Specific Amino Acid Sequences Research Articles

A CAIX Dual-Targeting Small-Molecule Probe for Noninvasive Imaging of ccRCC.

Carbonic anhydrase IX (CAIX), a zinc metal transmembrane protein, is highly expressed in 95% of clear cell renal cell carcinomas (ccRCCs). A positron emission tomography (PET) probe designed to target CAIX in nuclear medicine imaging technology can achieve precise positioning, is noninvasive, and can be used to monitor CAIX expression in lesions in real time. In this study, we constructed a novel acetazolamide dual-targeted small-molecule probe [68Ga]Ga-LF-4, which targets CAIX by binding to a specific amino acid sequence. After attenuation correction, the radiolabeling yield reached 66.95 ± 0.57% (n = 5) after 15 min of reaction and the radiochemical purity reached 99% (n = 5). [68Ga]Ga-LF-4 has good in vitro and in vivo stability, and in vivo safety and high affinity for CAIX, with a Kd value of 6.62 nM. Moreover, [68Ga]Ga-LF-4 could be quickly cleared from the blood in vivo. The biodistribution study revealed that the [68Ga]Ga-LF-4 signal was concentrated in the heart, lung, and kidney after administration, which was the same as that observed in the micro-PET/CT study. In a ccRCC patient-derived xenograft (PDX) model, the signal significantly accumulated in the tumor after administration, where it was retained for up to 4 h. After competitive blockade with LF-4, uptake at the tumor site was significantly reduced. The SUVmax of the probe [68Ga]Ga-LF-4 at the ccRCC tumor site was three times greater than that in the PC3 group with low CAIX expression at 30 min (ccRCC vs PC3:1.86 ± 0.03 vs 0.62 ± 0.01, t = 48.2, P < 0.0001). These results indicate that [68Ga]Ga-LF-4 is a novel small-molecule probe that targets CAIX and can be used to image localized and metastatic ccRCC lesions.

‘Main Mechanical Forces‐Chemical Interactions’ Interplay as a Tool to Elucidate Folding Mechanisms

ABSTRACTThe folding of peptides and proteins is rigidly reliant on the ‘chemical information’ carried by the specific amino acid sequence. In this study, three polypeptides (PDBs: 2jof, 1res and 1prv) were investigated as model systems to assess their folded features, thereby enabling further understanding of mechanisms that play a role in regulating the folding process more widely. A novel physico‐chemical approach of analysis is proposed herein, focusing on chemical interactions and their related mechanical forces that we trust are determinant to drive the folding. Through this methodology, we have predicted the conformations adopted by the three polypeptides and compared the outcomes to those experimentally determined, achieving a substantial structural agreement. Molecular dynamic simulations have been carried out to further support our calculations and structural results. Within the three models, we demonstrate that the interaction of each amino acid residue with its neighbour residues is a crucial determinant for the formation of the 3D stable native structures. This article provides initial evidence that the folding occurs by means of mechanical forces developed upon establishing chemical interactions amongst residues, which, in turn, are peculiar to each specific amino acid present in each position of the peptide chain.

Mutation of the peptide-regulated transcription factor ComR for amidated peptide specificity and heterologous function in Lactiplantibacillus plantarum WCFS1.

There is a growing interest in the use of probiotic bacteria as biosensors for the detection of disease. However, there is a lack of bacterial receptors developed for specific disease biomarkers. Here, we have investigated the use of the peptide-regulated transcription factor ComR from Streptococcus spp. for specific peptide biomarker detection. ComR exhibits a number of attractive features that are potentially exploitable to create a biomolecular switch for engineered biosensor circuitry within the probiotic organism Lactiplantibacillus plantarum WCFS1. Through iterative design-build-test cycles, we developed a genomically integrated, ComR-based biosensor circuit that allowed WCFS1 to detect low nanomolar concentrations of ComR's cognate peptide XIP. By screening a library of ComR proteins with mutant residues substituted at the K100 position, we identified mutations that increased the specificity of ComR toward an amidated version of its cognate peptide, demonstrating the potential for ComR to detect this important class of biomarker.IMPORTANCEUsing bacteria to detect disease is an exciting possibility under active study. Detecting extracellular peptides with specific amino acid sequences would be particularly useful as these are important markers of health and disease (biomarkers). In this work, we show that a probiotic bacteria (Lactiplantibacillus plantarum) can be genetically engineered to detect specific extracellular peptides using the protein ComR from Streptococcus bacteria. In its natural form, ComR allowed the probiotic bacteria to detect a specific peptide, XIP. We then modified XIP to be more like the peptide biomarkers found in humans and engineered ComR so that it activated with this modified XIP and not the original XIP. This newly engineered ComR also worked in the probiotic bacteria, as expected. This suggests that with additional engineering, ComR might be able to activate with human peptide biomarkers and be used by genetically engineered probiotic bacteria to better detect disease.

Enzyme-Inspired Ligand Engineering of Gold Nanoclusters for Electrocatalytic Microenvironment Manipulation.

Natural enzymes intricately regulate substrate accessibility through specific amino acid sequences and folded structures at their active sites. Achieving such precise control over the microenvironment has proven to be challenging in nanocatalysis, especially in the realm of ligand-stabilized metal nanoparticles. Here, we use atomically precise metal nanoclusters (NCs) as model catalysts to demonstrate an effective ligand engineering strategy to control the local concentration of CO2 on the surface of gold (Au) NCs during electrocatalytic CO2 reduction reactions (CO2RR). The precise incorporation of two 2-thiouracil-5-carboxylic acid (TCA) ligands within the pocket-like cavity of [Au25(pMBA)18]- NCs (pMBA = para-mercaptobenzoic acid) leads to a substantial acceleration in the reaction kinetics of CO2RR. This enhancement is attributed to a more favorable microenvironment in proximity to the active site for CO2, facilitated by supramolecular interactions between the nucleophilic Nδ- of the pyrimidine ring of the TCA ligand and the electrophilic Cδ+ of CO2. A comprehensive investigation employing absorption spectroscopy, mass spectrometry, isotopic labeling measurements, electrochemical analyses, and quantum chemical computation highlights the pivotal role of local CO2 enrichment in enhancing the activity and selectivity of TCA-modified Au25 NCs for CO2RR. Notably, a high Faradaic efficiency of 98.6% toward CO has been achieved. The surface engineering approach and catalytic fundamentals elucidated in this study provide a systematic foundation for the molecular-level design of metal-based electrocatalysts.

Cold atmospheric pressure plasma assisted rapid assembly of peptide-based structures: a molecular scaffold to form supramolecular architectures

The use of cold atmospheric pressure plasma (CAPP) technology in the production of peptide-based materials has shown great potential in modern technology. Herein, two aggregation-prone oligopeptides, GNNQQNY and KLVFFA, were subjected to CAPP treatment to form supramolecular assemblies/aggregates. Through peptide engineering and biophysical techniques, the effect of CAPP-generated reactive oxygen and nitrogen species on the oligopeptides were investigated for different treatment times revealing that the formation of these aggregates were primarily driven by electrostatic interactions without any chemical modifications. Field emission-scanning electron microscopy and Thioflavin T (ThT) binding assay confirmed the presence of distinct β-strands, particularly in the aggregates of the KLVFFA peptide upon CAPP irradiation. The combination of CAPP technology with peptide self-assembly process and the characterization techniques employed in this study holds promise for the development of such peptide supramolecular structures based on the specific amino acid sequences.

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation.

Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1's ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.

Peptide-Decorated Microneedles for the Detection of Microplastics.

The escalating utilization of plastics in daily life has resulted in pervasive environmental pollution and consequent health hazards. The challenge of detecting and capturing microplastics, which are imperceptible to the naked eye, is exacerbated by their diminutive size, hydrophobic surface properties, and capacity to absorb organic compounds. This study focuses on the application of peptides, constituted of specific amino acid sequences, and microneedles for the rapid and selective identification of microplastics. Peptides, due to their smaller size and greater environmental stability compared with antibodies, emerge as a potent solution to overcome the limitations inherent in existing detection methodologies. To immobilize peptides onto microneedles, this study employed microneedles embedded with gold nanorods, augmenting them with sulfhydryl (SH) groups at the peptides' termini. The sensor developed through this methodology exhibited efficient peptide binding to the microneedle tips, thereby facilitating the capture of microplastics. Raman spectroscopy was employed for the detection of microplastics, with the results demonstrating successful attachment to the microneedles. This novel approach not only facilitates localized analysis but also presents a viable strategy for the detection of microplastics across diverse environmental settings.

Characterization of Copper(II) and Zinc(II) Complexes of Peptides Mimicking the CuZnSOD Enzyme.

Antimicrobial peptides are short cationic peptides that are present on biological surfaces susceptible to infection, and they play an important role in innate immunity. These peptides, like other compounds with antimicrobial activity, often have significant superoxide dismutase (SOD) activity. One direction of our research is the characterization of peptides modeling the CuZnSOD enzyme and the determination of their biological activity, and these results may contribute to the development of novel antimicrobial peptides. In the framework of this research, we have synthesized 10, 15, and 16-membered model peptides containing the amino acid sequence corresponding to the Cu(II) and Zn(II) binding sites of the CuZnSOD enzyme, namely the Zn(II)-binding HVGD sequence (80-83. fragments), the Cu(II)-binding sequence HVH (fragments 46-48), and the histidine (His63), which links the two metal ions as an imidazolate bridge: Ac-FHVHEGPHFN-NH2 (L1(10)), Ac-FHVHAGPHFNGGHVG-NH2 (L2(15)), and Ac-FHVHEGPHFNGGHVGD-NH2 (L3(16)). pH-potentiometric, UV-Vis-, and CD-spectroscopy studies of the Cu(II), Zn(II), and Cu(II)-Zn(II) mixed complexes of these peptides were performed, and the SOD activity of the complexes was determined. The binding sites preferred by Cu(II) and Zn(II) were identified by means of CD-spectroscopy. From the results obtained for these systems, it can be concluded that in equimolar solution, the -(NGG)HVGD- sequence of the peptides is the preferred binding site for copper(II) ion. However, in the presence of both metal ions, according to the native enzyme, the -HVGD- sequence offers the main binding site for Zn(II), while the majority of Cu(II) binds to the -FHVH- sequence. Based on the SOD activity assays, complexes of the 15- and 16-membered peptide have a significant SOD activity. Although this activity is smaller than that of the native CuZnSOD enzyme, the complexes showed better performance in the degradation of superoxide anion than other SOD mimics. Thus, the incorporation of specific amino acid sequences mimicking the CuZnSOD enzyme increases the efficiency of model systems in the catalytic decomposition of superoxide anion.

Evaluating Coll2-1NO2 Level in Pre- and Post-menopausal Iraqi Women with Osteoarthritis

Osteoarthritis (OA) is not an autoimmune but a chronic inflammatory disease affecting the joints of humans due to mechanical stress mostly in the knees and the hip. In OA a modification of the cartilage is encountered causing a pain in the joints upon movement. Coll2-1NO2 is a specific sequence of amino acids produced from the degradation of type II collagen, which is abundantly found in the cartilage, followed by a nitration in the tyrosine residue as a consequence of stress-inflammatory events. In this study, Coll2-1NO2 is used as a biomarker of OA to predict the inflammatory stress condition as well as the cartilage degradation in pre- and post-menopausal women. The results showed a significant increase of serum Coll2-1NO2 in pre- and post-menopausal women with OA compared to pre- and post-menopausal health women, at similar body mass index category (overweight). Pearson’s correlation analysis showed non-significant association between Coll2-1NO2 and other studied parameters. Moreover, ROC analysis showed a very excellent sensitivity of Coll2-1NO2 as a prognostic biomarker for OA disease in pre- and post-menopausal women

HLA class I peptide polymorphisms contribute to class II DQβ0603:DQα0103 antibody specificity

Antibodies reactive to human leukocyte antigens (HLA) represent a barrier for patients awaiting transplantation. Based on reactivity patterns in single-antigen bead (SAB) assays, various epitope matching algorithms have been proposed to improve transplant outcomes. However, some antibody reactivities cannot be explained by amino acid motifs, leading to uncertainty about their clinical relevance. Antibodies against the HLA class II molecule, DQβ0603:DQα0103, present in some candidates, represent one such example. Here, we show that peptides derived from amino acids 119-148 of the HLA class I heavy chain are bound to DQβ0603:DQα0103 proteins and contribute to antibody reactivity through an HLA-DM-dependent process. Moreover, antibody reactivity is impacted by the specific amino acid sequence presented. In summary, we demonstrate that polymorphic HLA class I peptides, bound to HLA class II proteins, can directly or indirectly be part of the antibody binding epitope. Our findings have potential important implications for the field of transplant immunology and for our understanding of adaptive immunity.

Isolation of Cytosolic Ribosomes Associated with Plant Mitochondria and Chloroplasts.

Excluding the few dozen proteins encoded by the chloroplast and mitochondrial genomes, the majority of plant cell proteins are synthesized by cytosolic ribosomes. Most of these nuclear-encoded proteins are then targeted to specific cell compartments thanks to localization signals present in their amino acid sequence. These signals can be specific amino acid sequences known as transit peptides, or post-translational modifications, ability to interact with specific proteins or other more complex regulatory processes. Furthermore, in eukaryotic cells, protein synthesis can be regulated so that certain proteins are synthesized close to their destination site, thus enabling local protein synthesis in specific compartments of the cell. Previous studies have revealed that such locally translating cytosolic ribosomes are present in the vicinity of mitochondria and emerging views suggest that localized translation near chloroplasts could also occur. However, in higher plants, very little information is available on molecular mechanisms controlling these processes and there is a need to characterize cytosolic ribosomes associated with organelles membranes. To this goal, this protocol describes the purification of higher plant chloroplast and mitochondria and the organelle-associated cytosolic ribosomes.

Comprehensive Network and Structural Analysis of Bovine Papillomavirus, Squamous Cell Carcinoma Markers, and Elucidation of Efficacy Mechanisms of Phytochemicals from Thuja Occidentalis

Papillomaviruses infect cutaneous tissue in various species including bovines and from benign warts to malignant squamous cell carcinoma causing severe economic losses to the farmers. The mechanisms by which bovine papillomaviruses interact with host tissue are unclear. Hence in this study using classical network analysis tools, we evaluated interactions of Bovine papilloma (BPV) variants, with markers and receptors implicated in squamous cell carcinoma. Additionally, the Thuja phytoconstituents were also evaluated for its potential to target the BPV and squamous cell carcinoma network interactions to understand the mechanism of its clinical benefits. Various protein composition of 14 different virus variants of BPV were assessed against 24 markers of squamous cell carcinoma. Among these interactions EGFR consistently exhibited high-affinity interactions with the E1 protein in all isoforms of BPV. Type 4 BPV displayed the maximum number of binding sites (14) with a binding pocket score ranging from 15.47 to 141.34 and a probability score of 0.75 to 0.99. The comparison of the binding pockets identified that BPV types 2 and 13 had the highest number of common amino acid sequences. Further the alpha helix structure of specific common amino acid sequences contributes to a more robust and widespread affinity interaction with both E1 of various BPV types and EGFR. Analysis of Thuja phytochemicals suggested superior efficacy of Beyerene and Terpinene-4-ol towards all ten BPV targets and bEGFR. In conclusion, our comprehensive study leading to identification of E1 protein of BPV as a major interacting network with bEGFR, their key binding sites, and efficacy of Thuja phytoconstituents offer valuable insight into further experimental validation and development of novel therapeutic strategies against BPV-associated diseases.

Comparison of glutathione peroxidase-1 in free divers with their counterparts: A model study for sports informatics

Free diving is a popular sport because of many features such as sustainability, eco-friendly and challenges to nature. Due to increased interest in recent years, the number of competitions is also increasing gradually. On the other hand, scientific reports on the understanding of longer breath-holding mechanisms and metabolisms are still unclear. To provide contributions on this phenomenon, glutathione peroxidase was selected as a model enzyme because of its critical importance in breath-holding. The enzymes from both human and free diving animals were compared by using bioinformatics tools such as ProtParam, Swiss-Model, Clustal Omega and the results are discussed in the present paper. In conclusion, the specific amino acid sequences can be considered in the selection of elite free divers for international competitions to get the best results. However, it should be noted that special training methods should also be applied to have better breath-holding capacities.

Characterising the structural and cellular role of immunoglobulin C-terminal lysine in secretory pathways.

Improved understanding of expression of recombinant immunoglobulin (IgG)-based therapies can decrease manufacturing process costs and bring down costs to patients. Deletion of C-terminal Lysine (C-Lys) from IgG molecules has been shown to greatly impact yield. This study set out to characterise structural components of IgG C-terminal variants which modulate protein expression by examination of the consequences of mutations at the C-terminal of IgG on expression and by the use of fluorescent C-terminal fragment fusion proteins. Cell-based and cell-free experiments were also implemented to characterise how the C-terminal differentially engages with cellular pathways to modulate expression. IgG variants engineered by removal of the C-terminal Lys were expressed at significantly lower rates than control variants by CHO (and HEK) cells. Engineered constructs of mCherry fused with short regions of the C-terminal regions of IgG mimicked the ordering of expressability observed for IgG variants. These fluorescent C-terminal fragment fusions offered the potential to profile how sequences (and point mutations) modified expression. Via combinations of cell and cell-free systems, screening across a range of variants of IgG and mCherry reporter constructs has shown that interactions between specific C-terminal amino acid sequences and the ribosome can regulate the rate and extent of expression. This study highlights the importance of amino acid sequence regulatory events determining the efficiency of production of desirable recombinant proteins, showing that wildtype C-terminal lysine is a necessary capping molecule for IgG1 expression. From a wider perspective, these data are especially significant towards the design of novel entities. The approach has also provided information about novel short C-terminal tags which may be used to provide selective synthesis of specific subunits in the production of multisubunit products. Alternative strategies for removing C-terminal amino acid heterogeneity whilst maintaining efficient rates of expression have been provided.

Accelerating Lipid Flip-Flop at Low Concentrations: A General Mechanism for Membrane Binding Peptides.

We report a physicochemical investigation of the lipid transport properties of model lipid membranes in the presence of the antimicrobial peptide indolicidin through comparisons of experimental SANS/SAXS scattering techniques to fully atomistic molecular dynamics simulations. In agreement with the experiment, we show that upon peripheral binding of the peptides, even at low concentrations, lipid flip-flop dynamics is greatly accelerated. Computer modeling elucidates the interplay between structural changes and lipid dynamics induced by peptides and proposes a mechanism for the mode of action of antimicrobial peptides, assessing the major role of entropy for the catalysis of the flipping events. The mechanism introduced here is universal for all peptides with preferential peripheral binding to the membrane as it does not depend on the specific amino acid sequence.

Exploring binding positions and backbone conformations of peptide ligands of proteins with a backbone-centred statistical energy function.

When designing peptide ligands based on the structure of a protein receptor, it can be very useful to narrow down the possible binding positions and bound conformations of the ligand without the need to choose its amino acid sequence in advance. Here, we construct and benchmark a tool for this purpose based on a recently reported statistical energy model named SCUBA (Sidechain-Unknown Backbone Arrangement) for designing protein backbones without considering specific amino acid sequences. With this tool, backbone fragments of different local conformation types are generated and optimized with SCUBA-driven stochastic simulations and simulated annealing, and then ranked and clustered to obtain representative backbone fragment poses of strong SCUBA interaction energies with the receptor. We computationally benchmarked the tool on 111 known protein-peptide complex structures. When the bound ligands are in the strand conformation, the method is able to generate backbone fragments of both low SCUBA energies and low root mean square deviations from experimental structures of peptide ligands. When the bound ligands are helices or coils, low-energy backbone fragments with binding poses similar to experimental structures have been generated for approximately 50% of benchmark cases. We have examined a number of predicted ligand-receptor complexes by atomistic molecular dynamics simulations, in which the peptide ligands have been found to stay at the predicted binding sites and to maintain their local conformations. These results suggest that promising backbone structures of peptides bound to protein receptors can be designed by identifying outstanding minima on the SCUBA-modeled backbone energy landscape.

RNA-seq analysis reveals an immunomodulatory peptide from highland barley activating RAW264.7 macrophages via TNF/NF-κB signaling pathway.

Highland barley (HB) is an important cereal crop distributed in the plateau region. Bioactive peptides (BAPs) derived from cereal proteins have shown biological functions. However, the knowledge of highland barley peptide (HBP) is limited. This study aims to explore the immunomodulatory activity of HBP and the relationship between immunomodulatory activity and related gene expression through RNA-seq. Firstly, HBP is isolated from protease hydrolysates of HB protein, yielding 12.04% of crude HB protein. The molecular weight of HBP is about 1702 Da analyzed by gel filtration chromatography, and HBP has a specific amino acid sequence as Gln-Pro-Gln-Gln-Pro-Phe-Pro-Gln (QPQPFPQ) analyzed by LC-MS. Besides, HBP contains 42.20% hydrophobic amino acids and 10.86% basic amino acids. Next, the immunomodulatory activity of HBP in vitro shows that HBP enhances the phagocytosis of RAW264.7 macrophages, promotes nitric oxide (NO) production and the mRNA expression of pro-inflammatory genes including tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and inducible nitric oxide synthase (iNOS), and decreases the mRNA expression of anti-inflammatory gene, transforming growth factor β1 (TGF-β1). RNA-seq analysis reveals TNF and nuclear factor kappa B (NF-κB) pathways are upregulated, and RT-qPCR is performed to verify RNA-seq analysis. In conclusion, HBP activates RAW264.7 macrophages via TNF/NF-κB signaling pathway. HBP, as a significant immunomodulatory peptide, might be a promising resource for future functional foods.

Adsorption of pulmonary and exogeneous surfactants on SARS-CoV-2 spike protein

COVID-19 is transmitted by airborne particles containing virions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Coronavirus virions represent nanoparticles enveloped by a lipid bilayer decorated by a “crown” of Spike protein protrusions. Virus transmission into the cells is induced by binding of Spike proteins with ACE2 receptors of alveolar epithelial cells. Active clinical search is ongoing for exogenous surfactants and biologically active chemicals capable of hindering virion-receptor binding. Here, we explore by using coarse-grained molecular dynamics simulations the physico-chemical mechanisms of adsorption of selected pulmonary surfactants, zwitterionic dipalmitoyl phosphatidyl choline and cholesterol, and exogeneous anionic surfactant, sodium dodecyl sulfate, on the S1-domain of the Spike protein. We show that surfactants form micellar aggregates that selectively adhere to the specific regions of the S1-domain that are responsible for binding with ACE2 receptors. We find distinctly higher cholesterol adsorption and stronger cholesterol-S1 interactions in comparison with other surfactants, that is consistent with the experimental observations of the effects of cholesterol on COVID-19 infection. Distribution of adsorbed surfactant along the protein residue chain is highly specific and inhomogeneous with preferential adsorption around specific amino acid sequences. We observe preferential adsorption of surfactants on cationic arginine and lysine residues in the receptor-binding domain (RBD) that play an important role in ACE2 binding and are present in higher amounts in Delta and Omicron variants, which may lead to blocking direct Spike-ACE2 interactions. Our findings of strong selective adhesion of surfactant aggregates to Spike proteins have important implications for informing clinical search for therapeutic surfactants for curing and preventing COVID-19 caused by SARS-CoV-2 and its variants.

GAPDH Released from Lactobacillus johnsonii MG Enhances Barrier Function by Upregulating Genes Associated with Tight Junctions.

Extracellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has multiple interactions with various gut epithelial components. For instance, GAPDH in Lactobacillus johnsonii MG cells interacts with junctional adhesion molecule-2 (JAM-2) in Caco-2 cells and enhances tight junctions. However, the specificity of GAPDH toward JAM-2 and its role in the tight junctions in Caco-2 cells remain unclear. In the present study, we assessed the effect of GAPDH on tight junction regeneration and explored the GAPDH peptide fragments required for interaction with JAM-2. GAPDH was specifically bound to JAM-2 and rescued H2O2-damaged tight junctions in Caco-2 cells, with various genes being upregulated in the tight junctions. To understand the specific amino acid sequence of GAPDH that interacts with JAM-2, peptides interacting with JAM-2 and L. johnsonii MG cells were purified using HPLC and predicted using TOF-MS analysis. Two peptides, namely 11GRIGRLAF18 at the N-terminus and 323SFTCQMVRTLLKFATL338 at the C-terminus, displayed good interactions and docking with JAM-2. In contrast, the long peptide 52DSTHGTFNHEVSATDDSIVVDGKKYRVYAEPQAQNIPW89 was predicted to bind to the bacterial cell surface. Overall, we revealed a novel role of GAPDH purified from L. johnsonii MG in promoting the regeneration of damaged tight junctions and identified the specific sequences of GAPDH involved in JAM-2 binding and MG cell interaction.

Structural and functional analyses of the echinomycin resistance conferring protein Ecm16 from Streptomyces lasalocidi

Echinomycin is a natural product DNA bisintercalator antibiotic. The echinomycin biosynthetic gene cluster in Streptomyces lasalocidi includes a gene encoding the self-resistance protein Ecm16. Here, we present the 2.0 Å resolution crystal structure of Ecm16 bound to adenosine diphosphate. The structure of Ecm16 closely resembles that of UvrA, the DNA damage sensor component of the prokaryotic nucleotide excision repair system, but Ecm16 lacks the UvrB-binding domain and its associated zinc-binding module found in UvrA. Mutagenesis study revealed that the insertion domain of Ecm16 is required for DNA binding. Furthermore, the specific amino acid sequence of the insertion domain allows Ecm16 to distinguish echinomycin-bound DNA from normal DNA and link substrate binding to ATP hydrolysis activity. Expression of ecm16 in the heterologous host Brevibacillus choshinensis conferred resistance against echinomycin and other quinomycin antibiotics, including thiocoraline, quinaldopeptin, and sandramycin. Our study provides new insight into how the producers of DNA bisintercalator antibiotics fend off the toxic compounds that they produce.