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Fmp40 ampylase regulates cell survival upon oxidative stress by controlling Prx1 and Trx3 oxidation.Redox Biology Jul 2024Reactive oxygen species (ROS), play important roles in cellular signaling, nonetheless are toxic at higher concentrations. Cells have many interconnected, overlapped or...
Reactive oxygen species (ROS), play important roles in cellular signaling, nonetheless are toxic at higher concentrations. Cells have many interconnected, overlapped or backup systems to neutralize ROS, but their regulatory mechanisms remain poorly understood. Here, we reveal an essential role for mitochondrial AMPylase Fmp40 from budding yeast in regulating the redox states of the mitochondrial 1-Cys peroxiredoxin Prx1, which is the only protein shown to neutralize HO with the oxidation of the mitochondrial glutathione and the thioredoxin Trx3, directly involved in the reduction of Prx1. Deletion of FMP40 impacts a cellular response to HO treatment that leads to programmed cell death (PCD) induction and an adaptive response involving up or down regulation of genes encoding, among others the catalase Cta1, PCD inducing factor Aif1, and mitochondrial redoxins Trx3 and Grx2. This ultimately perturbs the reduced glutathione and NADPH cellular pools. We further demonstrated that Fmp40 AMPylates Prx1, Trx3, and Grx2 in vitro and interacts with Trx3 in vivo. AMPylation of the threonine residue 66 in Trx3 is essential for this protein's proper endogenous level and its precursor forms' maturation under oxidative stress conditions. Additionally, we showed the Grx2 involvement in the reduction of Trx3 in vivo. Taken together, Fmp40, through control of the reduction of mitochondrial redoxins, regulates the hydrogen peroxide, GSH and NADPH signaling influencing the yeast cell survival.
Topics: Oxidative Stress; Oxidation-Reduction; Saccharomyces cerevisiae Proteins; Thioredoxins; Saccharomyces cerevisiae; Peroxiredoxins; Hydrogen Peroxide; Glutathione; Mitochondria; Reactive Oxygen Species; Cell Survival; Apoptosis; Peroxidases; Glutaredoxins
PubMed: 38795545
DOI: 10.1016/j.redox.2024.103201 -
Vaccines May 2024Ad26.COV2.S vaccination can lead to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but severe adverse effect, characterized by thrombocytopenia and...
Ad26.COV2.S vaccination can lead to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but severe adverse effect, characterized by thrombocytopenia and thrombosis. The mechanism of VITT induction is unclear and likely multifactorial, potentially including the activation of platelets and endothelial cells mediated by the vaccine-encoded spike protein (S protein). Here, we investigated the biodistribution of the S protein after Ad26.COV2.S dosing in three animal models and in human serum samples. The S protein was transiently present in draining lymph nodes of rabbits after Ad26.COV2.S dosing. The S protein was detected in the serum in all species from 1 day to 21 days after vaccination with Ad26.COV2.S, but it was not detected in platelets, the endothelium lining the blood vessels, or other organs. The S protein S1 and S2 subunits were detected at different ratios and magnitudes after Ad26.COV2.S or COVID-19 mRNA vaccine immunization. However, the S1/S2 ratio did not depend on the Ad26 platform, but on mutation of the furin cleavage site, suggesting that the S1/S2 ratio is not VITT related. Overall, our data suggest that the S-protein biodistribution and kinetics after Ad26.COV2.S dosing are likely not main contributors to the development of VITT, but other S-protein-specific parameters require further investigation.
PubMed: 38793810
DOI: 10.3390/vaccines12050559 -
Vaccines May 2024() is a Gram-positive bacterium that causes substantial disease in pigs. is also an emerging zoonoses in humans, primarily in Asia, through the consumption of...
() is a Gram-positive bacterium that causes substantial disease in pigs. is also an emerging zoonoses in humans, primarily in Asia, through the consumption of undercooked pork and the handling of infected pig meat as well as carcasses. The complexity of epidemiology, characterized by the presence of multiple bacterial serotypes and strains with diverse sequence types, identifies a critical need for a universal vaccine with the ability to confer cross-protective immunity. Highly conserved immunogenic proteins are generally considered good candidate antigens for subunit universal vaccines. In this study, the cross-protection of the sugar ABC transporter substrate-binding protein (S-ABC), a surface-associated immunogenic protein of , was examined in mice for evaluation as a universal vaccine candidate. S-ABC was shown to be highly conserved, with 97% amino acid sequence identity across 31 strains deposited in GenBank. Recombinantly expressed S-ABC (rS-ABC) was recognized via rabbit sera specific to serotype 2. The immunization of mice with rS-ABC induced antigen-specific antibody responses, as well as IFN-γ and IL-4, in multiple organs, including the lungs. rS-ABC immunization conferred high (87.5% and 100%) protection against challenges with serotypes 2 and 9, demonstrating high cross-protection against these serotypes. Protection, albeit lower (50%), was also observed in mice challenged with serotype 7. These data identify S-ABC as a promising antigenic target within a universal subunit vaccine against .
PubMed: 38793795
DOI: 10.3390/vaccines12050544 -
Viruses Apr 2024The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing...
The cucumber mosaic virus (CMV) 2b protein is a suppressor of plant defenses and a pathogenicity determinant. Amongst the 2b protein's host targets is the RNA silencing factor Argonaute 1 (AGO1), which it binds to and inhibits. In , if 2b-induced inhibition of AGO1 is too efficient, it induces reinforcement of antiviral silencing by AGO2 and triggers increased resistance against aphids, CMV's insect vectors. These effects would be deleterious to CMV replication and transmission, respectively, but are moderated by the CMV 1a protein, which sequesters sufficient 2b protein molecules into P-bodies to prevent excessive inhibition of AGO1. Mutant 2b protein variants were generated, and red and green fluorescent protein fusions were used to investigate subcellular colocalization with AGO1 and the 1a protein. The effects of mutations on complex formation with the 1a protein and AGO1 were investigated using bimolecular fluorescence complementation and co-immunoprecipitation assays. Although we found that residues 56-60 influenced the 2b protein's interactions with the 1a protein and AGO1, it appears unlikely that any single residue or sequence domain is solely responsible. In silico predictions of intrinsic disorder within the 2b protein secondary structure were supported by circular dichroism (CD) but not by nuclear magnetic resonance (NMR) spectroscopy. Intrinsic disorder provides a plausible model to explain the 2b protein's ability to interact with AGO1, the 1a protein, and other factors. However, the reasons for the conflicting conclusions provided by CD and NMR must first be resolved.
Topics: Argonaute Proteins; Cucumovirus; Arabidopsis; Arabidopsis Proteins; Protein Binding; Viral Proteins; Host-Pathogen Interactions; Viral Replicase Complex Proteins; Plant Diseases; RNA-Dependent RNA Polymerase; Methyltransferases
PubMed: 38793558
DOI: 10.3390/v16050676 -
International Journal of Molecular... May 2024The subcellular location of a protein provides valuable insights to bioinformaticians in terms of drug designs and discovery, genomics, and various other aspects of...
The subcellular location of a protein provides valuable insights to bioinformaticians in terms of drug designs and discovery, genomics, and various other aspects of medical research. Experimental methods for protein subcellular localization determination are time-consuming and expensive, whereas computational methods, if accurate, would represent a much more efficient alternative. This article introduces an ab initio protein subcellular localization predictor based on an ensemble of Deep N-to-1 Convolutional Neural Networks. Our predictor is trained and tested on strict redundancy-reduced datasets and achieves 63% accuracy for the diverse number of classes. This predictor is a step towards bridging the gap between a protein sequence and the protein's function. It can potentially provide information about protein-protein interaction to facilitate drug design and processes like vaccine production that are essential to disease prevention.
Topics: Neural Networks, Computer; Computational Biology; Proteins; Software; Databases, Protein; Humans
PubMed: 38791479
DOI: 10.3390/ijms25105440 -
Bioengineering (Basel, Switzerland) May 2024Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for elucidating the molecular makeup of materials. It possesses the unique characteristics of...
Surface-enhanced Raman spectroscopy (SERS) is a powerful tool for elucidating the molecular makeup of materials. It possesses the unique characteristics of single-molecule sensitivity and extremely high specificity. However, the true potential of SERS, particularly in capturing the biochemical content of particles, remains underexplored. In this study, we harnessed transformer neural networks to interpret SERS spectra, aiming to discern the amino acid profiles within proteins. By training the network on the SERS profiles of 20 amino acids of human proteins, we explore the feasibility of predicting the predominant proteins within the µL-scale detection volume of SERS. Our results highlight a consistent alignment between the model's predictions and the protein's known amino acid compositions, deepening our understanding of the inherent information contained within SERS spectra. For instance, the model achieved low root mean square error (RMSE) scores and minimal deviation in the prediction of amino acid compositions for proteins such as Bovine Serum Albumin (BSA), ACE2 protein, and CD63 antigen. This novel methodology offers a robust avenue not only for protein analytics but also sets a precedent for the broader realm of spectral analyses across diverse material categories. It represents a solid step forward to establishing SERS-based proteomics.
PubMed: 38790349
DOI: 10.3390/bioengineering11050482 -
Nature Communications May 2024Stop codon readthrough events give rise to longer proteins, which may alter the protein's function, thereby generating short-lasting phenotypic variability from a single...
Stop codon readthrough events give rise to longer proteins, which may alter the protein's function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions.
Topics: Escherichia coli; Codon, Terminator; Protein Biosynthesis; Escherichia coli Proteins; Transcription, Genetic; Codon, Nonsense; DNA-Directed RNA Polymerases; Gene Expression Regulation, Bacterial
PubMed: 38789441
DOI: 10.1038/s41467-024-48387-x -
Pathogens (Basel, Switzerland) Apr 2024Fludioxonil is a highly effective phenylpyrrole fungicide for controlling . Although the field efficacy of fludioxonil remains high, isolates with reduced sensitivity...
Fludioxonil is a highly effective phenylpyrrole fungicide for controlling . Although the field efficacy of fludioxonil remains high, isolates with reduced sensitivity have been reported globally. The molecular target of fludioxonil still remains unknown; however, a mechanism of reduced sensitivity to fludioxonil underlies the overexpression of the ATP binding cassette (ABC) transporter AtrB in a dependent pathway of the Mrr1 transcription factor. Fludioxonil is a key player in controlling infection in table grapes in Chile. However, some isolates with a reduced sensitivity to fludioxonil were detected. This study observed endogenous overexpression in Chilean isolates with reduced sensitivity to fludioxonil ( = 22) compared to the sensitive isolates ( = 10). All isolates increased the expression of in a growth medium supplemented with fludioxonil (0.05 μg/mL). However, sensitive isolates showed lower expression than those with reduced fludioxonil sensitivity. Remarkably, a mutant version of the transcription factor Mrr1 carrying 21 amino acid modifications was identified in all isolates with reduced sensitivity to fludioxonil. These changes alter the protein's transcription factor domain and the C-terminal portion of the protein but not the Zn (2)-C6 fungal-type DNA-binding domain. These results suggest a direct relationship between the conserved and divergent mutant version of and sensitivity to fludioxonil. This study provides a new target for developing molecular diagnostic strategies to monitor 's sensitivity to fludioxonil in the field.
PubMed: 38787226
DOI: 10.3390/pathogens13050374 -
Journal of Microbiology and... Jun 2024Protein-specific antibodies are essential for various aspects of protein research, including detection, purification, and characterization. When specific antibodies are...
Protein-specific antibodies are essential for various aspects of protein research, including detection, purification, and characterization. When specific antibodies are unavailable, protein tagging is a useful alternative. Small epitope tags, typically less than 10 amino acids, are widely used in protein research due to the simple modification through PCR and reduced impact on the target protein's function compared to larger tags. The 2B8 epitope tag (RDPLPFFPP), reported by us in a previous study, has high specificity and sensitivity to the corresponding antibody. However, when attached to the C-terminus of the target protein in immunoprecipitation experiments, we observed a decrease in detection signal with reduced immunity and low protein recovery. This phenomenon was not unique to 2B8 and was also observed with the commercially available Myc tag. Our study revealed that C-terminal tagging of small epitope tags requires the addition of more than one extra amino acid to enhance (restore) antibody immunities. Moreover, among the amino acids we tested, serine was the best for the 2B8 tag. Our findings demonstrated that the interaction between a small epitope and a corresponding paratope of an antibody requires an extra amino acid at the C-terminus of the epitope. This result is important for researchers planning studies on target proteins using small epitope tags.
Topics: Epitopes; Amino Acids; Animals; Antibodies; Mice; Immunoprecipitation; Antibody Formation; Recombinant Fusion Proteins
PubMed: 38783697
DOI: 10.4014/jmb.2401.01036 -
The Journal of Headache and Pain May 2024The trigeminal system is key to the pathophysiology of migraine and cluster headache, two primary headache disorders that share many features. Recently, MER...
The trigeminal system is key to the pathophysiology of migraine and cluster headache, two primary headache disorders that share many features. Recently, MER proto-oncogene tyrosine kinase (MERTK), a cell surface receptor, was strongly associated with cluster headache through genetic studies. Further, the MERTK ligand galectin-3 has been found to be elevated in serum of migraine patients. In this study, MERTK and MERTK ligands were investigated in key tissue to better understand their potential implication in the pathophysiology of primary headache disorders. Immunohistochemistry was used to map MERTK and galectin-3 expression in rat trigeminal ganglia. RT-qPCR was used to assess MERTK gene expression in blood, and ELISA immunoassays were used for MERTK ligand quantification in serum from study participants with and without cluster headache. MERTK gene expression was elevated in blood samples from study participants with cluster headache compared to controls. In addition, MERTK ligand galectin-3 was found at increased concentration in the serum of study participants with cluster headache, whereas the levels of MERTK ligands growth arrest specific 6 and protein S unaffected. MERTK and galectin-3 were both expressed in rat trigeminal ganglia. Galectin-3 was primarily localized in smaller neurons and to a lesser extent in C-fibres, while MERTK was found in satellite glia cells and in the outer membrane of Schwann cells. Interestingly, a strong MERTK signal was found specifically in the region proximal to the nodes of Ranvier. The overexpression of MERTK and galectin-3 in tissue from study participants with cluster headache, as well as the presence of MERTK in rat peripheral satellite glia cells and Schwann cells in the trigeminal ganglia, further highlights MERTK signalling as an interesting potential future therapeutic target in primary headache.
Topics: Animals; Cluster Headache; c-Mer Tyrosine Kinase; Trigeminal Ganglion; Humans; Male; Rats; Female; Proto-Oncogene Mas; Adult; Middle Aged; Rats, Sprague-Dawley; Receptor Protein-Tyrosine Kinases; Blood Proteins; Galectins
PubMed: 38783191
DOI: 10.1186/s10194-024-01791-6