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PLoS Pathogens Jun 2024Coronavirus (CoV) nonstructural protein 1 (nsp1) is considered a pathogenic factor due to its ability to inhibit host antiviral responses by inducing general shutoff of...
Coronavirus (CoV) nonstructural protein 1 (nsp1) is considered a pathogenic factor due to its ability to inhibit host antiviral responses by inducing general shutoff of host protein synthesis. Nsp1 is expressed by α- and β-CoVs, but its functions and strategies to induce host shutoff are not fully elucidated. We compared the nsp1s from two β-CoVs (SARS-CoV and SARS-CoV-2) and two α-CoVs (NL63 and 229E) and found that NL63 nsp1 has the strongest shutoff activity. Unlike SARS-CoV nsp1s, which bind to 40S ribosomes and block translation of cellular mRNA, NL63 nsp1 did not inhibit translation of mRNAs transfected into cells. Instead, NL63 nsp1 localized to the nucleus and specifically inhibited transcription of genes under an RNA polymerase II (RNAPII) promoter. Further analysis revealed that NL63 nsp1 induces degradation of the largest subunit of RNAPII, Rpb1. This degradation was detected regardless of the phosphorylation state of Rpb1 and was blocked by the proteasome inhibitor MG132. We also found that Rpb1 was ubiquitinated in NL63-infected cells, and inhibition of ubiquitination by a ubiquitin activating enzyme inhibitor (TAK243) prevented degradation of Rpb1 in virus-infected cells. These data reveal an unrecognized strategy of host shutoff by human α-CoV NL63: targeting host transcription by inducing Rpb1 degradation to prevent host protein expression. Our study indicates that viruses within the same family can use completely distinct mechanisms to regulate host antiviral responses.
PubMed: 38900816
DOI: 10.1371/journal.ppat.1012329 -
Biological & Pharmaceutical Bulletin 2024Microglia, resident immune cells in the central nervous system (CNS), play a critical role in maintaining CNS homeostasis. However, microglia activated in response to...
Microglia, resident immune cells in the central nervous system (CNS), play a critical role in maintaining CNS homeostasis. However, microglia activated in response to brain injury produce various inflammatory mediators, including nitric oxide (NO) and proinflammatory cytokines, leading to considerable neuronal damage. NO generated by inducible NO synthase (iNOS) rapidly reacts with superoxide to form a highly toxic product, peroxynitrite. Therefore, iNOS is considered to be a putative therapeutic target for cerebral ischemia. Here, we examined the effects of panobinostat (Pano), a histone deacetylase inhibitor, on lipopolysaccharide (LPS)-induced iNOS expression using rat immortalized microglia HAPI cells. Pano inhibited LPS-induced expression of iNOS mRNA and NO production in a dose-dependent manner; however, it had little effect on the LPS-induced activation of c-Jun N-terminal kinase (JNK) and p38 or nuclear translocation of nuclear factor-κB (NF-κB). The interferon-β (IFN-β)/signal transducer and activator of transcription (STAT) pathway is essential for LPS-induced iNOS expression in macrophages/microglia. We also examined the effects of Pano on LPS-induced IFN-β signaling. Pano markedly inhibited LPS-induced IFN-β expression and subsequent tyrosine phosphorylation of STAT1. However, the addition of IFN-β restored the decreased STAT1 phosphorylation but not the decreased iNOS expression. In addition, Pano inhibited the LPS-increased expression of octamer binding protein-2 and interferon regulatory factor 9 responsible for iNOS expression, but IFN-β addition also failed to restore the decreased expression of these factors. Thus, we conclude that the inhibitory effects of Pano are due not only to the inhibition of the IFN-β/STAT axis but also to the downregulation of other factors not involved in this axis.
Topics: Animals; Microglia; Lipopolysaccharides; Nitric Oxide Synthase Type II; Histone Deacetylase Inhibitors; Rats; Panobinostat; Nitric Oxide; NF-kappa B; Cell Line; Interferon-beta; STAT1 Transcription Factor; Signal Transduction
PubMed: 38897970
DOI: 10.1248/bpb.b24-00111 -
Applied Microbiology and Biotechnology Jun 2024Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to...
Chinese hamster ovary (CHO) cells are popular in the pharmaceutical industry for their ability to produce high concentrations of antibodies and their resemblance to human cells in terms of protein glycosylation patterns. Current data indicate the relevance of CHO cells in the biopharmaceutical industry, with a high number of product commendations and a significant market share for monoclonal antibodies. To enhance the production capabilities of CHO cells, a deep understanding of their cellular and molecular composition is crucial. Genome sequencing and proteomic analysis have provided valuable insights into the impact of the bioprocessing conditions, productivity, and product quality. In our investigation, we conducted a comparative analysis of proteomic profiles in high and low monoclonal antibody-producing cell lines and studied the impact of tunicamycin (TM)-induced endoplasmic reticulum (ER) stress. We examined the expression levels of different proteins including unfolded protein response (UPR) target genes by using label-free quantification techniques for protein abundance. Our results show the upregulation of proteins associated with protein folding mechanisms in low producer vs. high producer cell line suggesting a form of ER stress related to specific protein production. Further, Hspa9 and Dnaja3 are notable candidates activated by the mitochondria UPR and play important roles in protein folding processes in mitochondria. We identified significant upregulation of Nedd8 and Lgmn proteins in similar levels which may contribute to UPR stress. Interestingly, the downregulation of Hspa5/Bip and Pdia4 in response to tunicamycin treatment suggests a low-level UPR activation. KEY POINTS: • Proteome profiling of recombinant CHO cells under mild TM treatment. • Identified protein clusters are associated with the unfolded protein response (UPR). • The compared cell lines revealed noticeable disparities in protein expression levels.
Topics: CHO Cells; Cricetulus; Tunicamycin; Animals; Antibodies, Monoclonal; Proteomics; Endoplasmic Reticulum Stress; Unfolded Protein Response; Proteome; Cricetinae
PubMed: 38896138
DOI: 10.1007/s00253-024-13223-1 -
BioRxiv : the Preprint Server For... Jun 2024An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two...
An alternative to lifelong antiretroviral therapy (ART) is needed to achieve durable control of HIV-1. Here we show that adeno-associated virus (AAV)-delivery of two rhesus macaque antibodies to the SIV envelope glycoprotein (Env) with potent neutralization and antibody-dependent cellular cytotoxicity can prevent viral rebound in macaques infected with barcoded SIV239M after discontinuing suppressive ART. Following AAV administration, sustained antibody expression with minimal anti-drug antibody responses was achieved in all but one animal. After ART withdrawal, SIV replication rebounded within two weeks in all of the control animals but remained below the threshold of detection in plasma (<15 copies/mL) for more than a year in four of the eight animals that received AAV vectors encoding Env-specific antibodies. Viral sequences from animals with delayed rebound exhibited restricted barcode diversity and antibody escape. Thus, sustained expression of antibodies with potent antiviral activity can afford durable, ART-free containment of pathogenic SIV infection.
PubMed: 38895320
DOI: 10.1101/2024.05.30.593694 -
BioRxiv : the Preprint Server For... Jun 2024Occludin (ocln) is one of the main regulatory cells of the blood-brain barrier (BBB). Ocln silencing resulted in alterations of the gene expression signatures of a...
Occludin (ocln) is one of the main regulatory cells of the blood-brain barrier (BBB). Ocln silencing resulted in alterations of the gene expression signatures of a variety of genes of the innate immunity system, including IFN-stimulated genes (ISGs) and the antiviral retinoic acid-inducible gene-1 (RIG-1) signaling pathway, which functions as a regulator of the cytoplasmic sensors upstream of the mitochondrial antiviral signaling protein (MAVS). Indeed, we observed dysfunctional mitochondrial bioenergetics, dynamics, and autophagy in our system. Alterations of mitochondrial bioenergetics and innate immune protection translated into worsened ischemic stroke outcomes in EcoHIV-infected ocln deficient mice. Overall, these results allow for a better understanding of the molecular mechanisms of viral infection in the brain and describe a previously unrecognized role of ocln as a key factor in the control of innate immune responses and mitochondrial dynamics, which affect cerebral vascular diseases such as ischemic stroke.
PubMed: 38895303
DOI: 10.1101/2024.06.07.598027 -
Molecules (Basel, Switzerland) Jun 2024COVID-19 continues to spread around the world. This is mainly because new variants of the SARS-CoV-2 virus emerge due to genomic mutations, evade the immune system and...
COVID-19 continues to spread around the world. This is mainly because new variants of the SARS-CoV-2 virus emerge due to genomic mutations, evade the immune system and result in the effectiveness of current therapeutics being reduced. We previously established a series of detection platforms, comprising computational docking analysis, S-protein-based ELISA, pseudovirus entry, and 3CL protease activity assays, which allow us to screen a large library of phytochemicals from natural products and to determine their potential in blocking the entry of SARS-CoV-2. In this new screen, rutaecarpine (an alkaloid from ) was identified as exhibiting anti-SARS-CoV-2 activity. Therefore, we conducted multiple rounds of structure-activity-relationship (SAR) studies around this phytochemical and generated several rutaecarpine analogs that were subjected to in vitro evaluations. Among these derivatives, RU-75 and RU-184 displayed remarkable inhibitory activity when tested in the 3CL protease assay, S-protein-based ELISA, and pseudovirus entry assay (for both wild-type and omicron variants), and they attenuated the inflammatory response induced by SARS-CoV-2. Interestingly, RU-75 and RU-184 both appeared to be more potent than rutaecarpine itself, and this suggests that they might be considered as lead candidates for future pharmacological elaboration.
Topics: Indole Alkaloids; SARS-CoV-2; Molecular Docking Simulation; Quinazolines; Humans; Antiviral Agents; Drug Design; Structure-Activity Relationship; COVID-19 Drug Treatment; Coronavirus 3C Proteases; Virus Internalization; Quinazolinones
PubMed: 38893512
DOI: 10.3390/molecules29112636 -
International Journal of Molecular... Jun 2024() is the causative agent of bovine tuberculosis (bTb). Genetic selection aiming to identify less susceptible animals has been proposed as a complementary measure in...
Genome-Wide Association Study Reveals Quantitative Trait Loci and Candidate Genes Associated with High Interferon-gamma Production in Holstein Cattle Naturally Infected with .
() is the causative agent of bovine tuberculosis (bTb). Genetic selection aiming to identify less susceptible animals has been proposed as a complementary measure in ongoing programs toward controlling infection. However, individual animal phenotypes for bTb based on interferon-gamma (IFNɣ) and its use in bovine selective breeding programs have not been explored. In the current study, IFNɣ production was measured using a specific IFNɣ ELISA kit in bovine purified protein derivative (bPPD)-stimulated blood samples collected from Holstein cattle. DNA isolated from the peripheral blood samples collected from the animals included in the study was genotyped with the EuroG Medium Density bead Chip, and the genotypes were imputed to whole-genome sequences. A genome-wide association analysis (GWAS) revealed that the IFNɣ in response to bPPD was associated with a specific genetic profile (heritability = 0.23) and allowed the identification of 163 SNPs, 72 quantitative trait loci (QTLs), 197 candidate genes, and 8 microRNAs (miRNAs) associated with this phenotype. No negative correlations between this phenotype and other phenotypes and traits included in the Spanish breeding program were observed. Taken together, our results define a heritable and distinct immunogenetic profile associated with strong production of IFNɣ in response to .
Topics: Animals; Cattle; Quantitative Trait Loci; Mycobacterium bovis; Genome-Wide Association Study; Interferon-gamma; Tuberculosis, Bovine; Polymorphism, Single Nucleotide; Phenotype; Genotype
PubMed: 38892353
DOI: 10.3390/ijms25116165 -
International Journal of Molecular... Jun 2024Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, with an increasing number of targeted therapies available. While biologics to treat...
Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases, with an increasing number of targeted therapies available. While biologics to treat AD exclusively target the key cytokines of type 2 immunity, Janus kinase inhibitors target a broad variety of cytokines, including IFN-γ. To better stratify patients for optimal treatment outcomes, the identification and characterization of subgroups, especially with regard to their IFNG expression, is of great relevance, as the role of IFNG in AD has not yet been fully clarified. This study aims to define AD subgroups based on their lesional IFNG expression and to characterize them based on their gene expression, T cell secretome and clinical attributes. RNA from the lesional and non-lesional biopsies of 48 AD patients was analyzed by RNA sequencing. Based on IFNG gene expression and the release of IFN-γ by lesional T cells, this cohort was categorized into three IFNG groups (high, medium, and low) using unsupervised clustering. The low IFNG group showed features of extrinsic AD with a higher prevalence of atopic comorbidities and impaired epidermal lipid synthesis. In contrast, patients in the high IFNG group had a higher average age and an activation of additional pro-inflammatory pathways. On the cellular level, higher amounts of M1 macrophages and natural killer cell signaling were detected in the high IFNG group compared to the low IFNG group by a deconvolution algorithm. However, both groups shared a common dupilumab response gene signature, indicating that type 2 immunity is the dominant immune shift in both subgroups. In summary, high and low IFNG subgroups correspond to intrinsic and extrinsic AD classifications and might be considered in the future for evaluating therapeutic efficacy or non-responders.
Topics: Dermatitis, Atopic; Humans; Interferon-gamma; Female; Male; Adult; Middle Aged; Antibodies, Monoclonal, Humanized; Macrophages; T-Lymphocytes; Killer Cells, Natural
PubMed: 38892346
DOI: 10.3390/ijms25116158 -
International Journal of Molecular... May 2024N-acetylcysteine (NAC) is a mucolytic agent with antioxidant and anti-inflammatory properties. The respiratory syncytial virus (RSV) is one of the most important... (Review)
Review
N-acetylcysteine (NAC) is a mucolytic agent with antioxidant and anti-inflammatory properties. The respiratory syncytial virus (RSV) is one of the most important etiological factors of lower respiratory tract infections, and exposure to air pollution appears to be additionally associated with higher RSV incidence and disease severity. We aimed to systematically review the existing literature to determine which molecular mechanisms mediate the effects of NAC in an RSV infection and air pollution, and to identify the knowledge gaps in this field. A search for original studies was carried out in three databases and a calibrated extraction grid was used to extract data on the NAC treatment (dose, timing), the air pollutant type, and the most significant mechanisms. We identified only 28 studies conducted in human cellular models ( = 18), animal models ( = 7), and mixed models ( = 3). NAC treatment improves the barrier function of the epithelium damaged by RSV and air pollution, and reduces the epithelial permeability, protecting against viral entry. NAC may also block RSV-activated phosphorylation of the epidermal growth factor receptor (EGFR), which promotes endocytosis and facilitates cell entry. EGFR also enhances the release of a mucin gene, MUC5AC, which increases mucus viscosity and causes goblet cell metaplasia; the effects are abrogated by NAC. NAC blocks virus release from the infected cells, attenuates the cigarette smoke-induced shift from necrosis to apoptosis, and reverses the block in IFN-γ-induced antiviral gene expression caused by the inhibited Stat1 phosphorylation. Increased synthesis of pro-inflammatory cytokines and chemokines is induced by both RSV and air pollutants and is mediated by the nuclear factor kappa-B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways that are activated in response to oxidative stress. MCP-1 (monocyte chemoattractant protein-1) and RANTES (regulated upon activation, expressed and secreted by normal T cells) partially mediate airway hyperresponsiveness (AHR), and therapeutic (but not preventive) NAC administration reduces the inflammatory response and has been shown to reduce ozone-induced AHR. Oxidative stress-induced DNA damage and cellular senescence, observed during RSV infection and exposure to air pollution, can be partially reversed by NAC administration, while data on the emphysema formation are disputed. The review identified potential common molecular mechanisms of interest that are affected by NAC and may alleviate both the RSV infection and the effects of air pollution. Data are limited and gaps in knowledge include the optimal timing or dosage of NAC administration, therefore future studies should clarify these uncertainties and verify its practical use.
Topics: Humans; Acetylcysteine; Respiratory Syncytial Virus Infections; Animals; Air Pollution; ErbB Receptors
PubMed: 38892239
DOI: 10.3390/ijms25116051 -
International Journal of Molecular... May 2024Microglia-mediated inflammatory response is one key cause of many central nervous system diseases, like Alzheimer's disease. We hypothesized that a novel C15orf39 (MAPK1...
Microglia-mediated inflammatory response is one key cause of many central nervous system diseases, like Alzheimer's disease. We hypothesized that a novel C15orf39 (MAPK1 substrate) plays a critical role in the microglial inflammatory response. To confirm this hypothesis, we used lipopolysaccharide (LPS)-and interferon-gamma (IFN-γ)-induced human microglia HMC3 cells as a representative indicator of the microglial in vitro inflammatory response. We found that C15orf39 was down-regulated when interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) expression increased in LPS/IFN-γ-stimulated HMC3 cells. Once C15orf39 was overexpressed, IL-6 and TNFα expression were reduced in LPS/IFN-γ-stimulated HMC3 cells. In contrast, C15orf39 knockdown promoted IL-6 and TNFα expression in LPS/IFN-γ-stimulated HMC3 cells. These results suggest that C15orf39 is a suppressive factor in the microglial inflammatory response. Mechanistically, C15orf39 interacts with the cytoplasmic protein arginine methyltransferase 2 (PRMT2). Thus, we termed C15orf39 a PRMT2 interaction protein (PRMT2 IP). Furthermore, the interaction of C15orf39 and PRMT2 suppressed the activation of NF-κB signaling via the PRMT2-IκBα signaling axis, which then led to a reduction in transcription of the inflammatory factors IL6 and TNF-α. Under inflammatory conditions, NF-κBp65 was found to be activated and to suppress C15orf39 promoter activation, after which it canceled the suppressive effect of the C15orf39-PRMT2-IκBα signaling axis on IL-6 and TNFα transcriptional expression. In conclusion, our findings demonstrate that in a steady condition, the interaction of C15orf39 and PRMT2 stabilizes IκBα to inhibit IL-6 and TNFα expression by suppressing NF-κB signaling, which reversely suppresses C15orf39 transcription to enhance IL-6 and TNFα expression in the microglial inflammatory condition. Our study provides a clue as to the role of C15orf39 in microglia-mediated inflammation, suggesting the potential therapeutic efficacy of C15orf39 in some central nervous system diseases.
Topics: Humans; Microglia; Protein-Arginine N-Methyltransferases; Lipopolysaccharides; Inflammation; Cell Line; Interleukin-6; Tumor Necrosis Factor-alpha; Interferon-gamma; Signal Transduction; NF-kappa B
PubMed: 38892217
DOI: 10.3390/ijms25116025