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Mts1 (S100A4) and Its Peptide Demonstrate Cytotoxic Activity in Complex with Tag7 (PGLYRP1) Peptide.International Journal of Molecular... Jun 2024Receptors of cytokines are major regulators of the immune response. In this work, we have discovered two new ligands that can activate the TNFR1 (tumor necrosis factor...
Receptors of cytokines are major regulators of the immune response. In this work, we have discovered two new ligands that can activate the TNFR1 (tumor necrosis factor receptor 1) receptor. Earlier, we found that the peptide of the Tag (PGLYRP1) protein designated 17.1 can interact with the TNFR1 receptor. Here, we have found that the Mts1 (S100A4) protein interacts with this peptide with a high affinity (K = 1.28 × 10 M), and that this complex is cytotoxic to cancer cells that have the TNFR1 receptor on their surface. This complex induces both apoptosis and necroptosis in cancer cells with the involvement of mitochondria and lysosomes in cell death signal transduction. Moreover, we have succeeded in locating the Mts1 fragment that is responsible for protein-peptide interaction, which highly specifically interacts with the Tag7 protein (K = 2.96 nM). The isolated Mts1 peptide M7 also forms a complex with 17.1, and this peptide-peptide complex also induces the TNFR1 receptor-dependent cell death. Molecular docking and molecular dynamics experiments show the amino acids involved in peptide binding and that may be used for peptidomimetics' development. Thus, two new cytotoxic complexes were created that were able to induce the death of tumor cells via the TNFR1 receptor. These results may be used in therapy for both cancer and autoimmune diseases.
Topics: Humans; Receptors, Tumor Necrosis Factor, Type I; Apoptosis; Protein Binding; Molecular Docking Simulation; Cell Line, Tumor; Peptides; Molecular Dynamics Simulation; Signal Transduction; Necroptosis; Oligopeptides; Cytokines
PubMed: 38928339
DOI: 10.3390/ijms25126633 -
International Journal of Molecular... Jun 2024Aflatoxin B (AFB) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from ANSB821 previously reported by our laboratory showed...
Aflatoxin B (AFB) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from ANSB821 previously reported by our laboratory showed great potential to degrade AFB without redox mediators. However, the use of this CotA-laccase to remove AFB in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (/K) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.
Topics: Aflatoxin B1; Bacillus licheniformis; Bacterial Proteins; Laccase; Mutagenesis, Site-Directed; Recombinant Proteins; Saccharomycetales
PubMed: 38928160
DOI: 10.3390/ijms25126455 -
International Journal of Molecular... Jun 2024The downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk...
The downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three β strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.
Topics: src Homology Domains; Drosophila Proteins; Molecular Dynamics Simulation; Protein Binding; Animals; Humans; Receptor Protein-Tyrosine Kinases; GRB2 Adaptor Protein; Molecular Docking Simulation; Binding Sites; Amino Acid Sequence; Magnetic Resonance Spectroscopy
PubMed: 38928093
DOI: 10.3390/ijms25126386 -
BMC Biotechnology Jun 2024Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with...
BACKGROUND
Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS
In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS
The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS
This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Topics: Animals; CHO Cells; Integrases; Cricetulus; Nuclear Localization Signals; Cell Nucleus; Serine; Green Fluorescent Proteins; Cricetinae; Xenopus laevis
PubMed: 38926833
DOI: 10.1186/s12896-024-00871-4 -
Scientific Reports Jun 2024Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations...
Heterozygous de novo mutations in the Activity-Dependent Neuroprotective Homeobox (ADNP) gene underlie Helsmoortel-Van der Aa syndrome (HVDAS). Most of these mutations are situated in the last exon and we previously demonstrated escape from nonsense-mediated decay by detecting mutant ADNP mRNA in patient blood. In this study, wild-type and ADNP mutants are investigated at the protein level and therefore optimal detection of the protein is required. Detection of ADNP by means of western blotting has been ambiguous with reported antibodies resulting in non-specific bands without unique ADNP signal. Validation of an N-terminal ADNP antibody (Aviva Systems) using a blocking peptide competition assay allowed to differentiate between specific and non-specific signals in different sample materials, resulting in a unique band signal around 150 kDa for ADNP, above its theoretical molecular weight of 124 kDa. Detection with different C-terminal antibodies confirmed the signals at an observed molecular weight of 150 kDa. Our antibody panel was subsequently tested by immunoblotting, comparing parental and homozygous CRISPR/Cas9 endonuclease-mediated Adnp knockout cell lines and showed disappearance of the 150 kDa signal, indicative for intact ADNP. By means of both a GFPSpark and Flag-tag N-terminally fused to a human ADNP expression vector, we detected wild-type ADNP together with mutant forms after introduction of patient mutations in E. coli expression systems by site-directed mutagenesis. Furthermore, we were also able to visualize endogenous ADNP with our C-terminal antibody panel in heterozygous cell lines carrying ADNP patient mutations, while the truncated ADNP mutants could only be detected with epitope-tag-specific antibodies, suggesting that addition of an epitope-tag possibly helps stabilizing the protein. However, western blotting of patient-derived hiPSCs, immortalized lymphoblastoid cell lines and post-mortem patient brain material failed to detect a native mutant ADNP protein. In addition, an N-terminal immunoprecipitation-competent ADNP antibody enriched truncating mutants in overexpression lysates, whereas implementation of the same method failed to enrich a possible native mutant protein in immortalized patient-derived lymphoblastoid cell lines. This study aims to shape awareness for critical assessment of mutant ADNP protein analysis in Helsmoortel-Van der Aa syndrome.
Topics: Humans; Homeodomain Proteins; Nerve Tissue Proteins; Mutation; HEK293 Cells; Autism Spectrum Disorder; Heart Diseases; Facies; Neurodevelopmental Disorders
PubMed: 38926592
DOI: 10.1038/s41598-024-65608-x -
Pathogens (Basel, Switzerland) May 2024is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid...
is the main pathogen of chicken necrotic enteritis (NE) causing huge economic losses in the poultry industry. Although dietary secondary bile acid deoxycholic acid (DCA) reduced chicken NE, the accumulation of conjugated tauro-DCA (TDCA) raised concerns regarding DCA efficacy. In this study, we aimed to deconjugate TDCA by bile salt hydrolase (BSH) to increase DCA efficacy against the NE pathogen . Assays were conducted to evaluate the inhibition of growth, hydrogen sulfide (HS) production, and virulence gene expression by TDCA and DCA. BSH activity and sequence alignment were conducted to select the gene for cloning. The gene from was PCR-amplified and cloned into plasmids pET-28a (pET-BSH) and pDR111 (pDR-BSH) for expressing the BSH protein in BL21 and 168 (BSH), respectively. His-tag-purified BSH from BL21 cells was evaluated by SDS-PAGE, Coomassie blue staining, and a Western blot (WB) assays. Secretory BSH from was analyzed by a Dot-Blot. -BSH was evaluated for the inhibition of growth. growth reached 7.8 log10 CFU/mL after 24 h culture. growth was at 8 vs. 7.4, 7.8 vs. 2.6 and 6 vs. 0 log10 CFU/mL in 0.2, 0.5, and 1 mM TDCA vs. DCA, respectively. Compared to TDCA, DCA reduced HS production and the virulence gene expression of , , , and . BSH activity was observed in and under anaerobe but not under 10% CO air. After the sequence alignment of from ten bacteria, from was selected, cloned into pET-BSH, and sequenced at 951 bp. After pET-BSH was transformed in BL21, BSH expression was assessed around 35 kDa using Coomassie staining and verified for His-tag using WB. After the subcloned and amylase signal peptide sequence was inserted into pDR-BSH, was transformed and named -BSH. The transformation was evaluated using PCR with around 3 kb and BSH around 5 kb. Secretory BSH expressed from -BSH was determined for His-tag using Dot-Blot. Importantly, growth was reduced greater than 59% log10 CFU/mL in the -BSH media precultured with 1 vs. 0 mM TDCA. In conclusion, TDCA was less potent than DCA against virulence, and recombinant secretory BSH from -BSH reduced growth, suggesting a new potential intervention against the pathogen-induced chicken NE.
PubMed: 38921762
DOI: 10.3390/pathogens13060464 -
Journal of Xenobiotics Jun 2024Contamination with plastics of small dimensions (<1 µm) represents a health concern for many terrestrial and aquatic organisms. This study examined the use of...
Contamination with plastics of small dimensions (<1 µm) represents a health concern for many terrestrial and aquatic organisms. This study examined the use of plastic-binding peptides as a coating probe to detect various types of plastic using a plasmon nano-gold sensor. Plastic-binding peptides were selected for polyethylene (PE), polyethylene terephthalate (PET), polypropylene (PP), and polystyrene (PS) based on the reported literature. Using nAu with each of these peptides to test the target plastics revealed high signal, at 525/630 nm, suggesting that the target plastic limited HCl-induced nAu aggregation. Testing with other plastics revealed some lack of specificity but the signal was always lower than that of the target plastic. This suggests that these peptides, although reacting mainly with their target plastic, show partial reactivity with the other target plastics. By using a multiple regression model, the relative levels of a given plastic could be corrected by the presence of other plastics. This approach was tested in freshwater mussels caged for 3 months at sites suspected to release plastic materials: in rainfall overflow discharges, downstream a largely populated city, and in a municipal effluent dispersion plume. The data revealed that the digestive glands of the mussels contained higher levels of PP, PE, and PET plastic particles at the rainfall overflow and downstream city sites compared to the treated municipal effluent site. This corroborated earlier findings that wastewater treatment could remove nanoparticles, at least in part. A quick and inexpensive screening test for plastic nanoparticles in biological samples with plasmonic nAu-peptides is proposed.
PubMed: 38921649
DOI: 10.3390/jox14020040 -
Current Issues in Molecular Biology May 2024Melanocytes, located in the epidermis' basal layer, are responsible for melanin pigment production, crucial for skin coloration and protection against UV...
Melanocytes, located in the epidermis' basal layer, are responsible for melanin pigment production, crucial for skin coloration and protection against UV radiation-induced damage. Melanin synthesis is intricately regulated by various factors, including the Wnt signaling pathway, particularly mediated by the microphthalmia-associated transcription factor (MITF). While MITF is recognized as a key regulator of pigmentation, its regulation by the Wnt pathway remains poorly understood. This study investigates the role of Sfrp5pepD, a peptide antagonist of the Wnt signaling pathway, in modulating melanogenesis and its potential therapeutic implications for pigmentary disorders. To tackle this issue, we investigated smaller peptides frequently utilized in cosmetics or pharmaceuticals. Nevertheless, there is a significant scarcity of reports on peptides associated with melanin-related signal modulation or inhibiting melanin production. Results indicate that Sfrp5pepD effectively inhibits Wnt signaling by disrupting the interaction between Axin-1 and β-catenin, thus impeding downstream melanogenic processes. Additionally, Sfrp5pepD suppresses the interaction between MITF and β-catenin, inhibiting their nuclear translocation and downregulating melanogenic enzyme expression, ultimately reducing melanin production. These inhibitory effects are validated in cell culture models suggesting potential clinical applications for hyperpigmentation disorders. Overall, this study elucidates the intricate interplay between Wnt signaling and melanogenesis, highlighting Sfrp5pepD as a promising therapeutic agent for pigmentary disorders. Sfrp5pepD, with a molecular weight of less than 500 Da, is anticipated to penetrate the skin unlike SFRPs. This suggests a strong potential for their use as cosmetics or transdermal absorption agents. Additional investigation into its mechanisms and clinical significance is necessary to enhance its effectiveness in addressing melanin-related skin conditions.
PubMed: 38920996
DOI: 10.3390/cimb46060324 -
Nature Communications Jun 2024Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including...
Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fibre photometry enabled direct recording of NOPLight binding to exogenous N/OFQ receptor ligands, as well as detection of endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA) during natural behaviors and chemogenetic activation of PNOC neurons. In summary, we show here that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely behaving animals.
Topics: Animals; Opioid Peptides; Nociceptin; Receptors, Opioid; Neurons; Humans; Mice; Male; Ventral Tegmental Area; Nociceptin Receptor; HEK293 Cells; Brain; Mice, Inbred C57BL; Ligands; Biosensing Techniques
PubMed: 38918403
DOI: 10.1038/s41467-024-49712-0 -
PloS One 2024SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at...
SlyD is a widely-occurring prokaryotic FKBP-family prolyl isomerase with an additional chaperone domain. Often, such as in Escherichia coli, a third domain is found at its C-terminus that binds nickel and provides it for nickel-enzyme biogenesis. SlyD has been found to bind signal peptides of proteins that are translocated by the Tat pathway, a system for the transport of folded proteins across membranes. Using peptide arrays to analyze these signal peptide interactions, we found that SlyD interacted only with positively charged peptides, with a preference for arginines over lysines, and large hydrophobic residues enhanced binding. Especially a twin-arginine motif was recognized, a pair of highly conserved arginines adjacent to a stretch of hydrophobic residues. Using isothermal titration calorimetry (ITC) with purified SlyD and a signal peptide-containing model Tat substrate, we could show that the wild type twin-arginine signal peptide was bound with higher affinity than an RR>KK mutated variant, confirming that positive charges are recognized by SlyD, with a preference of arginines over lysines. The specific role of negative charges of the chaperone domain surface and of hydrophobic residues in the chaperone active site was further analyzed by ITC of mutated SlyD variants. Our data show that the supposed key hydrophobic residues of the active site are indeed crucial for binding, and that binding is influenced by negative charges on the chaperone domain. Recognition of positive charges is likely achieved by a large negatively charged surface region of the chaperone domain, which is highly conserved although individual positions are variable.
Topics: Escherichia coli Proteins; Peptidylprolyl Isomerase; Escherichia coli; Protein Binding; Molecular Chaperones; Protein Sorting Signals; Hydrophobic and Hydrophilic Interactions; Calorimetry; Arginine; Amino Acid Sequence
PubMed: 38917203
DOI: 10.1371/journal.pone.0305823