SEM). Immobilization increased the temperature optimum from 60 to 70 °C for both free and immobilized enzymes, while the optimal pH of the enzymes did not change post-immobilization (pH 8). The immobilized biocatalyst significantly enhanced thermal stability, as well as enzyme stability at significant pH ranges. After 30 days of storage, the immobilized enzymes exhibited approximately 83 % of their relative activity, while the free protease retained only 56 % of its initial activity. Stabilization also altered the kinetic parameters (increasing Km, decreasing Kcat/Km, and Vmax) and thermodynamic parameters (increasing enzyme half-life and activation energy). The study's outcomes represent a significant advancement in the realm of enzyme synthesis and its stabilization using several combined technologies, including enzyme production with recombinant DNA technology based on gene synthesis, and its stabilization using a hybrid substrate synthesized from nanomaterials. Based on these findings, the immobilized recombinant enzyme has high potential for industrial use as an efficient and stable biocatalyst.
Leishmaniasis are a group of diseases caused by parasitic protozoa of the genus
. Current treatments are limited by difficult administration, high cost, poor efficacy, toxicity, and growing ...resistance. New agents, with new mechanisms of action, are urgently needed to treat the disease. Although extensively studied in other organisms, serine proteases (SPs) have not been widely explored as antileishmanial drug targets. Herein, we report for the first time an activity-based protein profiling (ABPP) strategy to investigate new therapeutic targets within the SPs of the
parasites. Active-site directed fluorophosphonate probes (rhodamine and biotin-conjugated) were used for the detection and identification of active
serine hydrolases (SHs). Significant differences were observed in the SHs expression levels throughout the
life cycle and between different
species. Using iTRAQ-labelling-based quantitative proteomic mass spectrometry, we identified two targetable SPs in
: carboxypeptidase LmxM.18.0450 and prolyl oligopeptidase LmxM.36.6750. Druggability was ascertained by selective inhibition using the commercial serine protease inhibitors chymostatin, lactacystin and ZPP, which represent templates for future anti-leishmanial drug discovery programs. Collectively, the use of ABPP method complements existing genetic methods for target identification and validation in
.
The lifespan of T cells is determined by continuous interactions of their T cell receptors (TCR) with self-peptide-MHC (self-pMHC) complexes presented by different subsets of antigen-presenting cells ...(APC). In the thymus, developing thymocytes are positively selected through recognition of self-pMHC presented by cortical thymic epithelial cells (cTEC). They are subsequently negatively selected by medullary thymic epithelial cells (mTEC) or thymic dendritic cells (DC) presenting self-pMHC complexes. In the periphery, the homeostasis of mature T cells is likewise controlled by the interaction of their TCR with self-pMHC complexes presented by lymph node stromal cells while they may be tolerized by DC presenting tissue-derived self-antigens. To perform these tasks, the different subsets of APC are equipped with distinct combination of antigen processing enzymes and consequently present specific repertoire of self-peptides. Here, we discuss one such antigen processing enzyme, the thymus-specific serine protease (TSSP), which is predominantly expressed by thymic stromal cells. In thymic DC and TEC, TSSP edits the repertoire of peptide presented by class II molecules and thus shapes the CD4 T cell repertoire.
Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium ...possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.
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•Mannheimiahaemolytica expresses a 70 kDa secreted serine protease at 37 °C or 39 °C.•This serine protease degrades sheep fibrinogen but not bovine IgG neither hemoglobin nor casein.•This 70 kDa protease is immune recognized by sera from bovines suffering acute or chronic pneumonia.•This protease was identified as protease IV.
In insects, serine proteases (SPs) and serine protease homologs (SPHs) constitute a large family of proteins involved in multiple physiological processes such as digestion, development, and immunity. ...Here we identified 145 SPs and 38 SPHs in the genome of an endoparasitoid, Pteromalus puparum. Gene duplication and tandem repeats were observed in this large SPs/SPHs family. We then analyzed the expression profiles of SP/SPH genes in response to different microbial infections (Gram-positive bacterium Micrococcus luteus, Gram-negative bacterium Escherichia coli, and entomopathogenic fungus Beauveria bassiana), as well as in different developmental stages and tissues. Some SPs/SPHs also displayed distinct expression patterns in venom gland, suggesting their specific physiological functions as venom proteins. Our finding lays groundwork for further research of SPs and SPHs expressed in the venom glands.
•One hundred and eighty-three serine proteases (SPs) and serine protease homologs (SPHs) were identified.•Detailed information of SPs and SPHs was revealed by genome scale analysis.•Tandem repeats of SPs and SPHs with similar genomic structures indicated gene duplication events happened.•Six SPs and two SPHs were predicted as venom proteins.
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