FRET Peptide Research Articles

Biochemical characterization, stability, and kinetics of three substrates of the recombinant TMPRSS2 serine protease domain

Transmembrane serine protease 2 (TMPRSS2) is a membrane-bound protease belonging to the type II transmembrane serine protease (TTSP) family. It is a multidomain protein, including a serine protease domain responsible for its self-activation. The protein has been implicated as an oncogenic transcription factor and for its ability to cleave (prime) the SARS-CoV-2 spike protein. In order to characterize the TMPRSS2 biochemical properties, we expressed the serine protease domain (rTMPRSS2_SP) in Komagataella phaffii using the pPICZαA vector and purified it using immobilized metal affinity (Ni Sepharose™ excel) and size exclusion (Superdex 75) chromatography. We explored operational fluorescence resonance energy transfer FRET peptides as substrates. We chose the peptide Abz-QARK-(Dnp)-NH2 (Abz = ortho-aminobenzoic acid, the fluorescence donor, and Dnp = 2,4-dinitrophenyl, the quencher group) as a substrate to find the optimal conditions for maximum enzymatic activity. We found that metallic ions such as Ca2+ and Na+ increased enzymatic activity, but ionic surfactants and reducing agents decreased catalytic capacity. Finally, we determined the rTMPRSS2_SP stability for long-term storage. Altogether, our results represent the first comprehensive characterization of TMPRSS2’s biochemical properties, providing valuable insights into its serine protease domain.

Leptolysin, a Leptospira secreted metalloprotease of the pappalysin family with broad-spectrum activity.

Extracellular proteolytic enzymes are produced by a variety of pathogenic microorganisms, and contribute to host colonization by modulating virulence. Here, we present a first characterization of leptolysin, a Leptospira metalloprotease of the pappalysin family identified in a previous exoproteomic study. Comparative molecular analysis of leptolysin with two other pappalysins from prokaryotes, ulilysin and mirolysin, reveals similarities regarding calcium, zinc, and arginine -binding sites conservation within the catalytic domain, but also discloses peculiarities. Variations observed in the primary and tertiary structures may reflect differences in primary specificities. Purified recombinant leptolysin of L. interrogans was obtained as a ~50 kDa protein. The protease exhibited maximal activity at pH 8.0 and 37°C, and hydrolytic activity was observed in the presence of different salts with maximum efficiency in NaCl. Substrate specificity was assessed using a small number of FRET peptides, and showed a marked preference for arginine residues at the P1 position. L. interrogans leptolysin proteolytic activity on proteinaceous substrates such as proteoglycans and plasma fibronectin was also evaluated. All proteins tested were efficiently degraded over time, confirming the protease´s broad-spectrum activity in vitro. In addition, leptolysin induced morphological alterations on HK-2 cells, which may be partially attributed to extracellular matrix (ECM) degradation. Hemorrhagic foci were observed in the dorsal skin of mice intradermally injected with leptolysin, as a plausible consequence of ECM disarray and vascular endothelium glycocalyx damage. Assuming that leptospiral proteases play an important role in all stages of the infectious process, characterizing their functional properties, substrates and mechanisms of action is of great importance for therapeutic purposes.

A fluorogenic probe for granzyme B enables in-biopsy evaluation and screening of response to anticancer immunotherapies

Immunotherapy promotes the attack of cancer cells by the immune system; however, it is difficult to detect early responses before changes in tumor size occur. Here, we report the rational design of a fluorogenic peptide able to detect picomolar concentrations of active granzyme B as a biomarker of immune-mediated anticancer action. Through a series of chemical iterations and molecular dynamics simulations, we synthesize a library of FRET peptides and identify probe H5 with an optimal fit into granzyme B. We demonstrate that probe H5 enables the real-time detection of T cell-mediated anticancer activity in mouse tumors and in tumors from lung cancer patients. Furthermore, we show image-based phenotypic screens, which reveal that the AKT kinase inhibitor AZD5363 shows immune-mediated anticancer activity. The reactivity of probe H5 may enable the monitoring of early responses to anticancer treatments using tissue biopsies.

Molecular Basis for Antiviral Action of EDP‐235: A Potent and Selective SARS‐CoV‐2 3CLpro Inhibitor for the Treatment of Covid 19

To date, there are no approved oral antiviral therapies that can be administered early in the course of COVID‐19 to suppress progression of the disease or for prophylaxis. EDP‐235 is a potent and selective inhibitor of SARS‐CoV‐2 3C‐like protease (3CLpro). EDP‐235 inhibits SARS‐CoV‐2 3CLpro protease activity with an IC50 of 5.8 ± 3.7 nM and retains its activity against variant 3CLpro proteins from multiple SARS‐CoV‐2 lineages (IC50 range of 2.8­–5.8 nM). 3CLpro protease activity progress curves showed significant curvature in a time‐ and EDP‐235‐concentration‐dependent manner indicative of slow‐onset inhibition. Slow reversal of inhibition of SARS‐CoV‐2 3CLpro enzyme activity was observed in a jump dilution experiment. Michaelis‐Menten kinetic studies with a FRET peptide substrate in the presence of EDP‐235 indicated that EDP‐235 is a substrate‐competitive inhibitor of SARS‐CoV‐2 3CLpro with an overall dissociation constant Ki of 3.0 ± 1.6 nM. SARS‐CoV‐2 3CLpro was crystallized bound to a close analog of EDP‐235 and structure elucidation revealed that the ligand bound at the active site and interacted with side chains of conserved residues Cys‐145, His‐163, and Glu‐166. EDP‐235 also potently inhibits 3CLpro enzymes from other α‐coronaviruses (IC50 range of 2–4 nM) and β‐coronaviruses (SARS‐CoV IC50 of 5.4 nM, MERS‐CoV IC50 of 70 nM) which cause disease in humans to date. EDP‐235 resistance mutations in HCoV‐229E map to the active site of 3CLpro close to the predicted binding site and offer additional support to the mechanism of inhibition. EDP‐235 also showed a favorable selectivity profile (>300 selectivity index) when tested against a panel of 30 mammalian proteases. In summary, EDP‐235 acts as a slow‐onset, slow‐reversible, substrate‐competitive inhibitor of SARS‐CoV‐2 3CLpro. The outstanding preclinical profile of EDP‐235 supports its further evaluation as an oral therapeutic for the management of COVID‐19.

FRET-Based Assays to Determine Calpain Activity.

Calpains are signaling proteases that have relatively little sequence specificity but some preferences for certain residues on either side of the scissile bond. As with most proteases, they mainly cut unstructured or extended regions of their target proteins. The tendency for concentrated calpain to rapidly autoproteolyze when activated by calcium complicates the kinetic assessment of calpain activity. As calpain autoproteolyzes, the amount of fully active enzyme continuously decreases until all of the calpain molecules have been cut and their activity reduced to a tiny fraction of the starting rate. To accurately measure calpain kinetics, only the initial rate of substrate hydrolysis, where autoproteolysis is minimal, can be used. To accomplish this, a method for rapid, quantifiable determination of substrate cleavage is required. Many of the existing assays are lacking in their sensitivity to accurately quantify calpain activity within this timeframe. However, the FRET peptide substrates developed by Cuerrier et al. have been shown to have sufficiently high affinity between substrate and enzyme to accurately measure the initial enzyme reaction velocity at substrate concentrations above the Km value. With a suitably sensitive fluorimeter, sufficient data can be obtained to evaluate calpain kinetics and inhibition. Here we describe a facile, reliable calpain assay based on the continuous monitoring of FRET fluorescence from the highly sensitive calpain-specific substrate, (EDANS)-EPLFAERK-(DABCYL). We illustrate some difficulties associated with determining kinetic constants of whole calpains that are simultaneously undergoing autoproteolysis and how the assay can be used to help characterize calpain-specific inhibitors. We also present a variation of this fluorescence-based assay for high-throughput screening using the calpain protease core and a fluorescence plate reader.

Metallo-Organozymes with Specific Proteolytic Activity.

Metallopeptides that show efficiency and selectivity in peptide bond cleavage in water at room temperature and neutral conditions are presented. These small and versatile organozymes take advantage of metal-coordinating building blocks that are strategically positioned centrally in a peptide backbone or in a peptide macrocycle. This approach provided peptide-metal complexes with scaffolds capable of utilizing the peptide functionality for productive binding of fluorogenic FRET peptide substrates, subsequently leading to highly selective peptide bond cleavage. The ligand chemistry has been optimized to provide an easy access to new metallo-peptides with the ability to cleave previously inaccessible peptide bonds. Evolutionary principles of stepwise selection and variation offered by combinatorial methods were used and were guided by molecular modeling to develop catalytic metallo-peptides that mimic metalloproteases.

Super-silent FRET Sensor Enables Live Cell Imaging and Flow Cytometric Stratification of Intracellular Serine Protease Activity in Neutrophils

Serine proteases are released by neutrophils to act primarily as antimicrobial proteins but excessive and unbalanced serine protease activity results in serious host tissue damage. Here the synthesis of a novel chemical sensor based on a multi-branched fluorescence quencher is reported. It is super-silent, exhibiting no fluorescence until de-quenched by the exemplar serine protease human neutrophil elastase, rapidly enters human neutrophils, and is inhibited by serine protease inhibitors. This sensor allows live imaging of intracellular serine protease activity within human neutrophils and demonstrates that the unique combination of a multivalent scaffold combined with a FRET peptide represents a novel and efficient strategy to generate super-silent sensors that permit the visualisation of intracellular proteases and may enable point of care whole blood profiling of neutrophils.

A Tropical Composting Operation Unit at São Paulo Zoo as a Source of Bacterial Proteolytic Enzymes

Composting operation systems are valuable sources of microorganisms and enzymes. This work reports the assessment of proteolytic enzymes from cultivable bacteria isolated from a composting facility of the São Paulo Zoo Park (SPZPF), São Paulo, Brazil. Three hundred bacterial isolates were obtained and identified based on 16S rRNA gene as belonging to 13 different genera. The most common genus among the isolates was Bacillus (67%); some of which show high proteolytic activity in their culture media. Biochemical assays of hydrolytic activities using FRET peptides as substrates allowed the characterization of a repertoire of serine proteases and metalloproteases with different molecular weights secreted by Bacillus strains isolated from composting. Furthermore, thermostable serine and metalloproteases were detected in the composting leachate, which might be of interest for industrial applications.

Specificity profiling of human trypsin-isoenzymes.

In humans, three different trypsin-isoenzymes have been described. Of these, trypsin-3 appears to be functionally different from the others. In order to systematically study the specificity of the trypsin-isoenzymes, we utilized proteome-derived peptide libraries and quantitative proteomics. We found similar specificity profiles dominated by the well-characterized preference for cleavage after lysine and arginine. Especially, trypsin-1 slightly favored lysine over arginine in this position, while trypsin-3 did not discriminate between them. In the P1' position, which is the residue C-terminal to the cleavage site, we noticed a subtle enrichment of alanine and glycine for all three trypsins and for trypsin-3 there were additional minor P1' and P2' preferences for threonine and aspartic acid, respectively. These findings were confirmed by FRET peptide substrates showing different susceptibility to cleavage by different trypsins. The preference of trypsin-3 for aspartic acid in P2' is explained by salt bridge formation with the unique Arg193. This salt bridge enables and stabilizes a canonical oxyanion conformation by the amides of Ser195 and Arg193, thus manifesting a selective substrate-assisted catalysis. As trypsin-3 has been proposed to be a therapeutic target and marker for cancers, our results may aid the development of specific inhibitors for cancer therapy and diagnostic probes.

Kallikrein-related peptidase 6 can cleave human-muscle-type 6-phosphofructo-1-kinase into highly active shorter fragments

PurposeCancer cells consume more glucose than normal human cells and convert most glucose into lactate. It has been proposed that deregulated glycolysis is triggered by the posttranslational modification of 85 kDa muscle-type 6-phosphofructo-1-kinase (PFK-M) which is cleaved by a specific protease to form shorter, highly active, feedback-inhibition-resistant PFK-M fragments. Principal resultsTo find the protease involved in PFK-M modification, analyses of the protease target sites on the human PFK-M enzyme yielding 45–47 kDa fragments were performed in silico. The results suggested that an enzyme in the kallikrein (KLK) family may be involved. Kallikreins can be self-activated in the cytosol and are often overexpressed in cancer cells. After incubating the internally quenched FRET peptide with a sequence characteristic of the target site, along with the active KLK6, the cleavage of the peptide was observed. The ability of KLK6 to cleave native PFK-M and form highly active citrate-resistant 45 kDa fragments was further confirmed by enzymatic tests and SDS-PAGE. A role of KLK6 in the posttranslational modification of native PFK-M was ultimately confirmed in vivo. A yeast strain that encoded native human PFK-M as the only PFK1 enzyme was additionally transformed with proKLK6 or KLK6 genes under the control of an inducible promoter. The transformants growth rate was found to increase after the induction of proKLK6 gene expression as compared to the strain with the native PFK-M enzyme. ConclusionKLK6 may be the key protease involved in the modification of PFK-M and trigger deregulated glycolytic flux in cancer cells.

Activity of human kallikrein-related peptidase 6 (KLK6) on substrates containing sequences of basic amino acids. Is it a processing protease?

Human kallikrein 6 (KLK6) is highly expressed in the central nervous system and with elevated level in demyelinating disease. KLK6 has a very restricted specificity for arginine (R) and hydrolyses myelin basic protein, protein activator receptors and human ionotropic glutamate receptor subunits. Here we report a previously unreported activity of KLK6 on peptides containing clusters of basic amino acids, as in synthetic fluorogenic peptidyl-Arg-7-amino-4-carbamoylmethylcoumarin (peptidyl-ACC) peptides and FRET peptides in the format of Abz-peptidyl-Q-EDDnp (where Abz=ortho-aminobenzoic acid and Q-EDDnp=glutaminyl-N-(2,4-dinitrophenyl) ethylenediamine), in which pairs or sequences of basic amino acids (R or K) were introduced. Surprisingly, KLK6 hydrolyzed the fluorogenic peptides Bz-A-R↓R-ACC and Z-R↓R-MCA between the two R groups, resulting in non-fluorescent products. FRET peptides containing furin processing sequences of human MMP-14, nerve growth factor (NGF), Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4) were cleaved by KLK6 at the same position expected by furin. Finally, KLK6 cleaved FRET peptides derived from human proenkephalin after the KR, the more frequent basic residues flanking enkephalins in human proenkephalin sequence. This result suggests the ability of KLK6 to release enkephalin from proenkephalin precursors and resembles furin a canonical processing proteolytic enzyme. Molecular models of peptides were built into the KLK6 structure and the marked preference of the cut between the two R of the examined peptides was related to the extended conformation of the substrates.

Interface between breast cancer cells and the tumor microenvironment using platelet-rich plasma to promote tumor angiogenesis - influence of platelets and fibrin bundles on the behavior of breast tumor cells.

Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-β, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment.

Structure-Activity Relationship of Chlorotoxin-Like Peptides.

Animal venom (e.g., scorpion) is a rich source of various protein and peptide toxins with diverse physio-/pharmaco-logical activities, which generally exert their action via target-specific modulation of different ion channel functions. Scorpion venoms are among the most widely-known source of peptidyl neurotoxins used for callipering different ion channels, such as; Na+, K+, Ca+, Cl−, etc. A new peptide of the chlorotoxin family (i.e., Bs-Tx7) has been isolated, sequenced and synthesized from scorpion Buthus sindicus (family Buthidae) venom. This peptide demonstrates 66% with chlorotoxin (ClTx) and 82% with CFTR channel inhibitor (GaTx1) sequence identities reported from Leiurus quinquestriatus hebraeus venom. The toxin has a molecular mass of 3821 Da and possesses four intra-chain disulphide bonds. Amino acid sequence analysis of Bs-Tx7 revealed the presence of a scissile peptide bond (i.e., Gly-Ile) for human MMP2, whose activity is increased in the case of tumour malignancy. The effect of hMMP2 on Bs-Tx7, or vice versa, observed using the FRET peptide substrate with methoxycoumarin (Mca)/dinitrophenyl (Dnp) as fluorophore/quencher, designed and synthesized to obtain the lowest Km value for this substrate, showed approximately a 60% increase in the activity of hMMP2 upon incubation of Bs-Tx7 with the enzyme at a micromolar concentration (4 µM), indicating the importance of this toxin in diseases associated with decreased MMP2 activity.

Characterization of Tiki, a New Family of Wnt-specific Metalloproteases

The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn2+/Co2+ but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.

Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay.

Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min(-1) × 10(-3)), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail.

Mapping molecular differences and extracellular matrix gene expression in segmental outflow pathways of the human ocular trabecular meshwork.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma, and lowering IOP remains the only effective treatment for glaucoma. The trabecular meshwork (TM) in the anterior chamber of the eye regulates IOP by generating resistance to aqueous humor outflow. Aqueous humor outflow is segmental, but molecular differences between high and low outflow regions of the TM are poorly understood. In this study, flow regions of the TM were characterized using fluorescent tracers and PCR arrays. Anterior segments from human donor eyes were perfused at physiological pressure in an ex vivo organ culture system. Fluorescently-labeled microspheres of various sizes were perfused into anterior segments to label flow regions. Actively perfused microspheres were segmentally distributed, whereas microspheres soaked passively into anterior segments uniformly labeled the TM and surrounding tissues with no apparent segmentation. Cell-tracker quantum dots (20 nm) were localized to the outer uveal and corneoscleral TM, whereas larger, modified microspheres (200 nm) localized throughout the TM layers and Schlemm’s canal. Distribution of fluorescent tracers demonstrated a variable labeling pattern on both a macro- and micro-scale. Quantitative PCR arrays allowed identification of a variety of extracellular matrix genes differentially expressed in high and low flow regions of the TM. Several collagen genes (COL16A1, COL4A2, COL6A1 and 2) and MMPs (1, 2, 3) were enriched in high, whereas COL15A1, and MMP16 were enriched in low flow regions. Matrix metalloproteinase activity was similar in high and low regions using a quantitative FRET peptide assay, whereas protein levels in tissues showed modest regional differences. These gene and protein differences across regions of the TM provide further evidence for a molecular basis of segmental flow routes within the aqueous outflow pathway. New insight into the molecular mechanisms of segmental aqueous outflow may aid in the design and delivery of improved treatments for glaucoma patients.

ACE I/D genotype-related increase in ACE plasma activity is a better predictor for schizophrenia diagnosis than the genotype alone

BackgroundAngiotensin-I converting enzyme (ACE) is a key component of the renin–angiotensin system (RAS). Although the several contradictory data, ACE has been associated with schizophrenia (SCZ) pathophysiology. Here the ACE activity of SCZ patients and healthy controls (HCs), and its possible correlations with the ACE polymorphism genotype and symptomatic dimensions, was investigated. MethodologyACE activity of 86 SCZ patients and 100 HCs paired by age, gender and educational level was measured, using the FRET peptide substrate and the specific inhibitor lisinopril. The ACE insertion/deletion (I/D) genotypes were assessed by the restriction fragment length polymorphism (RFLP) technique. ResultsSignificantly higher ACE activity was observed in SCZ patients compared to HCs (t=−5.09; p<0.001). The area under the receiver operating characteristic (ROC) curve was 0.701. Mean ACE activity levels were higher for the D-allele carriers (F=5.570; p=0.005), but no significant difference was found among SCZ patients and HCs for genotypes frequencies (Chi-squared=2.08; df=2; p=0.35). Interestingly, we found that the difference between the measured ACE activity for each SCZ patient and the expected average mean value for each respective genotype group (for control subjects) was a better predictor of SCZ than the ACE dichotomized values (high/low) or ACE I/D. ConclusionOur results suggest that higher levels of ACE activity are associated with SCZ with stronger impact when the genetic background of each individual is considered. This may explain the heterogeneity of the results on ACE previously reported.

Substrate specificity of mitochondrial intermediate peptidase analysed by a support-bound peptide library

The substrate specificity of recombinant human mitochondrial intermediate peptidase (hMIP) using a synthetic support-bound FRET peptide library is presented. The collected fluorescent beads, which contained the hydrolysed peptides generated by hMIP, were sequenced by Edman degradation. The results showed that this peptidase presents a remarkable preference for polar uncharged residues at P1 and P1′ substrate positions: Ser=Gln>Thr at P1 and Ser>Thr at P1′. Non-polar residues were frequent at the substrate P3, P2, P2′ and P3′ positions. Analysis of the predicted MIP processing sites in imported mitochondrial matrix proteins shows these cleavages indeed occur between polar uncharged residues. Previous analysis of these processing sites indicated the importance of positions far from the MIP cleavage site, namely the presence of a hydrophobic residue (Phe or Leu) at P8 and a polar uncharged residue (Ser or Thr) at P5. To evaluate this, additional kinetic analyses were carried out, using fluorogenic substrates synthesized based on the processing sites attributed to MIP. The results described here underscore the importance of the P1 and P1′ substrate positions for the hydrolytic activity of hMIP. The information presented in this work will help in the design of new substrate-based inhibitors for this peptidase.

A simple, rapid and sensitive FRET assay for botulinum neurotoxin serotype B detection.

Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63–85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection.

Use of a Synthetic Biosensor for Neutralizing Activity-Biased Selection of Monoclonal Antibodies against Atroxlysin-I, an Hemorrhagic Metalloproteinase from Bothrops atrox Snake Venom

BackgroundThe snake Bothrops atrox is responsible for the majority of envenomings in the northern region of South America. Severe local effects, including hemorrhage, which are mainly caused by snake venom metalloproteinases (SVMPs), are not fully neutralized by conventional serum therapy. Little is known about the immunochemistry of the P-I SVMPs since few monoclonal antibodies (mAbs) against these molecules have been obtained. In addition, producing toxin-neutralizing mAbs remains very challenging.Methodology/Principal FindingsHere, we report on the set-up of a functional screening based on a synthetic peptide used as a biosensor to select neutralizing mAbs against SVMPs and the successful production of neutralizing mAbs against Atroxlysin-I (Atr-I), a P-I SVMP from B. atrox. Hybridomas producing supernatants with inhibitory effect against the proteolytic activity of Atr-I towards the FRET peptide Abz-LVEALYQ-EDDnp were selected. Six IgG1 Mabs were obtained (named mAbatr1 to mAbatr6) and also two IgM. mAbatrs1, 2, 3 and 6 were purified. All showed a high specific reactivity, recognizing only Atr-I and B. atrox venom in ELISA and a high affinity, showing equilibrium constants in the nM range for Atr-I. These mAbatrs were not able to bind to Atr-I overlapping peptides, suggesting that they recognize conformational epitopes.Conclusions/SignificanceFor the first time a functional screening based on a synthetic biosensor was successfully used for the selection of neutralizing mAbs against SVMPs.