scholarly journals Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease

2016 ◽  
Vol 311 (3) ◽  
pp. G571-G580 ◽  
Author(s):  
Guoxian Wei ◽  
Na Tian ◽  
Roland Siezen ◽  
Detlef Schuppan ◽  
Eva J. Helmerhorst
Keyword(s):  
Celiac Disease ◽  
Serine Proteases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Triad ◽  
Local Alignment ◽  
Peptide Substrates ◽  
Food Grade ◽  
Search Tool ◽  
Rothia Mucilaginosa

Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD.

scholarly journals Screening of HIV-1 Protease Using a Combination of an Ultra-High-Throughput Fluorescent-Based Assay and RapidFire Mass Spectrometry

2015 ◽  
Vol 20 (5) ◽  
pp. 606-615 ◽  
Author(s):  
Juncai Meng ◽  
Ming-Tain Lai ◽  
Vandna Munshi ◽  
Jay Grobler ◽  
John McCauley ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Antiviral Agents ◽  
Sampling Rate ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Label Free ◽  
Substrate Selection ◽  
Peptide Substrates ◽  
Hiv 1

HIV-1 protease (PR) represents one of the primary targets for developing antiviral agents for the treatment of HIV-infected patients. To identify novel PR inhibitors, a label-free, high-throughput mass spectrometry (HTMS) assay was developed using the RapidFire platform and applied as an orthogonal assay to confirm hits identified in a fluorescence resonance energy transfer (FRET)–based primary screen of > 1 million compounds. For substrate selection, a panel of peptide substrates derived from natural processing sites for PR was evaluated on the RapidFire platform. As a result, KVSLNFPIL, a new substrate measured to have a ~ 20- and 60-fold improvement in kcat/ Km over the frequently used sequences SQNYPIVQ and SQNYPIV, respectively, was identified for the HTMS screen. About 17% of hits from the FRET-based primary screen were confirmed in the HTMS confirmatory assay including all 304 known PR inhibitors in the set, demonstrating that the HTMS assay is effective at triaging false-positives while capturing true hits. Hence, with a sampling rate of ~7 s per well, the RapidFire HTMS assay enables the high-throughput evaluation of peptide substrates and functions as an efficient tool for hits triage in the discovery of novel PR inhibitors.

scholarly journals Fluorescence Lifetime–Based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro

2014 ◽  
Vol 19 (6) ◽  
pp. 870-877 ◽  
Author(s):  
Andreas Boettcher ◽  
Nathalie Gradoux ◽  
Edwige Lorthiois ◽  
Trixi Brandl ◽  
David Orain ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Serine Proteases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Competitive Binding ◽  
Binding Assays ◽  
Time Resolved ◽  
Design And Synthesis ◽  
Competitive Binding Assays

Fluorescence lifetime (FLT)–based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site–directed low-molecular-weight inhibitors conjugated to PT14) are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer–based competitive binding assays.

scholarly journals Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification

2013 ◽  
Vol 18 (6) ◽  
pp. 726-735 ◽  
Author(s):  
Tanja Ouimet ◽  
Sophie Duquesnoy ◽  
Hervé Poras ◽  
Marie-Claude Fournié-Zaluski ◽  
Bernard P. Roques
Keyword(s):  
Peptide Mapping ◽  
Resonance Energy Transfer ◽  
Medicinal Preparation ◽  
Resonance Energy ◽  
Peptide Sequence ◽  
Alternative Methods ◽  
Peptide Substrates ◽  
Active Moiety ◽  
Detection And Quantification ◽  
Better Than

Detection and quantification of low doses of botulinum toxin serotype A (BoNT/A) in medicinal preparations require precise and sensitive methods. With mounting pressure from governmental authorities to replace the mouse LD50 assay, interest in alternative methods such as the endopeptidase assay, quantifying the toxin active moiety, is growing. Using internal collision-induced fluorescence quenching, Pharmaleads produced peptides encompassing the SNAP-25 cleavage site: a 17-mer (PL63) and a 48-mer (PL50) reaching the previously identified α-exosite, with PL50 showing higher apparent affinity for BoNT/A. Peptide mapping experiments revealed that this increased affinity is mainly due to a connecting peptide sequence between the N-terminus of PL63 and the α-exosite, identifying a new cooperative exosite on BoNT/A. Other endopeptidase substrates available, including SNAPTide and BoTest A/E, are both based on fluorescent resonance energy transfer (FRET) technology. To compare these assays, their limits of detection and quantification were determined using light chain or 150-kDa BoNT/A. Detection limits of PL50 and BoTest were over 100 times better than those using SNAPtide in standard conditions. Although the BoTest possessed a detection limit around 0.2 pM for either BoNT/A form, its quantification limit (9.7 pM) using purified BoNT/A was 12 times inferior to PL50, estimated at 0.8 pM, suitable for medicinal preparation quantification.

scholarly journals A FRET Approach to Detect Paraoxon among Organophosphate Pesticides Using a Fluorescent Biosensor

Sensors ◽  
2022 ◽  
Vol 22 (2) ◽  
pp. 561
Author(s):  
Andreia C. M. Rodrigues ◽  
Maria Vittoria Barbieri ◽  
Marco Chino ◽  
Giuseppe Manco ◽  
Ferdinando Febbraio
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Triad ◽  
Thermostable Enzyme ◽  
In Situ Monitoring ◽  
Promising Candidate ◽  
Fluorescent Biosensor ◽  
Alicyclobacillus Acidocaldarius ◽  
Significant Interference

The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser–His–Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.

scholarly journals Characterization of ecotin homologs from Campylobacter rectus and Campylobacter showae

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0244031
Author(s):  
Cody Thomas ◽  
Harald Nothaft ◽  
Ruchi Yadav ◽  
Christopher Fodor ◽  
Abofu Alemka ◽  
...  
Keyword(s):  
Oral Cavity ◽  
Campylobacter Jejuni ◽  
Serine Proteases ◽  
Resonance Energy Transfer ◽  
Factor Xa ◽  
Resonance Energy ◽  
Deficient Mutant ◽  
Campylobacter Rectus

Ecotin, first described in Escherichia coli, is a potent inhibitor of a broad range of serine proteases including those typically released by the innate immune system such as neutrophil elastase (NE). Here we describe the identification of ecotin orthologs in various Campylobacter species, including Campylobacter rectus and Campylobacter showae residing in the oral cavity and implicated in the development and progression of periodontal disease in humans. To investigate the function of these ecotins in vitro, the orthologs from C. rectus and C. showae were recombinantly expressed and purified from E. coli. Using CmeA degradation/protection assays, fluorescence resonance energy transfer and NE activity assays, we found that ecotins from C. rectus and C. showae inhibit NE, factor Xa and trypsin, but not the Campylobacter jejuni serine protease HtrA or its ortholog in E. coli, DegP. To further evaluate ecotin function in vivo, an E. coli ecotin-deficient mutant was complemented with the C. rectus and C. showae homologs. Using a neutrophil killing assay, we demonstrate that the low survival rate of the E. coli ecotin-deficient mutant can be rescued upon expression of ecotins from C. rectus and C. showae. In addition, the C. rectus and C. showae ecotins partially compensate for loss of N-glycosylation and increased protease susceptibility in the related pathogen, Campylobacter jejuni, thus implicating a similar role for these proteins in the native host to cope with the protease-rich environment of the oral cavity.

scholarly journals Fluorogenic peptide substrates for carboxydipeptidase activity of cathepsin B.

2004 ◽  
Vol 51 (1) ◽  
pp. 81-92 ◽  
Author(s):  
Krystyna Stachowiak ◽  
Monika Tokmina ◽  
Anna Karpińska ◽  
Renata Sosnowska ◽  
Wiesław Wiczk
Keyword(s):  
Amino Acid ◽  
Cysteine Protease ◽  
Cathepsin B ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinetic Studies ◽  
Amino Acid Sequences ◽  
Peptide Substrates ◽  
Donor And Acceptor ◽  
Specificity Constant

Cathepsin B is a lysosomal cysteine protease exhibiting mainly dipeptidyl carboxypeptidase activity, which decreases dramatically above pH 5.5, when the enzyme starts acting as an endopeptidase. Since the common cathepsin B assays are performed at pH 6 and do not distinguish between these activities, we synthesized a series of peptide substrates specifically designed for the carboxydipeptidase activity of cathepsin B. The amino-acid sequences of the P(5)-P(1) part of these substrates were based on the binding fragments of cystatin C and cystatin SA, the natural reversible inhibitors of papain-like cysteine protease. The sequences of the P'(1)-P'(2) dipeptide fragments of the substrates were chosen on the basis of the specificity of the S'(1)-S'(2) sites of the cathepsin B catalytic cleft. The rates of hydrolysis by cathepsin B and papain, the archetypal cysteine protease, were monitored by a continuous fluorescence assay based on internal resonance energy transfer from an Edans to a Dabcyl group. The fluorescence energy donor and acceptor were attached to the C- and the N-terminal amino-acid residues, respectively. The kinetics of hydrolysis followed the Michaelis-Menten model. Out of all the examined peptides Dabcyl-R-L-V-G-F- E(Edans) turned out to be a very good substrate for both papain and cathepsin B at both pH 6 and pH 5. The replacement of Glu by Asp turned this peptide into an exclusive substrate for cathepsin B not hydrolyzed by papain. The substitution of Phe by Nal in the original substrate caused an increase of the specificity constant for cathepsin B at pH 5, and a significant decrease at pH 6. The results of kinetic studies also suggest that Arg in position P(4) is not important for the exopeptidase activity of cathepsin B, and that introducing Glu in place of Val in position P(2) causes an increase of the substrate preference towards this activity.

Engineering resonance energy transfer for advanced immunoassays: The case of celiac disease

2012 ◽  
Vol 425 (1) ◽  
pp. 13-17 ◽  
Author(s):  
Sabato D’Auria ◽  
Elisa Apicella ◽  
Maria Staiano ◽  
Stefano Di Giovanni ◽  
Giuseppe Ruggiero ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Celiac Disease ◽  
Resonance Energy Transfer ◽  
Resonance Energy

scholarly journals High-Throughput Screening of Low Molecular Weight NS3-NS4A Protease Inhibitors Using a Fluorescence Resonance Energy Transfer Substrate

2005 ◽  
Vol 16 (6) ◽  
pp. 385-392 ◽  
Author(s):  
Kenji Sudo ◽  
Kayo Yamaji ◽  
Kouich Kawamura ◽  
Tomoko Nishijima ◽  
Naoko Kojima ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Serine Proteases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Compound Libraries ◽  
Fluorescence Resonance ◽  
Potent Inhibitory Activity

Hepatitis C virus (HCV) NS3-NS4A protease is an attractive target for anti-HCV agents because of its important role in replication. An optimized fluorescence resonance energy transfer (FRET) substrate for NS3-NS4A protease, based on the sequence of the NS5A-5B cleavage site, was designed and synthesized. High-throughput screening of in-house compound libraries was performed using a FRET substrate FS10 (MOCAc-DKIVPC-SMSYK-Dnp) and MBP-NS3-NS4A fusion protein. Several hit compounds were found, including YZ-9577 (2-oxido-1,2,5-oxadiazole-3,4-diyl) bis (phenylmethanone) with potent inhibitory activity (IC50=1.6 μM) and good selectivity against other human serine proteases.

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