Abstract Philasterides dicentrarchi is a causative agent of scuticociliatosis in olive flounder Paralichthys olivaceus , aquaculture in Korea. In this study, a cDNA encoding a cathepsin L-like ...cysteine protease (PdCtL) of P. dicentrarchi (synonym Miamiensis avidus ) was identified. To express the PdCtL recombinant protein in a heterologous system, 10 codons were redesigned to conform to the standard eukaryotic genetic code using polymerase chain reaction (PCR)-based site-directed mutagenesis. The recombinant P. dicentrarchi procathepsin L (proPdCtL) was expressed at high levels in E. coli Rosetta (DE3) pLysS with a pPET21a vector, and successfully refolded, purified, and activated into a functional and enzymatically active form. The optimal pH for protease activity was 5. Similar to other cysteine proteases, enzyme activity was inhibited by E64 and leupeptin. Immunogenicity of recombinant PdCtL was assessed by enzyme-linked immunosorbent assay, western blot, and specific anti-recombinant PdCtL antibodies were detected. Our results suggest that the biochemical characteristics of the recombinant ciliate proPdCtL protein are similar to those of the cathepsin L-like cysteine protease, that the PCR-based site-direct mutated ciliate gene was successfully expressed in a biochemically active form, and that the recombinant PdCtL acted as a specific epitope in olive flounder.
Cystatin C (CysC) is a protease encoded by housekeeping genes. Although its prognostic value in heart failure (HF) is well known, it is debatable whether this value is due to the greater accuracy of ...CysC in calculating the glomerular filtration rate or to its involvement in pathological ventricular remodeling. The aim of this study was to determine whether CysC expression changes in the myocardium of fetuses of different ages and in the myocardium of adults with various cardiovascular diseases, as well as to analyze the correlation between its serum concentrations and cardiac structure and morphology in a patient group with HF.
We analyzed the correlations (Pearson's r and Spearman's test) between the serum CysC levels and echocardiographic parameters of 351 patients with HF. We also performed immunohistochemical staining for CysC, metalloproteinase-9 (MMP-9) and desmin in 9 cardiac tissue samples from autopsies of 4 fetuses of different gestational ages and 5 healthy adults or adults with cardiovascular disease.
For the patients with HF, there was no correlation between the CysC concentrations and the cardiac parameters measured by 2D echocardiography. The immunohistochemistry showed a weak background staining for CysC in all samples, regardless of age and the presence or absence of cardiovascular diseases.
Our results suggest that CysC does not have a significant role in the pathological remodeling of the left ventricle in HF.
La cistatina C (CisC) es una proteasa codificada por genes de mantenimiento («housekeeping genes»). Aunque su valor pronóstico en la insuficiencia cardiaca (IC) es bien conocido, se debate si es debido a su mayor precisión en la estimación del filtrado glomerular, o a su implicación en el remodelado ventricular patológico. El propósito de este estudio fue comprobar si la expresión de CisC se modificaba en el miocardio de fetos de diferentes edades y en el de adultos con diversas enfermedades cardiovasculares, así como analizar la correlación entre sus concentraciones séricas y la estructura y morfología cardiaca en un grupo de pacientes con IC.
Se analizaron las correlaciones (test de Pearson y Spearman) entre la CisC sérica y los parámetros ecocardiográficos de 351 pacientes con IC. También se realizó una tinción inmunohistoquímica para CisC, metaloproteinasa 9 (MMP-9) y desmina en 9 muestras de tejido cardiaco procedentes de las autopsias de 4 fetos con diferente edad gestacional y 5 adultos sanos o con enfermedad cardiovascular.
En pacientes con IC no se encontró correlación entre las concentraciones de CisC y los parámetros cardiacos medidos por ecocardiografía 2 D. La inmunohistoquímica mostró una débil tinción de fondo para CisC en todas las muestras, independientemente de la edad y la presencia o no de enfermedades cardiovasculares.
Nuestros resultados sugieren que la CisC no tiene un papel significativo en el remodelado patológico del ventrículo izquierdo en la IC.
The interrelationship of the multiple cysteine proteases (CPs) found characteristically at high activity in Leishmania mexicana amastigotes has been investigated. The mature forms of the five enzymes ...of groups B and C, which have subtly different substrate preferences, are the same size. Enzymically deglycosylated group A CP proteins also have the same molecular mass. Proteases of all three groups are specifically recognized by antisera raised against the group B or group C CPs. In addition, CPs of groups A, B and C have highly similar N-terminal amino acid sequences. The consensus sequence matches that predicted from the sequenced Imcpb gene, which occurs in a tandem array of over ten similar genes. Thus, the results are consistent with the groups A, B and C CPs being products of different Imcpb genes within the array, the different genes encoding CPs with identical N-termini, but with limited amino acid substitutions within the mature enzyme accounting for the different properties of the CPs. Evidence is also presented to indicate membrane-association of proteolytically active but less processed forms of Imcpb products.
Autoimmune diseases are characterized by multiple autoantibodies and/or autoreactive T cells that recognize a large number of antigens. Many of these antigens undergo extensive post-translational ...modifications during apoptosis and act as substrates for the pro-apoptotic cystein proteases. Here, Mauro Piacentini and Vittorio Colizzi discuss the effects on autoimmunity produced by post-translational modifications of proteins catalysed by the pro-apoptotic enzyme tissue transglutaminase.
We review the structural diversification of metacaspases, proteases that are involved in various physiological and pathological processes in plant cells, and consider how increasing information ...regarding their structure is aiding the interpretation of their distinct functional properties.
Abstract
Caspases are metazoan proteases, best known for their involvement in programmed cell death in animals. In higher plants genetically controlled mechanisms leading to the selective death of individual cells also involve the regulated interplay of various types of proteases. Some of these enzymes are structurally homologous to caspases and have therefore been termed metacaspases. In addition to the two well-studied metacaspase variants found in higher plants, type I and type II, biochemical data have recently become available for metacaspases of type III and metacaspase-like proteases, which are present only in certain algae. Although increasing in vitro and in vivo data suggest the existence of further sub-types, a lack of structural information hampers the interpretation of their distinct functional properties. However, the identification of key amino acid residues involved in the proteolytic mechanism of metacaspases, as well as the increased availability of plant genomic and transcriptomic data, is increasingly enabling in-depth analysis of all metacaspase types found in plastid-containing organisms. Here, we review the structural distribution and diversification of metacaspases and in doing so try to provide comprehensive guidelines for further analyses of this versatile family of proteases in organisms ranging from simple unicellular species to flowering plants.
Malaria is considered one of the life-threatening mosquito-borne infectious diseases responsible for approximately more than 4,00,000 deaths every year all over the world. Plasmodium falciparum and ...Plasmodium vivax are widespread species, but infections caused by the former are of great concern.
Among the various forms of infections associated with Plasmodium falciparum, cerebral malaria (CM) is the most severe neurological complication, accounting for almost 13% of all malariarelated mortality. The development of effective therapeutics is urgently needed to overcome the fatality of this dreadful disease.
The present work attempted to design and virtually screen a chemical library of 75 molecules (N-Mannich base derivatives of primaquine bearing isatin moiety as heterocyclic) by molecular docking studies against anti-malarial target proteins-Cystein Protease Falcipain-2; Dipeptidyl Aminopeptidase- 1; Dipeptidyl Aminopeptidase-3 and Glycogen synthase Kinase-3β receptors, for evaluating their anti-malarial potential. Among all studied anti-malarial target receptors, the designed molecules showed an overall higher affinity for Dipeptidyl Aminopeptidase-3. Furthermore, the molecules were analyzed for binding affinity and drug-like properties using Lipinski rules, and 30 best hits were shortlisted and analyzed for the pharmacokinetic profile.
Two of these hits were found to be more toxic than primaquine, hence were omitted in further analysis. Later, these 28 hits were docked against two target proteins, (a) Plasmodium falciparum erythrocyte membrane protein-1 and (b) Intracellular adhesion molecule-1, to determine their efficiency against cerebral malaria, and the results were recorded. Analysis of docking results led to the identification of the 8 studied molecules as lead molecules which were selected for chemical synthesis, in vivo studies, and further preclinical evaluation.
The molecule DSR 11 was predicted as the most appropriate lead molecule for anti-CM activity in the present investigation apart from the other seven molecules (DSR4, DSR26, DSR38, DSR40, DSR49, DSR56, and DSR70).
The significant role of papain-like cysteine proteases, including papain, cathepsin L and SARS-CoV-2 PLpro, in biomedicine and biotechnology makes them interesting model systems for sensor ...development. These enzymes have a free thiol group that is suitable for many sensor designs including strong binding to gold nanoparticles or low-molecular-weight inhibitors. Focusing on the importance of the preservation of native protein structure for inhibitor-binding and molecular-imprinting, which has been applied in some efficient examples of sensor development, the aim of this work was to examine the effects of the free-thiol-group's reversible blocking on papain denaturation that is the basis of its activity loss and aggregation. To utilize biophysical methods common in protein structural transitions characterization, such as fluorimetry and high-resolution infrared spectroscopy, low-molecular-weight electrophilic thiol blocking reagent S-Methyl methanethiosulfonate (MMTS) was used in solution. MMTS binding led to a two-fold increase in 8-Anilinonaphthalene-1-sulfonic acid fluorescence, indicating increased hydrophobic residue exposure. A more in-depth analysis showed significant transitions on the secondary structure level upon MMTS binding, mostly characterized by the lowered content of α-helices and unordered structures (either for approximately one third), and the increase in aggregation-specific β-sheets (from 25 to 52%) in a dose-dependant manner. The recovery of this inhibited protein showed that reversibility of inhibition is accompanied by reversibility of protein denaturation. Nevertheless, a 100-fold molar excess of the inhibitor led to the incomplete recovery of proteolytic activity, which can be explained by irreversible denaturation. The structural stability of the C-terminal β-sheet rich domain of the papain-like cysteine protease family opens up an interesting possibility to use its foldamers as a strategy for sensor development and other multiple potential applications that rely on the great commercial value of papain-like cysteine proteases.
This study aims to explore possible involvement of Sesbania grandiflora leaves extract on haemostatic pathway. The crude dialyzed fraction (SgCDF) hydrolyses casein and exhibiting a protease activity ...(19.7 ± 1.5U/Hr) at 100μg/ml. Zymography (casein) studies further reveal the presence of two high molecular weight protease(s). Presence of possible protease(s) was confirmed by inhibition of Proteolytic activity by mercury chloride (HgCl2) suggesting the presence of Cysteine protease. The stability of protease activity against various parameters viz., temperature, pH, salt (NaCl) has been evaluated. These protease(s) exhibiting a promising Fibrinogenolytic activity by hydrolyzing Aα and Bβ subunit of fibrinogen at 25μg and 50μg concentration, whereas SgCDF fails to degrade the γ-subunit at the same concentration. The protease shows a promising recalcification time up to 80 ± 2% against Trypsin. These findings suggest the possible role of proteolytic fraction of Sesbania grandiflora in haemostatic and wound healing process.
•Marasmius oreades agglutinin (MOA) is toxic to NIH/3T3 cells.•The toxic effect of MOA leads to the arrest of protein and DNA synthesis.•The proteolytic activity of MOA is the main, yet not the sole ...source of its cytotoxic effect.•A peptide-array based substrate profiling of MOA provides a list of potential targets.•The substrate screening data point to BAX as a potential mediator of the cytotoxic effect.
The Marasmius oreades mushroom agglutinin (MOA) is a blood group B-specific lectin carrying an active proteolytic domain. Its enzymatic activity has recently been shown to be critical for toxicity of MOA toward the fungivorous soil nematode Caenorhabditis elegans. Here we present evidence that MOA also induces cytotoxicity in a cellular model system (murine NIH/3T3 cells), by inhibiting protein synthesis, and that cytotoxicity correlates, at least in part, with proteolytic activity. A peptide-array screen identified the apoptosis mediator BAX as a potential proteolytic substrate and further suggests a variety of bacterial and fungal peptides as potential substrates. These findings are in line with the suggestion that MOA and related proteases may play a role for host defense.
PDF
