SB-PEPTIDE’s recent citations

 

SB-PEPTIDE which belongs to the Smartox group is a French company specialized in peptide chemistry. The company offers various services including:

Peptide synthesis

 Peptide modifications

Synthesis of peptide libraries

Protein synthesis

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SB-PEPTIDE / Smartox are honored to have been cited in many and various scientific publications. Here are few examples  :

Mar. Drugs 23 mai 2024, 22(6), 244. doi: https://doi.org/10.3390/md22060244

The Identification of Peptide Inhibitors of the Coronavirus 3CL Protease from a Fucus ceranoides L. Hydroalcoholic Extract Using a Ligand-Fishing Strategy

Brown seaweeds of the Fucus genus represent a rich source of natural antiviral products. In this study, a Fucus ceranoides hydroalcoholic extract (FCHE) was found to inhibit 74.2 ± 1.3% of the proteolytic activity of the free SARS-CoV-2 3CL protease (3CLpro), an enzyme that plays a pivotal role in polyprotein processing during coronavirus replication and has been identified as a relevant drug discovery target for SARS- and MERS-CoVs infections. To purify and identify 3CLpro ligands with potential inhibitory activity using a one-step approach, we immobilized the enzyme onto magnetic microbeads (3CLpro-MPs), checked that the enzymatic activity was maintained after grafting, and used this bait for a ligand-fishing strategy followed by a high-resolution mass spectrometry analysis of the fished-out molecules. Proof of concept for the ligand-fishing capacity of the 3CLpro-MPs was demonstrated by doping the FCHE extract with the substrate peptide TSAVLQ-pNA, resulting in the preferential capture of this high-affinity peptide within the macroalgal complex matrix. Ligand fishing in the FCHE alone led to the purification and identification via high-resolution mass spectrometry (HRMS) of seven hepta-, octa-, and decapeptides in an eluate mix that significantly inhibited the free 3CLpro more than the starting FCHE (82.7 ± 2.2% inhibition). Molecular docking simulations of the interaction between each of the seven peptides and the 3CLpro demonstrated a high affinity for the enzyme’s proteolytic active site surpassing that of the most affine peptide ligand identified so far (a co-crystallographic peptide). Testing of the corresponding synthetic peptides demonstrated that four out of seven significantly inhibited the free 3CLpro (from 46.9 ± 6.4 to 76.8 ± 3.6% inhibition at 10 µM). This study is the first report identifying peptides from Fucus ceranoides with high inhibitory activity against the SARS-CoV-2 3CLprotease which bind with high affinity to the protease’s active site. It also confirms the effectiveness of the ligand-fishing strategy for the single-step purification of enzyme inhibitors from complex seaweed matrices.
Int. J. Mol. Sci. 26 Jan 2024;25:1539. doi: https://doi.org/10.3390/ijms25031539

Non-Mature miRNA-Encoded Micropeptide miPEP166c Stimulates Anthocyanin and Proanthocyanidin Synthesis in Grape Berry Cells

The phenylpropanoid and flavonoid pathways exhibit intricate regulation, not only influenced by environmental factors and a complex network of transcription factors but also by post-transcriptional regulation, such as silencing by microRNAs and miRNA-encoded micropeptides (miPEPs). VviMYBC2-L1 serves as a transcriptional repressor for flavonoids, playing a crucial role in coordinating the synthesis of anthocyanin and proanthocyanidin. It works in tandem with their respective transcriptional activators, VviMYBA1/2 and VviMYBPA1, to maintain an equilibrium of flavonoids. We have discovered a miPEP encoded by miR166c that appears to target VviMYBC2-L1. We conducted experiments to test the hypothesis that silencing this transcriptional repressor through miPEP166c would stimulate the synthesis of anthocyanins and proanthocyanidins. Our transcriptional analyses by qPCR revealed that the application of exogenous miPEP166c to Gamay Fréaux grape berry cells resulted in a significant upregulation in flavonoid transcriptional activators (VviMYBA1/2 and VviMYBPA1) and structural flavonoid genes (VviLDOX and VviDFR), as well as genes involved in the synthesis of proanthocyanidins (VviLAR1 and VviANR) and anthocyanins (VviUFGT1). These findings were supported by the increased enzyme activities of the key enzymes UFGT, LAR, and ANR, which were 2-fold, 14-fold, and 3-fold higher, respectively, in the miPEP166c-treated cells. Ultimately, these changes led to an elevated total content of anthocyanins and proanthocyanidins.
J Immunol. 12 Jan 2024;ji2300704. doi: https://doi.org/10.4049/jimmunol.2300704

Development of a New Off-the-Shelf Plasmacytoid Dendritic Cell–Based Approach for the Expansion and Characterization of SARS-CoV-2–Specific T Cells

Global vaccination against COVID-19 has been widely successful; however, there is a need for complementary immunotherapies in severe forms of the disease and in immunocompromised patients. Cytotoxic CD8+ T cells have a crucial role in disease control, but their function can be dysregulated in severe forms of the disease. We report here a cell-based approach using a plasmacytoid dendritic cell line (PDC*line) to expand in vitro specific CD8+ responses against COVID-19 Ags. We tested the immunogenicity of eight HLA-A*02:01 restricted peptides derived from diverse SARS-Cov-2 proteins, selected by bioinformatics analyses in unexposed and convalescent donors. Higher ex vivo frequencies of specific T cells against these peptides were found in convalescent donors compared with unexposed donors, suggesting in situ T cell expansion upon viral infection. The peptide-loaded PDC*line induced robust CD8+ responses with total amplification rates that led up to a 198-fold increase in peptide-specific CD8+ T cell frequencies for a single donor. Of note, six of eight selected peptides provided significant amplifications, all of which were conserved between SARS-CoV variants and derived from the membrane, the spike protein, the nucleoprotein, and the ORF1ab. Amplified and cloned antiviral CD8+ T cells secreted IFN-γ upon peptide-specific activation. Furthermore, specific TCR sequences were identified for two highly immunogenic Ags. Hence, PDC*line represents an efficient platform to identify immunogenic viral targets for future immunotherapies.

Curr. Issues Mol. Biol. 28 Sep 2023;45(10):7944-7955. doi: https://doi.org/10.3390/cimb45100502

Identification of T-Cell Epitopes Using a Combined In-Silico and Experimental Approach in a Mouse Model for SARS-CoV-2

Following viral infection, T-cells are crucial for an effective immune response to intracellular pathogens, including respiratory viruses. During the COVID-19 pandemic, diverse assays were required in pre-clinical trials to evaluate the immune response following vaccination against SARS-CoV-2 and assess the response following exposure to the virus. To assess the nature and potency of the cellular response to infection or vaccination, a reliable and specific activity assay was needed. A cellular activity assay based on the presentation of short peptides (epitopes) allows the identification of T cell epitopes displayed on different alleles of the MHC, shedding light on the strength of the immune response towards antigens and aiding in antigen design for vaccination. In this report, we describe two approaches for scanning T cell epitopes on the surface glycoprotein of the SARS-CoV-2 (spike), which is utilized for attachment and entry and serves as an antigen in many vaccine candidates. We demonstrate that epitope scanning is feasible using peptide libraries or computational scanning combined with a cellular activity assay. Our scans identified four CD8 T cell epitopes, including one novel undescribed epitope. These epitopes enabled us to establish a reliable T-cell response assay, which was examined and used in various experimental mouse models for SARS-CoV-2 infection and vaccination. These approaches could potentially aid in future antigen design for vaccination and establish cellular activity assays against uncharacterized antigens of emerging pathogens.
Photodiagnosis Photodyn Ther. 2023 Jul 25;103725. doi: https://doi.org/10.1016/j.pdpdt.2023.103725

Chlorin-e6 conjugated to the antimicrobial peptide ll-37 loaded nanoemulsion enhances photodynamic therapy against multi-species biofilms related to periodontitis

In our previous studies, Chlorin-e6 (Ce6) demonstrated a significant reduction of microorganisms’ viability against multi-species biofilm related to periodontitis while irradiated with blue light. However, the conjugation of Ce6 and antimicrobial peptides, and the incorporation of this photosensitizer in a nanocarrier, is still poorly explored. We hypothesized that chlorin-e6 conjugated to the antimicrobial peptide LL-37 loaded nanoemulsion could inhibit a multi-species biofilm related to periodontitis during photodynamic therapy (PDT), the pre-treatment with hydrogen peroxide was also tested. The nanoemulsion (NE) incorporated with Ce6 was characterized regarding the physiochemical parameters. Images were obtained by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Later, the Ce6 and LL-37 incorporated in NE was submitted to UV-Vis analysis and Reactive Oxygen Species (ROS) assay. Finally, the combined formulation (Ce6+LL-37 in nanoemulsion) was tested against multi-species biofilm related to periodontitis. The formed nanoformulation was kinetically stable, optically transparent with a relatively small droplet diameter (134.2 unloaded and 146.9 loaded), and weak light scattering. The NE system did not impact the standard UV-VIS spectra of Ce6, and the ROS production was improved while Ce6 was incorporated in the NE. The combination of Ce6 and LL-37 in NE was effective to reduce the viability of all bacteria tested. The treatment with hydrogen peroxide previous to PDT significantly impacted bacterial viability. The current aPDT regimen was the best already tested against periodontal biofilm by our research team. Our results suggest that this combined protocol must be exploited for clinical applications in localized infections such as periodontal disease. – Nanoemulsion demonstrated to be an excellent nanocarrier for photodynamic application. – Chlorin-e6 incorporated in nanoemulsion showed great physicochemical and biophotonic parameters. – The combination of chlorin-e6 and LL-37 peptide in nanoemulsion is effective to eliminate periodontal pathogenic bacteria. – The treatment with hydrogen peroxide previous to PDT significantly impacted bacterial viability.

Research Square. 2023 Jul 25. doi: https://doi.org/10.21203/rs.3.rs-3170915/v1

Recombinant production of human antimicrobial peptide LL- 37 and its secondary structure

Antimicrobial peptides, including the human cathelicidin LL-37, offer a possible solution to the global problem of bacterial resistance to antibiotics. LL-37 peptide has potent antimicrobial effects against current multidrug-resistant bacterial strains. The peptide itself is also characterized by a very diverse range of immunomodulatory effects. The aim of this study was to produce antimicrobially active peptide LL-37 in E. coli in high yields using an own expression system pUbEx100 with the fusion protein ubiquitin. The results showed that the peptide GLL-37 could be produced in high amounts, but this peptide did not have antimicrobial activity compared to synthetically produced LL-37. The synthetic peptide LL-37 (sLL-37) alone showed potent activity against multidrug-resistant strains Acinetobacter baumannii and Pseudomonas aeruginosa. CD spectroscopy results showed that the produced peptide GLL-37 is in α-helix form in contrast to the sLL-37 (random-coil form). The recombinant peptide GLL-37 can not bind to the membrane in the α-helix form, it would have to be in the form of a random-coil. This study confirms by CD spectroscopy the previously observed mechanism of access of LL-37 peptide to the bacterial membrane obtained by NMR.
Cell Metabolism. 2023 Apr 04;35(4):P633-650.e9. doi: https://doi.org/10.1016/j.cmet.2023.02.013

Linoleic acid potentiates CD8+ T cell metabolic fitness and antitumor immunity

The metabolic state represents a major hurdle for an effective adoptive T cell therapy (ACT). Indeed, specific lipids can harm CD8+ T cell (CTL) mitochondrial integrity, leading to defective antitumor responses. However, the extent to which lipids can affect the CTL functions and fate remains unexplored. Here, we show that linoleic acid (LA) is a major positive regulator of CTL activity by improving metabolic fitness, preventing exhaustion, and stimulating a memory-like phenotype with superior effector functions. We report that LA treatment enhances the formation of ER-mitochondria contacts (MERC), which in turn promotes calcium (Ca2+) signaling, mitochondrial energetics, and CTL effector functions. As a direct consequence, the antitumor potency of LA-instructed CD8 T cells is superior in vitro and in vivo. We thus propose LA treatment as an ACT potentiator in tumor therapy.

Nature Communications. 2023 Jan 17; Vol 14:254. doi: https://doi.org/10.1038/s41467-023-35951-0

Complementary peptides represent a credible alternative to agrochemicals by activating translation of targeted proteins

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) from any gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.

Nat Immunol. 2023;24:84–95. doi: https://doi.org/10.1038/s41590-022-01374-0

ETV3 and ETV6 enable monocyte differentiation into dendritic cells by repressing macrophage fate commitment

In inflamed tissues, monocytes differentiate into macrophages (mo-Macs) or dendritic cells (mo-DCs). In chronic nonresolving inflammation, mo-DCs are major drivers of pathogenic events. Manipulating monocyte differentiation would therefore be an attractive therapeutic strategy. However, how the balance of mo-DC versus mo-Mac fate commitment is regulated is not clear. In the present study, we show that the transcriptional repressors ETV3 and ETV6 control human monocyte differentiation into mo-DCs. ETV3 and ETV6 inhibit interferon (IFN)-stimulated genes; however, their action on monocyte differentiation is independent of IFN signaling. Instead, we find that ETV3 and ETV6 directly repress mo-Mac development by controlling MAFB expression. Mice deficient for Etv6 in monocytes have spontaneous expression of IFN-stimulated genes, confirming that Etv6 regulates IFN responses in vivo. Furthermore, these mice have impaired mo-DC differentiation during inflammation and reduced pathology in an experimental autoimmune encephalomyelitis model. These findings provide information about the molecular control of monocyte fate decision and identify ETV6 as a therapeutic target to redirect monocyte differentiation in inflammatory disorders.

Pestic Biochem Physiol. 2023 Feb;190/105317. doi: https://doi.org/10.1016/j.pestbp.2022.105317

Cell penetrating peptides are versatile tools for enhancing multimodal uptake into cells from pest insects

Cell penetrating peptides (CPPs) are small peptides defined by their ability to deliver molecular cargo into cells. While the subject of frequent investigation for pharmaceutical drug delivery, little consideration has been given to the possibility of CPPs for use as insecticides or insecticide enhancers. Here, we characterize the entry of four fluorescently tagged CPPs into two insect cell lines and dissected midgut tissues in terms of both total quantity and mode of penetration. Fluorescent microscopy showed that substantial amounts of CPPs penetrate the plasma membrane via endosomal uptake in ovarian (Sf9) and midgut derived (AW1) lepidopteran cells and that this process was sensitive to selected endocytosis inhibitors. Differences in the quantity of uptake was observed between CPPs, and further differences were found in the ability CPP-1838 to efficiently penetrate membranes through passive diffusion. These findings were extended to primary midgut derived cells and dissected tissues suggesting that CPPs show a preference for goblet cells and that CPP-1838 shows far higher rates of penetration. CPP-1838 thus shows extraordinary abilities to penetrate cells efficiency in both a diffusional and endocytotic manner. From these results more sophisticated delivery methods based on the utilization of CPPs can be developed.

PLoS Pathog. 2023 Apr 24;19(4):e1011339. doi: https://doi.org/10.1371/journal.ppat.1011339

Neutralization of zoonotic retroviruses by human antibodies: Genotype-specific epitopes within the receptor-binding domain from simian foamy virus

Infection with viruses of animal origin pose a significant threat to human populations. Simian foamy viruses (SFVs) are frequently transmitted to humans, in which they establish a lifelong infection, with the persistence of replication-competent virus. However, zoonotic SFVs do not induce severe disease nor are they transmitted between humans. Thus, SFVs represent a model of zoonotic retroviruses that lead to a chronic infection successfully controlled by the human immune system. We previously showed that infected humans develop potent neutralizing antibodies (nAbs). Within the viral envelope (Env), the surface protein (SU) carries a variable region that defines two genotypes, overlaps with the receptor binding domain (RBD), and is the exclusive target of nAbs. However, its antigenic determinants are not understood. Here, we characterized nAbs present in plasma samples from SFV-infected individuals living in Central Africa. Neutralization assays were carried out in the presence of recombinant SU that compete with SU at the surface of viral vector particles. We defined the regions targeted by the nAbs using mutant SU proteins modified at the glycosylation sites, RBD functional subregions, and genotype-specific sequences that present properties of B-cell epitopes. We observed that nAbs target conformational epitopes. We identified three major epitopic regions: the loops at the apex of the RBD, which likely mediate interactions between Env protomers to form Env trimers, a loop located in the vicinity of the heparan binding site, and a region proximal to the highly conserved glycosylation site N8. We provide information on how nAbs specific for each of the two viral genotypes target different epitopes. Two common immune escape mechanisms, sequence variation and glycan shielding, were not observed. We propose a model according to which the neutralization mechanisms rely on the nAbs to block the Env conformational change and/or interfere with binding to susceptible cells. As the SFV RBD is structurally different from known retroviral RBDs, our data provide fundamental knowledge on the structural basis for the inhibition of viruses by nAbs.

Nature Communications. 2023 Jan 10;14:254. doi: https://doi.org/10.1038/s41467-023-35951-0

Complementary peptides represent a credible alternative to agrochemicals by activating translation of targeted proteins

The current agriculture main challenge is to maintain food production while facing multiple threats such as increasing world population, temperature increase, lack of agrochemicals due to health issues and uprising of weeds resistant to herbicides. Developing novel, alternative, and safe methods is hence of paramount importance. Here, we show that complementary peptides (cPEPs) fromany gene can be designed to target specifically plant coding genes. External application of synthetic peptides increases the abundance of the targeted protein, leading to related phenotypes. Moreover, we provide evidence that cPEPs can be powerful tools in agronomy to improve plant traits, such as growth, resistance to pathogen or heat stress, without the needs of genetic approaches. Finally, by combining their activity they can also be used to reduce weed growth.

J. ACS Nano. 2022 Feb 17;16(3):4444-4457. doi: https://doi.org/10.1021/acsnano.1c10734

Surfactant-like Peptide Self-Assembled into Hybrid Nanostructures for Electronic Nose Applications

An electronic nose (e-nose) utilizes a multisensor array, which relies on the vector contrast of combinatorial responses, to effectively discriminate between volatile organic compounds (VOCs). In recent years, hierarchical structures made of nonbiological materials have been used to achieve the required sensor diversity. With the advent of self-assembling peptides, the ability to tune nanostructuration, surprisingly, has not been exploited for sensor array diversification. In this work, a designer surfactant-like peptide sequence, CG7–NH2, is used to fabricate morphologically and physicochemically heterogeneous “biohybrid” surfaces on Au-covered chips. These multistructural sensing surfaces, containing immobilized hierarchical nanostructures surrounded by self-assembled monolayers, are used for the detection and discrimination of VOCs. Through a simple and judicious design process, involving changes in pH and water content of peptide solutions, a five-element biohybrid sensor array coupled with a gas-phase surface plasmon resonance imaging system is shown to achieve sufficient discriminatory capabilities for four VOCs. Moreover, the limit of detection of the multiarray system is bench-marked at <1 and 6 ppbv for hexanoic acid and phenol (esophago-gastric biomarkers), respectively. Finally, the humidity effects are characterized, identifying the dissociation rate constant as a robust descriptor for classification, further exemplifying their efficacy as biomaterials in the field of artificial olfaction.

J. Cell Reports. 2022 Feb 08;38(6):110339. doi: https://doi.org/10.1016/j.celrep.2022.110339

Characterization of plant microRNA-encoded peptides (miPEPs) reveals molecular mechanisms from the translation to activity and specificity

MicroRNAs (miRNAs) are transcribed as long primary transcripts (pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs encode regulatory peptides called miPEPs, which specifically enhance the transcription of the pri-miRNA from which they originate. However, paradoxically, whereas miPEPs have been identified in different plant species, they are poorly conserved, raising the question of the mechanisms underlying their specificity. To address this point, we identify and re-annotate multiple Arabidopsis thaliana pri-miRNAs in order to identify ORF encoding miPEPs. The study of several identified miPEPs in different species show that non-conserved miPEPs are only active in their plant of origin, whereas conserved ones are active in different species. Finally, we find that miPEP activity relies on the presence of its own miORF, explaining both the lack of selection pressure on miPEP sequence and the ability for non-conserved peptides to play a similar role, i.e., to activate the expression of their corresponding miRNA.

https://www.sb-peptide.com/2022/03/04/microrna-peptide/

J of Neuro Met. 2022 Feb 01;367:109443. doi: https://doi.org/10.1016/j.jneumeth.2021.109443

An optimized and validated protocol for inducing chronic experimental autoimmune encephalomyelitis in C57BL/6J mice

Background: Myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis (EAE) is the most commonly used animal model of multiple sclerosis. However, variations in the induction protocol can affect EAE progression, and may reduce the comparability of data.

Optimized method: In the present study, we investigated the influence of the different components used for EAE induction in C57BL/6J mice on disease progression. In the present study, MOG35–55-induced chronic EAE in C57BL/6J mice has been applied as a model to challenge optimal pertussis toxin (PTx) dosing, while considering variations in batch potency.

Results: We demonstrate that the dosage of PTx, adjusted to its potency, influences EAE development in a dosedependent manner. Our data show that with our protocol, which considers PTx potency, C57BL/6J mice consistently develop symptoms of EAE. The mice show a typical chronic course with symptom onset after 10.5 ± 1.08 days and maximum severity around day 16 postimmunization followed by a mild remission of symptoms.

Comparison with existing methods: Previously studies reveal that alterations in PTx dosing directly modify EAE progression. Our present study highlights that PTx batches differ in potency, resulting in inconsistent EAE induction. We also provide a clear protocol that allows a reduction in the number of mice used in EAE experiments, while maintaining consistent results.

Conclusion: Higher standards for comparability and reproducibility are needed to ensure and maximize the generation of reliable EAE data. Specifically, consideration of PTx potency. With our method of establishing consistent EAE pathogenesis, improved animal welfare standards and a reduction of mice used in experimentation can be achieved.

J. Nature. 2021 Oct 31;24:84-95. doi: https://www.nature.com/articles/s41590-022-01374-0

Cold adaptation in the environmental bacterium Shewanella oneidensis is controlled by a J-domain co-chaperone protein network

In inflamed tissues, monocytes differentiate into macrophages (mo-Macs) or dendritic cells (mo-DCs). In chronic nonresolving inflammation, mo-DCs are major drivers of pathogenic events. Manipulating monocyte differentiation would therefore be an attractive therapeutic strategy. However, how the balance of mo-DC versus mo-Mac fate commitment is regulated is not clear. In the present study, we show that the transcriptional repressors ETV3 and ETV6 control human monocyte differentiation into mo-DCs. ETV3 and ETV6 inhibit interferon (IFN)-stimulated genes; however, their action on monocyte differentiation is independent of IFN signaling. Instead, we find that ETV3 and ETV6 directly repress mo-Mac development by controlling MAFB expression. Mice deficient for Etv6 in monocytes have spontaneous expression of IFN stimulated genes, confirming that Etv6 regulates IFN responses in vivo. Furthermore, these mice have impaired mo-DC differentiation during inflammation and reduced pathology in an experimental autoimmune encephalomyelitis model. These findings provide information about the molecular control of monocyte fate decision and identify ETV6 as a therapeutic target to redirect monocyte differentiation in inflammatory disorders.

Nat Commun. 2020 Mar 27;11(1):1591. doi: https://doi.org/10.1038/s41467-020-15392-9

Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA

BACKGROUND:

Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD–PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach — combining cryo-EM, X-ray crystallography, protein–protein interaction measurements, and activity assays — we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication.

Talanta. 2019 Oct 1;203:322-327. doi: https://doi.org/10.1016/j.talanta.2019.05.062

Antimicrobial peptide arrays for wide spectrum sensing of pathogenic bacteria

BACKGROUND:

Fast detection of bacteria in samples presumed to be un-contaminated, such as blood, is of great importance. Indeed, rapid diagnosis allows the set-up of appropriate antibiotic treatment. Besides clinical issues, there are many other domains, such as food processing or drug manufacturing, where the strict absence of any bacteria has to be assessed. Because the bacterial load found in most contaminated samples is often below the limit of detection for currently validated assays, a preliminary enrichment step is required to allow bacterial multiplication before proceeding to the analysis step, whatever it might be – cultural, immunological or molecular methods. In this study, we describe the use of a biosensor for single-step bacteria detection. The whole analysis is performed in less than 20 h, during the growth phase of the micro-organisms, using an array of antimicrobial peptides (AMPs) coupled with a surface plasmon resonance imager (SPRI). A wide range of bacterial strains are assayed, showing differentiated affinity patterns with the immobilized peptides, which are confirmed by multivariate analysis. This work establishes the evidence that antimicrobial peptides, mostly used so far in the antibiotic drug industry, are suited for the wide-spectrum detection of unknown bacteria in samples, even at very low initial loads. Moreover, the small set of AMPs that were assayed provided a specific affinity profile for each pathogen, as confirmed by multivariate analyses. Furthermore, this work opens up the possibility of applying this method in more complex and relevant samples such as foodstuff, urine or blood.

Biomacromolecules. 2019 Dec 07;21:680–687. doi: https://doi.org/10.1021/acs.biomac.9b01390

Shape-Memory Effect by Sequential Coupling of Functions over Different Length Scales in an Architectured Hydrogel

BACKGROUND:

The integration of functions in materials in order to gain macroscopic effects in response to environmental changes is an ongoing challenge in material science. Here, functions on different hierarchical levels are sequentially linked to translate a pH-triggered conformational transition from the molecular to the macroscopic level to induce directed movements in hydrogels. When the pH is increased, lysine-rich peptide molecules change their conformation into a β-hairpin structure because of the reduced electrostatic repulsion among the deprotonated amino groups. Coupled to this conformation change is the capability of the β-hairpin motifs to subsequently assemble into aggregates acting as reversible cross-links, which are used as controlling units to fix a temporary macroscopic shape. A structural function implemented into the hydrogel by a microporous architecture-enabled nondisruptive deformation upon compression by buckling of pore walls and their elastic recovery. Coupled to this structural function is the capability of the porous material to enhance the diffusion of ions into the hydrogel and to keep the dimension of the macroscopic systems almost constant when the additional cross-links are formed or cleaved as it limits the dimensional change of the pore walls. Covalent cross-linking of the hydrogel into a polymer network acted as gear shift to ensure translation of the function on the molecular level to the macroscopic dimension. In this way, the information of a directed shape-shift can be programmed into the material by mechanical deformation and pH-dependent formation of temporary net points. The information could be read out by lowering the pH. The peptides reverted back into their original random coil conformation and the porous polymer network could recover from the previously applied elastic deformation. The level of multifunctionality of the hydrogels can be increased by implementation of additional orthogonal functions such as antimicrobicity by proper selection of multifunctional peptides, which could enable sophisticated biomedical devices.

Commun Biol. 2019 Aug 29;2-323. doi: https://doi.org/10.1038/s42003-019-0567-3

Cold adaptation in the environmental bacterium Shewanella oneidensis is controlled by a J-domain co-chaperone protein network

BACKGROUND:

DnaK (Hsp70) is a major ATP-dependent chaperone that functions with two co-chaperones, a J-domain protein (JDP) and a nucleotide exchange factor to maintain proteostasis in most organisms. Here, we show that the environmental bacterium Shewanella oneidensis possesses a previously uncharacterized short JDP, AtcJ, dedicated to cold adaptation and composed of a functional J-domain and a C-terminal extension of 21 amino acids. We showed that atcJ is the first gene of an operon encoding also AtcA, AtcB and AtcC, three proteins of unknown functions. Interestingly, we found that the absence of AtcJ, AtcB or AtcC leads to a dramatically reduced growth at low temperature. In addition, we demonstrated that AtcJ interacts via its C-terminal extension with AtcC, and that AtcC binds to AtcB. Therefore, we identified a previously uncharacterized protein network that involves the DnaK system with a dedicated JDP to allow bacteria to survive to cold environment.

Acta Neuropathol Commun. 2019 Aug 20;7(1):134. doi: https://doi.org/10.1186/s40478-019-0761-z

Retinal ischemia induces α-SMA-mediated capillary pericyte contraction coincident with perivascular glycogen depletion

BACKGROUND:

Increasing evidence indicates that pericytes are vulnerable cells, playing pathophysiological roles in various neurodegenerative processes. Microvascular pericytes contract during cerebral and coronary ischemia and do not relax after re-opening of the occluded artery, causing incomplete reperfusion. However, the cellular mechanisms underlying ischemia-induced pericyte contraction, its delayed emergence, and whether it is pharmacologically reversible are unclear. Here, we investigate i) whether ischemia-induced pericyte contractions are mediated by alpha-smooth muscle actin (α-SMA), ii) the sources of calcium rise in ischemic pericytes, and iii) if peri-microvascular glycogen can support pericyte metabolism during ischemia. Thus, we examined pericyte contractility in response to retinal ischemia both in vivo, using adaptive optics scanning light ophthalmoscopy and, ex vivo, using an unbiased stereological approach. We found that microvascular constrictions were associated with increased calcium in pericytes as detected by a genetically encoded calcium indicator (NG2-GCaMP6) or a fluoroprobe (Fluo-4). Knocking down α-SMA expression with RNA interference or fixing F-actin with phalloidin or calcium antagonist amlodipine prevented constrictions, suggesting that constrictions resulted from calcium- and α-SMA-mediated pericyte contractions. Carbenoxolone or a Cx43-selective peptide blocker also reduced calcium rise, consistent with involvement of gap junction-mediated mechanisms in addition to voltage-gated calcium channels. Pericyte calcium increase and capillary constrictions became significant after 1 h of ischemia and were coincident with depletion of peri-microvascular glycogen, suggesting that glucose derived from glycogen granules could support pericyte metabolism and delay ischemia-induced microvascular dysfunction. Indeed, capillary constrictions emerged earlier when glycogen breakdown was pharmacologically inhibited. Constrictions persisted despite recanalization but were reversible with pericyte-relaxant adenosine administered during recanalization. Our study demonstrates that retinal ischemia, a common cause of blindness, induces α-SMA- and calcium-mediated persistent pericyte contraction, which can be delayed by glucose driven from peri-microvascular glycogen. These findings clarify the contractile nature of capillary pericytes and identify a novel metabolic collaboration between peri-microvascular end-feet and pericytes.

J. Virol. 2019 May 15;93(11):e00068-19. doi: https://doi.org/10.1128/jvi.00068-19

An Immunodominant and Conserved B-Cell Epitope in the Envelope of Simian Foamy Virus Recognized by Humans Infected with Zoonotic Strains from Apes

BACKGROUND:

Cross-species transmission of simian foamy viruses (SFVs) from nonhuman primates (NHPs) to humans is currently ongoing. These zoonotic retroviruses establish lifelong persistent infection in their human hosts. SFV are apparently nonpathogenic in vivo, with ubiquitous in vitro tropism. Here, we aimed to identify envelope B-cell epitopes that are recognized following a zoonotic SFV infection. We screened a library of 169 peptides covering the external portion of the envelope from the prototype foamy virus (SFVpsc_huHSRV.13) for recognition by samples from 52 Central African hunters (16 uninfected and 36 infected with chimpanzee, gorilla, or Cercopithecus SFV). We demonstrate the specific recognition of peptide N96-V110 located in the leader peptide, gp18LP. Forty-three variant peptides with truncations, alanine substitutions, or amino acid changes found in other SFV species were tested. We mapped the epitope between positions 98 and 108 and defined six amino acids essential for recognition. Most plasma samples from SFV-infected humans cross-reacted with sequences from apes and Old World monkey SFV species. The magnitude of binding to peptide N96-V110 was significantly higher for samples of individuals infected with a chimpanzee or gorilla SFV than those infected with a Cercopithecus SFV. In conclusion, we have been the first to define an immunodominant B-cell epitope recognized by humans following zoonotic SFV infection.

Br J Pharmacol. 2019 May;176(9):1298-1314. doi: https://doi.org/10.1111/bph.14628

From identification to functional characterization of cyriotoxin-1a, an antinociceptive toxin from the spider Cyriopagopus schioedtei

BACKGROUND:

Purpose: The NaV 1.7 channel is highly expressed in dorsal root ganglia of the sensory nervous system and plays a central role in the pain signalling process. We investigated a library prepared from original venoms of 117 different animals to identify new selective inhibitors of this target.

Experimental approach: We used high throughput screening of a large venom collection using automated patch-clamp experiments on human voltage-gated sodium channel subtypes and then in vitro and in vivo electrophysiological experiments to characterize the active peptides that have been purified, sequenced, and chemically synthesized. Analgesic effects were evaluated in vivo in mice models.

Key results: We identified cyriotoxin-1a (CyrTx-1a), a novel peptide isolated from Cyriopagopus schioedtei spider venom, as a candidate for further characterization. This 33 amino acids toxin belongs to the inhibitor cystine knot structural family and inhibits hNaV 1.1-1.3 and 1.6-1.7 channels in the low nanomolar range, compared to the micromolar range for hNaV 1.4-1.5 and 1.8 channels. CyrTx-1a was 920 times more efficient at inhibiting tetrodotoxin (TTX)-sensitive than TTX-resistant sodium currents recorded from adult mouse dorsal root ganglia neurons and in vivo electrophysiological experiments showed that CyrTx-1a was approximately 170 times less efficient than huwentoxin-IV at altering mouse skeletal neuromuscular excitability properties. CyrTx-1a exhibited an analgesic effect in mice by increasing reaction time in the hot-plate assay.

Conclusions and implications: The pharmacological profile of CyrTx-1a paves the way for further molecular engineering aimed to optimize the potential antinociceptive properties of this peptide.

Sci Rep. 2019 Mar 18;9(1):4771. doi: https://doi.org/10.1038/s41598-019-41404-w

New Therapeutic Approach for Targeting Hippo Signalling Pathway

BACKGROUND:

Nuclear localization signals are short amino acid sequences that target proteins for nuclear import. In this manuscript, we have generated a chimeric tri-functional peptide composed of a cell penetrating peptide (CPP), a nuclear localization sequence and an interfering peptide blocking the interaction between TEAD and YAP, two transcription factors involved in the Hippo signalling pathway, whose deregulation is related to several types of cancer. We have validated the cell penetration and nuclear localization by flow cytometry and fluorescence microscopy and shown that the new generated peptide displays an apoptotic effect in tumor cell lines thanks to the specific nuclear delivery of the cargo, which targets a protein/protein interaction in the nucleus. In addition, the peptide has an anti-tumoral effect in vivo in xenograft models of breast cancer. The chimeric peptide designed in the current study shows encouraging prospects for developing nuclear anti- neoplastic drugs.

Toxicon X. 2019 Feb 23;2:100010. doi: https://doi.org/10.1016/j.toxcx.2019.100010

Fluorescent analogues of BeKm-1 with high and specific activity against the hERG channel

BACKGROUND:

Peptidic toxins that target specifically mammalian channels and receptors can be found in the venom of animals. These toxins are rarely used directly as tools for biochemical experiments, and need to be modified via the attachment of chemical groups (e.g., radioactive or fluorescent moieties). Ideally, such modifications should maintain the toxin specificity and affinity for its target. With the goal of obtaining fluorescent derivatives of BeKm-1, a toxin from the scorpion species Buthus eupeus that selectively inhibits the voltage-gated potassium ion channel hERG, we produced four active analogues using a model of BeKm-1 docking to the outer mouth of the channel. In these BeKm-1 analogues, the natural peptide was linked to the fluorescent cyanine 5 (Cy5) probe via four different linkers at Arg1 or Arg/Lys27. All analogues retained their specificity towards the hERG channel in electrophysiological experiments but displayed a lesser affinity. These results validate our strategy for designing toxin analogues and demonstrate that different chemical groups can be attached to different residues of BeKm-1.

Cell. 2019 Feb 7;176(4):702-715.e14. doi: https://doi.org/10.1016/j.cell.2018.12.018

Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin

BACKGROUND:

Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable ∼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.

J. Analyst. 2018 Apr 30;143(9):2165-2173. doi: https://doi.org/10.1126/science.aav8573

A new functional membrane protein microarray based on tethered phospholipid bilayers

BACKGROUND:

A new prototype of a membrane protein biochip is presented in this article. This biochip was created by the combination of novel technologies of peptide-tethered bilayer lipid membrane (pep-tBLM) formation and solid support micropatterning. Pep-tBLMs integrating a membrane protein were obtained in the form of microarrays on a gold chip. The formation of the microspots was visualized in real-time by surface plasmon resonance imaging (SPRi) and the functionality of a GPCR (CXCR4), reinserted locally into microwells, was assessed by ligand binding studies. In brief, to achieve micropatterning, P19-4H, a 4 histidine-possessing peptide spacer, was spotted inside microwells obtained on polystyrene-coated gold, and Ni-chelating proteoliposomes were injected into the reaction chamber. Proteoliposome binding to the peptide was based on metal–chelate interaction. The peptide-tethered lipid bilayer was finally obtained by addition of a fusogenic peptide (AH peptide) to promote proteoliposome fusion. The CXCR4 pep-tBLM microarray was characterized by surface plasmon resonance imaging (SPRi) throughout the building-up process. This new generation of membrane protein biochip represents a promising method of developing a screening tool for drug discovery.

J. MRS Advances. 2017;2(47):2571-2579. doi: https://doi.org/10.1557/adv.2017.491

PH-sensitivity and Conformation Change of the N-terminal Methacrylated Peptide VK20

BACKGROUND:

N-terminal methacrylation of peptide MAX1, which is capable of conformational changes by variation of the pH, results in a peptide, named VK20. Increasing the reactivity of this terminal group enables further coupling reactions or chemical modifications of the peptide. However, this end group functionalization may influence the ability of conformational changes of VK20, as well as its properties. In this paper, the influence of pH on the transition between random coil and ß-sheet conformation of VK20, including the transition kinetics, were investigated. At pH values of 9 and higher, the kinetics of ß-sheet formation increased for VK20, compared to MAX1. The self-assembly into ß-sheets recognized by the formation of a physically crosslinked gel was furthermore indicated by a significant increase of G’. An increase in pH (from 9 to 9.5) led to a faster gelation of the peptide VK20. Simultaneously, G’ was increased from 460 ± 70 Pa (at pH 9) to 1520 ± 180 Pa (at pH 9.5). At the nanoscale, the gel showed a highly interconnected fibrillary network structure with uniform fibril widths of approximately 3.4 ± 0.5 nm (N=30). The recovery of the peptide conformation back to random coil resulted in the dissolution of the gel, whereby the kinetics of the recovery depended on the pH. Conclusively, the ability of MAX1 to undergo conformational changes was not affected by N-terminal methacrylation whereas the kinetics of pH-sensitive ß-sheet formations has been increased.

J. Nature Sci. Rep. 2016 Jan 27;6:19725. doi: https://doi.org/10.1038/srep19725

TCTP contains a BH3-like domain, which instead of inhibiting, activates Bcl-xL

BACKGROUND:

Translationally Controlled Tumor Protein (TCTP) is anti-apoptotic, key in development and cancer, however without the typical Bcl2 family members’ structure. Here we report that TCTP contains a BH3-like domain and forms heterocomplexes with Bcl-xL. The crystal structure of a Bcl-xL deletion variant-TCTP11–31 complex reveals that TCTP refolds in a helical conformation upon binding the BH3-groove of Bcl-xL, although lacking the h1-subregion interaction. Experiments using in vitro-vivo reconstituted systems and TCTP+/− mice indicate that TCTP activates the anti-apoptotic function of Bcl-xL, in contrast to all other BH3-proteins. Replacing the non-conserved h1 of TCTP by that of Bax drastically increases the affinity of this hybrid for Bcl-xL, modifying its biological properties. This work reveals a novel class of BH3-proteins potentiating the anti-apoptotic function of Bcl-xL.

J. Science. 2015 Nov 27;350(6264):1079-1084. doi: https://doi.org/10.1126/science.aad1329

Anticancer immunotherapy by CTLA-4 blockade relies on the gut microbiota

BACKGROUND:

Antibodies targeting CTLA-4 have been successfully used as cancer immunotherapy. We find that the antitumor effects of CTLA-4 blockade depend on distinct Bacteroides species. In mice and patients, T cell responses specific for B. thetaiotaomicron or B. fragilis were associated with the efficacy of CTLA-4 blockade. Tumors in antibiotic-treated or germ-free mice did not respond to CTLA blockade. This defect was overcome by gavage with B. fragilis, by immunization with B. fragilis polysaccharides, or by adoptive transfer of B. fragilis-specific T cells. Fecal microbial transplantation from humans to mice confirmed that treatment of melanoma patients with antibodies against CTLA-4 favored the outgrowth of B. fragilis with anticancer properties. This study reveals a key role for Bacteroidales in the immunostimulatory effects of CTLA-4 blockade.

J. Biosens. & Bioelectro. 2015 May 15;67:634-641. doi: https://doi.org/10.1016/j.bios.2014.09.084

Peptide conjugated chitosan foam as a novel approach for capture-purification and rapid detection of hapten – Example of ochratoxin A

BACKGROUND:

A novel bioassay for the detection and monitoring of Ochratoxin A (OTA), a natural carcinogenic mycotoxin produced by Aspergillus and Penicillium fungi, has been developed and applied for the screening of red wine. Here we report the immobilization and orientation of NOF4, a synthetic peptide, onto 3-D porous chitosan supports using a N-terminal histidine tag to allow binding to M++ ions that were previously adsorbed onto the high surface area biopolymer. Three divalent cations (M++=Zn++, Co++, Ni++) were evaluated and were found to adsorb via a Langmuir model and to have binding capacities in the order Zn++>Co++>Ni++. Following Zn++ saturation and washing, C-terminus vs. the N-terminus His-tagged NOF4 was evaluated. At 1000 µg L-1 OTA the N-terminus immobilization was more efficient (2.5 times) in the capture of OTA. HRP labeled OTA was added to the antigen solutions (standards or samples) and together competitively incubated on biospecific chitosan foam. The chemiluminescence substrate luminol was then added and after 5 min of enzymatic reaction, light emission signals (λmax=425 nm) were analyzed. Calibration curves of %B/B0 vs. OTA concentration in PBS showed that half-inhibition occurred at 1.17 µg L−1, allowing a range of discrimination of 0.25 and 25 µg L−1. In red wine, the minimum concentration of OTA that the system can detect was 0.5 µg L−1 and could detect up to 5 µg L−1. Assay validation was performed against immunoaffinity column (IAC) tandem reversed-phase high pressure liquid chromatography with fluorescence detection (HPLC–FLD) and provided quite good agreement. The association of chitosan foam and specific peptide represents a new approach with potential for both purification–concentration and detection of small molecules. In the future this assay will be implemented in a solid-sate bioelectronic format.

J. Nature. 2015 Mar 25;520:90-93. doi: https://doi.org/10.1038/nature14346

Primary transcripts of microRNAs encode regulatory peptides

BACKGROUND:

MicroRNAs (miRNAs) are small regulatory RNA molecules that inhibit the expression of specific target genes by binding to and cleaving their messenger RNAs or otherwise inhibiting their translation into proteins1. miRNAs are transcribed as much larger primary transcripts (pri-miRNAs), the function of which is not fully understood. Here we show that plant pri-miRNAs contain short open reading frame sequences that encode regulatory peptides. The pri-miR171b of Medicago truncatula and the pri-miR165a of Arabidopsis thaliana produce peptides, which we term miPEP171b and miPEP165a, respectively, that enhance the accumulation of their corresponding mature miRNAs, resulting in downregulation of target genes involved in root development. The mechanism of miRNA-encoded peptide (miPEP) action involves increasing transcription of the pri-miRNA. Five other pri-miRNAs of A. thaliana and M. truncatula encode active miPEPs, suggesting that miPEPs are widespread throughout the plant kingdom. Synthetic miPEP171b and miPEP165a peptides applied to plants specifically trigger the accumulation of miR171b and miR165a, leading to reduction of lateral root development and stimulation of main root growth, respectively, suggesting that miPEPs might have agronomical applications.

J. EMBO. 2012 Sep 12;31:18. doi: https://doi.org/10.1038/emboj.2012.226

Cold adaptation in the environmental bacterium Shewanella oneidensis is controlled by a J-domain co-chaperone protein network

BACKGROUND:

Calcium current through voltage‐gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser10 histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.