scholarly journals Expression and characterization of cathepsin P

2004 ◽  
Vol 378 (2) ◽  
pp. 657-663 ◽  
Author(s):  
Robert W. MASON ◽  
Carolyn A. BERGMAN ◽  
Guizhen LU ◽  
Jennifer FRENCK HOLBROOK ◽  
Katia SOL-CHURCH
Keyword(s):  
Mouse Genome ◽  
Cathepsin L ◽  
Mammalian Species ◽  
Proteinase K ◽  
Protein Substrates ◽  
Optimal Activity ◽  
Neutral Ph ◽  
Cathepsin P ◽  
Mrna Analysis

The mouse genome contains a family of clan C1A proteases that appear to be restricted to rodents within Eutherian (placental) mammals. mRNA analysis has shown that these genes are expressed exclusively in placenta. Sequence analysis predicts that the expressed proteins will be functional and consequently it was proposed that this family of proteases may have evolved to perform subspecialized functions of the closely related protease, cathepsin L, that is expressed in placental tissues of all mammalian species. In the present study, it was shown that cathepsin P can be expressed in Pichia pastoris as an inactive zymogen that can be activated with proteinase K, chymotrypsin or pancreatic elastase at neutral pH. Unlike other mammalian cathepsins, cathepsin P could also be autoactivated at neutral pH, but not at acidic pH. The activated enzyme was capable of hydrolysing peptidyl substrates and the protein substrates azocasein and transferrin, with optimal activity at pH 6.5–7.5. Little activity could be detected at pH 5.0 and below. Salts such as Na2SO4 and hyaluronate stimulated the activity of the protease against peptidyl substrates. The properties of cathepsin P appear to be quite distinct from those of cathepsin L, indicating that the duplication that gave rise to cathepsin P has probably not yielded an enzyme that provides a subfunction of cathepsin L in rodents. It seems probable that cathepsin P has evolved to perform a function that is performed by an evolutionarily unrelated protease in other mammalian species.

scholarly journals Patterns of gene expression in schistosomes: localization by whole mountin situhybridization

Parasitology ◽  
2007 ◽  
Vol 134 (11) ◽  
pp. 1589-1597 ◽  
Author(s):  
G. P. DILLON ◽  
J. C. ILLES ◽  
H. V. ISAACS ◽  
R. A. WILSON
Keyword(s):  
Gene Expression ◽  
Cathepsin L ◽  
Expression Patterns ◽  
Proteinase K ◽  
Fluorescent Substrate ◽  
Rna Probes ◽  
Fast Red ◽  
Proteinase K Treatment

SUMMARYAs a consequence of comprehensive transcriptome analysis followed by sequencing and draft assembly of the genome, the emphasis of schistosome research is shifting from the identification of genes to the characterization of their functions and interactions. Developmental biologists have long used whole mountin situhybridization (WISH) to determine gene expression patterns, as a vital tool for formulating and testing hypotheses about function. This paper describes the application of WISH to the study of gene expression in larval and adult schistosomes. Fixed worms were permeablized by proteinase K treatment for hybridization with digoxygenin-labelled RNA probes, with binding being detected by alkaline phosphatase-coupled anti-digoxygenin antibodies, and BM Purple substrate. Discrete staining patterns for the transcripts of the molecules Sm29, cathepsin L, antigen 10.3 and chorion were observed in the tegument cell bodies, gut epithelium, oesophageal gland and vitelline lobules, respectively, of adult worms. Transcripts of the molecules SGTP4, GP18-22 and cathepsin L were localized to tegument cell bodies and embryonic gut, respectively, of lung schistosomula. We also showed that Fast Red TR fluorescent substrate can refine the pattern of localization permitting use of confocal microscopy. We believe that method of WISH will find broad application, in synergy with other emerging post-genomic techniques, such as RNA interference, to studies focused at increasing our molecular understanding of schistosomes.

In Silico Identification and Molecular Characterization of Extracellular Cathepsin L Proteases from Giardia duodenalis

2020 ◽  
Vol 17 (4) ◽  
pp. 342-351
Author(s):  
Sergio A. Durán-Pérez ◽  
José G. Rendón-Maldonado ◽  
Lucio de Jesús Hernandez-Diaz ◽  
Annete I. Apodaca-Medina ◽  
Maribel Jiménez-Edeza ◽  
...  
Keyword(s):  
In Silico ◽  
Cathepsin L ◽  
Three Dimensional ◽  
Giardia Duodenalis ◽  
Evolutionary Genetics ◽  
Extracellular Proteins ◽  
In Silico Identification ◽  
Dimensional Models ◽  
Three Dimensional Models

Background: The protozoan Giardia duodenalis, which causes giardiasis, is an intestinal parasite that commonly affects humans, mainly pre-school children. Although there are asymptomatic cases, the main clinical features are chronic and acute diarrhea, nausea, abdominal pain, and malabsorption syndrome. Little is currently known about the virulence of the parasite, but some cases of chronic gastrointestinal alterations post-infection have been reported even when the infection was asymptomatic, suggesting that the cathepsin L proteases of the parasite may be involved in the damage at the level of the gastrointestinal mucosa. Objective: The aim of this study was the in silico identification and characterization of extracellular cathepsin L proteases in the proteome of G. duodenalis. Methods: The NP_001903 sequence of cathepsin L protease from Homo sapienswas searched against the Giardia duodenalisproteome. The subcellular localization of Giardia duodenaliscathepsin L proteases was performed in the DeepLoc-1.0 server. The construction of a phylogenetic tree of the extracellular proteins was carried out using the Molecular Evolutionary Genetics Analysis software (MEGA X). The Robetta server was used for the construction of the three-dimensional models. The search for possible inhibitors of the extracellular cathepsin L proteases of Giardia duodenaliswas performed by entering the three-dimensional structures in the FINDSITEcomb drug discovery tool. Results: Based on the amino acid sequence of cathepsin L from Homo sapiens, 8 protein sequences were identified that have in their modular structure the Pept_C1A domain characteristic of cathepsins and two of these proteins (XP_001704423 and XP_001704424) are located extracellularly. Threedimensional models were designed for both extracellular proteins and several inhibitory ligands with a score greater than 0.9 were identified. In vitrostudies are required to corroborate if these two extracellular proteins play a role in the virulence of Giardia duodenalisand to discover ligands that may be useful as therapeutic targets that interfere in the mechanism of pathogenesis generated by the parasite. Conclusion: In silicoanalysis identified two proteins in the Giardia duodenalisprotein repertoire whose characteristics allowed them to be classified as cathepsin L proteases, which may be secreted into the extracellular medium to act as virulence factors. Three-dimensional models of both proteins allowed the identification of inhibitory ligands with a high score. The results suggest that administration of those compounds might be used to block the endopeptidase activity of the extracellular cathepsin L proteases, interfering with the mechanisms of pathogenesis of the protozoan parasite Giardia duodenalis.

scholarly journals Identification and characterization of a phospholipase C activity in resident mouse peritoneal macrophages. Inhibition of the enzyme by phenothiazines

1981 ◽  
Vol 197 (2) ◽  
pp. 523-526 ◽  
Author(s):  
Paul D. Wightman ◽  
Mary Ellen Dahlgren ◽  
James C. Hall ◽  
Philip Davies ◽  
Robert J. Bonney
Keyword(s):  
Phospholipase C ◽  
High Activity ◽  
Peritoneal Macrophages ◽  
Ph Optimum ◽  
Reaction Velocity ◽  
Neutral Ph ◽  
Maximum Reaction ◽  
Mouse Peritoneal Macrophages ◽  
Identification And Characterization

Resident mouse peritoneal macrophages contain a phospholipase C of high activity that is specific for phosphatidylinositol. The activity has a neutral pH optimum, is Ca2+-dependent and has a maximum reaction velocity of 525nmol/h per mg of protein. Certain phenothiazines are potent inhibitors of this activity.

Purification and partial amino acid sequence of thuricin S, a new anti-Listeriabacteriocin from Bacillus thuringiensis

2007 ◽  
Vol 53 (2) ◽  
pp. 284-290 ◽  
Author(s):  
Sonia Chehimi ◽  
François Delalande ◽  
Sophie Sablé ◽  
Mohamed-Rabeh Hajlaoui ◽  
Alain Van Dorsselaer ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Proteinase K ◽  
Terminal Sequence ◽  
Partial Amino Acid Sequence ◽  
Ph Values ◽  
Heat Stable ◽  
Isolation And Characterization ◽  
Terminal Amino ◽  
Edman Sequencing

We report the isolation and characterization of a new bacteriocin, thuricin S, produced by the Bacillus thuringiensis subsp. entomocidus HD198 strain. This antibacterial activity is sensitive to proteinase K, is heat-stable, and is stable at a variety of pH values (3–10.5). The monoisotopic mass of thuricin S purified by high perfomance liquid chromatography, as determined with mass spectrometry ESI-TOF-MS, is 3137.61 Da. Edman sequencing and NanoESI-MS/MS experiments provided the sequence of the 18 N-terminal amino acids. Interestingly, thuricin S has the same N-terminal sequence (DWTXWSXL) as bacthuricin F4 and thuricin 17, produced by B. thuringiensis strains BUPM4 and NEB17, respectively, and could therefore be classified as a new subclass IId bacteriocin.

scholarly journals Purification and characterization of bacteriocins-like inhibitory substances from food isolated Enterococcus faecalis OS13 with activity against nosocomial enterococci

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ahmed O. El-Gendy ◽  
Dag A. Brede ◽  
Tamer M. Essam ◽  
Magdy A. Amin ◽  
Shaban H. Ahmed ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Antimicrobial Agents ◽  
Food Samples ◽  
Proteinase K ◽  
Clinical Samples ◽  
Bile Salt Hydrolase ◽  
Antibiotic Resistant ◽  
Molecular Weights ◽  
Global Threat

AbstractNosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13β) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13β were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13β was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13β represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

Cleavage of immunoglobulin G by excretory–secretory cathepsin S-like protease of Spirometra mansoni plerocercoid

Parasitology ◽  
1994 ◽  
Vol 109 (5) ◽  
pp. 611-621 ◽  
Author(s):  
Y. Kong ◽  
Y.-B. Chung ◽  
S.-Y. Cho ◽  
S.-Y. Kang
Keyword(s):  
Immunoglobulin G ◽  
Cathepsin L ◽  
Immunoblot Analysis ◽  
Cathepsin S ◽  
Terminal Amino Acid ◽  
Optimal Activity ◽  
Spirometra Mansoni ◽  
Terminal Amino ◽  
Secretory Products ◽  
Ph Range

When immunoglobulin G (IgG) was incubated with Spirometra mansoni plerocercoid (sparganum), it was cleaved into Fab and Fc fragments. Fab/c fragments were also hydrolysed. The digestion was accelerated by dithiothreitol (DTT), indicating that cleavage of IgG heavy chain was due to a cysteine protease secreted into the medium. The responsible enzyme, of Mr 27 (± 0·8) kDa, was purified by a series of thiopropyl affinity, Sephacryl S-300 HR and DEAE-anion exchange chromatographies, either from worm extracts or from excretory–secretory products (ESP). The purified, thiol-dependent protease showed an optimal activity at pH 5·7 with 0·1 M sodium acetate but was active over the pH range 4·5–8·0. Its activity was inhibited completely by 10−5 M L-trans-epoxysuccinylleucylamido(4-guanidino) butane (E-64) and 1 mM iodoacetamide (IAA), but by only 53% using the specific cathepsin L inhibitor, Z-Phe-Phe-CHN2 (5 × 10−5 M). Partial NH2-terminal amino acid sequence was Leu-Pro-Asp-Ser-Val-Asn-Trp-Arg-Glu-Gly-Ala-Val-Thr-Ala-Val which showed 80% homology to human cathepsin S. Immunoblot analysis showed that sera from infected patients exhibited IgE antibody reaction. It is proposed that cleavage of immunoglobulin by an excreted–secreted, cathepsin S-like, allergenic protease is a mechanism of immune evasion used by the sparganum.

Biochemical characterization of human HIF hydroxylases using HIF protein substrates that contain all three hydroxylation sites

2011 ◽  
Vol 436 (2) ◽  
pp. 363-369 ◽  
Author(s):  
Melissa B. Pappalardi ◽  
Dean E. McNulty ◽  
John D. Martin ◽  
Kelly E. Fisher ◽  
Yong Jiang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Hypoxia Inducible Factor ◽  
Prolyl Hydroxylase ◽  
Transactivation Domain ◽  
Prolyl Hydroxylases ◽  
Proline Residue ◽  
Protein Substrates ◽  
Steady State Kinetic ◽  
Asparaginyl Hydroxylase

The HIF (hypoxia-inducible factor) plays a central regulatory role in oxygen homoeostasis. HIF proteins are regulated by three Fe(II)- and α-KG (α-ketoglutarate)-dependent prolyl hydroxylase enzymes [PHD (prolyl hydroxylase domain) isoenzymes 1–3 or PHD1, PHD2 and PHD3] and one asparaginyl hydroxylase [FIH (factor inhibiting HIF)]. The prolyl hydroxylases control the abundance of HIF through oxygen-dependent hydroxylation of specific proline residues in HIF proteins, triggering subsequent ubiquitination and proteasomal degradation. FIH inhibits the HIF transcription activation through asparagine hydroxylation. Understanding the precise roles and regulation of these four Fe(II)- and α-KG-dependent hydroxylases is of great importance. In the present paper, we report the biochemical characterization of the first HIF protein substrates that contain the CODDD (C-terminal oxygen-dependent degradation domain), the NODDD (N-terminal oxygen-dependent degradation domain) and the CAD (C-terminal transactivation domain). Using LC-MS/MS (liquid chromatography–tandem MS) detection, we show that all three PHD isoenzymes have a strong preference for hydroxylation of the CODDD proline residue over the NODDD proline residue and the preference is observed for both HIF1α and HIF2α protein substrates. In addition, steady-state kinetic analyses show differential substrate selectivity for HIF and α-KG in reference to the three PHD isoforms and FIH.

scholarly journals Characterization of bacteriocin and chitinase producing bacterial isolates with broad-spectrum antimicrobial activities

2021 ◽  
Vol 3 (8) ◽  
Author(s):  
Muhammad Yasir ◽  
Basit Zeshan ◽  
Nur Hardy A. Daud ◽  
Izzah Shahid ◽  
Hafza Khalid
Keyword(s):  
Antimicrobial Activity ◽  
Inhibitory Activity ◽  
Antifungal Drugs ◽  
Proteinase K ◽  
Diffusion Method ◽  
List Type ◽  
Bacterial Isolates ◽  
E Coli ◽  
Inhibitory Potential

Abstract There is a need for more efficient and eco-friendly approaches to overcome increasing microbial infections. Bacteriocins and chitinases from Bacillus spp. can be powerful alternatives to conventional antibiotics and antifungal drugs, respectively. The purpose of this study was to assess the inhibitory potential of bacteriocins and chitinase enzymes against multiple resistant bacterial and fungal pathogens. Bacterial isolates were selected by growth on minimal salts medium and after that were morphologically and biochemically characterized. The physiochemical characterization of bacteriocins was carried out. The inhibitory potential of bacteriocins towards six pathogenic bacteria was determined by the well diffusion assay while chitinase activity towards three fungal strains was determined by the dual plate culture assay. Two bacterial strains (WW2P1 and WRE4P2), out of nine showed inhibition of K. pneumonia, P. aeruginosa, E. coli and MRSA while WW4P2 was positive against S. typhimurium and E. coli and WRE10P2 against P. aeruginosa, S. pneumoniae. Two bacterial isolates (WW3P1 and WRE10P2) were chosen for further study on the basis of their antifungal activities. Of these, WW3P1 isolate was more effective against A. fumigatus as well as A. niger. The proteinaceous nature of the bacteriocins was confirmed by treatment of the crude extract with proteinase K. It was found that the inhibitory activity of strain WW3P1 against E. coli was highest at 20 °C, and against S. pneumoniae it was at 20 °C and pH 10 after treatment with EDTA. Inhibition by strain the WRE10P2 against P. aeruginosa was highest at 20 °C and pH 14. It was found that EDTA increased the inhibitory activity of strain WW2P1 against P. aeruginosa, K. pneumoniae and E. coli by 2 ± 0.235, 3.5 ± 0.288, 2.5 ± 1.040 times, respectively, of strain WRE4P2 against P. aeruginosa and E. coli by 2.5 ± 0.763, 2.7 ± 0.5 times, respectively, and of strain WRE10P2 against S. pneumoniae by 3 ± 0.6236 times. The isolates have promising inhibitory activity, which should be further analyzed for the commercial production of antimicrobials. Article highlights The current study aimed to isolate the microbiome from wheat plant (Triticum aestivum L.), to screen for bacteriocin production and to assess its antimicrobial activity against human pathogens. Forty-one phenotypically different bacterial colonies were subjected to bacteriocin purification from which 25 colonies showed positive reactions. These 25 bacterial isolates were screened against six different human bacterial pathogens using the well diffusion method to check the antimicrobial activity. Out of nine bacterial isolates, WW3P1 and WRE10P2 were able to degrade the chitin and utilize it as their sole energy source. Strain WRE4P2 exhibited partial inactivation in its activity against MRSA after treatment with proteinase K.

scholarly journals Expression, Purification, and Characterization of (R)-Sulfolactate Dehydrogenase (ComC) from the Rumen MethanogenMethanobrevibacter milleraeSM9

Archaea ◽  
2017 ◽  
Vol 2017 ◽  
pp. 1-6
Author(s):  
Yanli Zhang ◽  
Linley R. Schofield ◽  
Carrie Sang ◽  
Debjit Dey ◽  
Ron S. Ronimus
Keyword(s):  
Biosynthetic Pathway ◽  
Critical Role ◽  
Reduction Reaction ◽  
Coenzyme M ◽  
Purification And Characterization ◽  
The Third ◽  
Optimal Activity ◽  
Methyl Coenzyme M Reductase

(R)-Sulfolactate dehydrogenase (EC 1.1.1.337), termed ComC, is a member of an NADH/NADPH-dependent oxidoreductase family of enzymes that catalyze the interconversion of 2-hydroxyacids into their corresponding 2-oxoacids. The ComC reaction is reversible and in the biosynthetic direction causes the conversion of (R)-sulfolactate to sulfopyruvate in the production of coenzyme M (2-mercaptoethanesulfonic acid). Coenzyme M is an essential cofactor required for the production of methane by the methyl-coenzyme M reductase complex. ComC catalyzes the third step in the first established biosynthetic pathway of coenzyme M and is also involved in methanopterin biosynthesis. In this study, ComC fromMethanobrevibacter milleraeSM9 was cloned and expressed inEscherichia coliand biochemically characterized. Sulfopyruvate was the preferred substrate using the reduction reaction, with 31% activity seen for oxaloacetate and 0.2% seen forα-ketoglutarate. Optimal activity was observed at pH 6.5. The apparentKMfor coenzyme (NADH) was 55.1 μM, and for sulfopyruvate, it was 196 μM (for sulfopyruvate theVmaxwas 93.9 μmol min−1 mg−1andkcatwas 62.8 s−1). The critical role of ComC in two separate cofactor pathways makes this enzyme a potential means of developing methanogen-specific inhibitors for controlling ruminant methane emissions which are increasingly being recognized as contributing to climate change.

scholarly journals Characterization of CAA0225, a Novel Inhibitor Specific for Cathepsin L, as a Probe for Autophagic Proteolysis

2009 ◽  
Vol 32 (3) ◽  
pp. 475-479 ◽  
Author(s):  
Katsuyuki Takahashi ◽  
Takashi Ueno ◽  
Isei Tanida ◽  
Naoko Minematsu-Ikeguchi ◽  
Mitsuo Murata ◽  
...  
Keyword(s):  
Cathepsin L
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