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FASEB Journal : Official Publication of... Jul 2024N-glycosylation is the most common protein modification in the eukaryotic secretory pathway. It involves the attachment a high mannose glycan to Asn residues in the...
N-glycosylation is the most common protein modification in the eukaryotic secretory pathway. It involves the attachment a high mannose glycan to Asn residues in the context of Asn-X-Ser/Thr/Cys, a motif known as N-glycosylation sequon. This process is mediated by STT3A and STT3B, the catalytic subunits of the oligosaccharyltransferase complexes. STT3A forms part of complexes associated with the SEC61 translocon and functions co-translationally. Vacant sequons have another opportunity for glycosylation by complexes carrying STT3B. Local sequence information plays an important role in determining N-glycosylation efficiency, but non-local factors can also have a significant impact. For instance, certain proteins associated with human genetic diseases exhibit abnormal N-glycosylation levels despite having wild-type acceptor sites. Here, we investigated the effect of protein stability on this process. To this end, we generated a family of 40 N-glycan acceptors based on superfolder GFP, and we measured their efficiency in HEK293 cells and in two derived cell lines lacking STT3B or STT3A. Sequon occupancy was highly dependent on protein stability, improving as the thermodynamic stability of the acceptor proteins decreases. This effect is mainly due to the activity of the STT3B-based OST complex. These findings can be integrated into a simple kinetic model that distinguishes local information within sequons from global information of the acceptor proteins.
Topics: Humans; Glycosylation; HEK293 Cells; Protein Processing, Post-Translational; Hexosyltransferases; Membrane Proteins; Protein Stability; Polysaccharides
PubMed: 38934375
DOI: 10.1096/fj.202302267R -
Heliyon Jun 2024This study sheds light on a ground-breaking biochemical mechanotransduction pathway and reveals how Piezo1 channels orchestrate cell migration. We observed an increased...
This study sheds light on a ground-breaking biochemical mechanotransduction pathway and reveals how Piezo1 channels orchestrate cell migration. We observed an increased cell migration rate in HEK293T (HEK) cells treated with Yoda1, a Piezo1 agonist, or in HEK cells overexpressing Piezo1 (HEK + P). Conversely, a significant reduction in cell motility was observed in HEK cells treated with GsMTx4 (a channel inhibitor) or upon silencing Piezo1 (HEK-P). Our findings establish a direct correlation between alterations in cell motility, Piezo1 expression, abnormal F-actin microfilament dynamics, and the regulation of Cofilin1, a protein involved in severing F-actin microfilaments. Here, the conversion of inactive pCofilin1 to active Cofilin1, mediated by the serine/threonine-protein phosphatase 2A catalytic subunit C (PP2AC), resulted in increased severing of F-actin microfilaments and enhanced cell migration in HEK + P cells compared to HEK controls. However, this effect was negligible in HEK-P and HEK cells transfected with hsa-miR-133b, which post-transcriptionally inhibited PP2AC mRNA expression. In summary, our study suggests that Piezo1 regulates cell migration through a biochemical mechanotransduction pathway involving PP2AC-mediated Cofilin1 dephosphorylation, leading to changes in F-actin microfilament dynamics.
PubMed: 38933959
DOI: 10.1016/j.heliyon.2024.e32458 -
Frontiers in Veterinary Science 2024Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that targets pig intestines to cause disease. It is globally widespread and causes huge economic...
Nucleotide metabolism-related host proteins RNA polymerase II subunit and uridine phosphorylase 1 interacting with porcine epidemic diarrhea virus N proteins affect viral replication.
Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that targets pig intestines to cause disease. It is globally widespread and causes huge economic losses to the pig industry. PEDV N protein is the protein that constitutes the core of PEDV virus particles, and most of it is expressed in the cytoplasm, and a small part can also be expressed in the nucleus. However, the role of related proteins in host nucleotide metabolic pathways in regulating PEDV replication have not been fully elucidated. In this study, PEDV-N-labeled antibodies were co-immunoprecipitated and combined with LC-MS to screen for host proteins that interact with N proteins. Bioinformatics analyses showed that the selected host proteins were mainly enriched in metabolic pathways. Moreover, co-immunoprecipitation and confocal microscopy confirmed that the second-largest subunit of RNA polymerase II (RPB2) and uridine phosphorylase 1 (UPP1) interacted with the N protein. RPB2 is the main subunit of RNA polymerase II and plays an important role in eukaryotic transcription. UPP1 is an enzyme that catalyzes reversible phosphorylation of uridine to uracil and ribo-1-phosphate to promote catabolism and bio anabolism. RPB2 overexpression significantly promoted viral replication, whereas UPP1 overexpression significantly inhibited viral replication. Studies on interactions between the PEDV N and host proteins are helpful in elucidating the pathogenesis and immune escape mechanism of PEDV.
PubMed: 38933700
DOI: 10.3389/fvets.2024.1417348 -
Nucleic Acids Research Jun 2024The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ...
The replicative mitochondrial DNA polymerase, Polγ, and its protein regulation are essential for the integrity of the mitochondrial genome. The intricacies of Polγ regulation and its interactions with regulatory proteins, which are essential for fine-tuning polymerase function, remain poorly understood. Misregulation of the Polγ heterotrimer, consisting of (i) PolG, the polymerase catalytic subunit and (ii) PolG2, the accessory subunit, ultimately results in mitochondrial diseases. Here, we used single particle cryo-electron microscopy to resolve the structure of PolG in its apoprotein state and we captured Polγ at three intermediates within the catalytic cycle: DNA bound, engaged, and an active polymerization state. Chemical crosslinking mass spectrometry, and site-directed mutagenesis uncovered the region of LonP1 engagement of PolG, which promoted proteolysis and regulation of PolG protein levels. PolG2 clinical variants, which disrupted a stable Polγ complex, led to enhanced LonP1-mediated PolG degradation. Overall, this insight into Polγ aids in an understanding of mitochondrial DNA replication and characterizes how machinery of the replication fork may be targeted for proteolytic degradation when improperly functioning.
PubMed: 38932681
DOI: 10.1093/nar/gkae539 -
Journal of Medical Virology Jun 2024COVID-19 tended to be less aggressive in dengue endemic regions. Conversely, dengue cases plummeted in dengue endemic zones during the active years of the pandemic...
COVID-19 tended to be less aggressive in dengue endemic regions. Conversely, dengue cases plummeted in dengue endemic zones during the active years of the pandemic (2020-2021). We and others have demonstrated serological cross-reactivity between these two viruses of different families. We further demonstrated that COVID-19 serum samples that were cross-reactive in dengue virus (DV) serological tests, "cross-neutralized" all DV serotypes in Huh7 cells. Here we showed by co-immunoprecipitation (Co-IP) and atomic force microscopy (AFM) imaging that severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 (SARS-CoV-2) spike (S) protein subunit S1 and S2 monoclonal antibodies can indeed, bind to DV particles. Likewise, DV envelope antibodies (DV E Abs) showed high docking frequency with other human pathogenic beta-CoVs and murine hepatitis virus-1 (MHV-1). SARS-CoV-2 Ab didn't show docking or Co-IP with MHV-1 supporting poor cross-protection among CoVs. DV E Abs showed binding to MHV-1 (AFM, Co-IP, and immunofluorescence) and prepandemic dengue patients' serum samples even "cross-neutralized" MHV-1 plaques in cell culture. Furthermore, dengue serum samples showed marked inhibition potential in a surrogate virus-based competitive enzyme-linked immunosorbent assay, used for determining neutralizing Abs against SARS-CoV-2 S protein receptor-binding domain in COVID-19 serum samples. We therefore, provide multiple evidence as to why CoVs are epidemiologically less prevalent in highly dengue endemic regions globally.
Topics: Dengue Virus; Humans; Dengue; Cross Reactions; Antibodies, Viral; SARS-CoV-2; Spike Glycoprotein, Coronavirus; COVID-19; Antibodies, Neutralizing; Animals; Antibodies, Monoclonal; Endemic Diseases
PubMed: 38932494
DOI: 10.1002/jmv.29771 -
Vaccines Jun 2024Serovar Typhi Ty21a (Ty21a) is the only licensed oral vaccine against typhoid fever. Due to its excellent safety profile, it has been used as a promising vector strain...
Serovar Typhi Ty21a (Ty21a) is the only licensed oral vaccine against typhoid fever. Due to its excellent safety profile, it has been used as a promising vector strain for the expression of heterologous antigens for mucosal immunization. As the efficacy of any bacterial live vector vaccine correlates with its ability to express and present sufficient antigen, the genes for antigen expression are traditionally located on plasmids with antibiotic resistance genes for stabilization. However, for use in humans, antibiotic selection of plasmids is not applicable, leading to segregational loss of the antigen-producing plasmid. Therefore, we developed an oral Ty21a-based vaccine platform technology, the JMU-SalVac-system (Julius-Maximilians-Universität Würzburg) in which the antigen delivery plasmids (pSalVac-plasmid-series) are stabilized by a Δ/-based balanced-lethal system (BLS). The system is made up of the chromosomal knockout of the essential tyrosyl-tRNA-synthetase gene () and the in trans complementation of on the pSalVac-plasmid. Further novel functional features of the pSalVac-plasmids are the presence of two different expression cassettes for the expression of protein antigens. In this study, we present the construction of vaccine strains with BLS plasmids for antigen expression. The expression of cytosolic and secreted mRFP and cholera toxin subunit B (CTB) proteins as model antigens is used to demonstrate the versatility of the approach. As proof of concept, we show the induction of previously described in vivo inducible promoters cloned into pSalVac-plasmids during infection of primary macrophages and demonstrate the expression of model vaccine antigens in these relevant human target cells. Therefore, antigen delivery strains developed with the JMU-SalVac technology are promising, safe and stable vaccine strains to be used against mucosal infections in humans.
PubMed: 38932416
DOI: 10.3390/vaccines12060687 -
Vaccines Jun 2024Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of infants and older people. There is an urgent need for safe and...
Immune Responses and Protection Profiles in Mice Induced by Subunit Vaccine Candidates Based on the Extracellular Domain Antigen of Respiratory Syncytial Virus G Protein Combined with Different Adjuvants.
Respiratory syncytial virus (RSV) is a leading cause of severe lower respiratory tract disease of infants and older people. There is an urgent need for safe and effective vaccines against RSV infection. In this study, we analyzed the effects of the immune response and protection with the RSV recombinant G protein extracellular domain (G) combined with various adjuvants as novel subunit vaccines in mice. All groups receiving RSV G combined with adjuvants exhibited robust humoral and cellular immunity compared to those receiving an adjuvant alone or inactivated RSV vaccine. The greatest effect was observed in mice receiving G combined with a CpG ODN + Alum salt adjuvant, resulting in the highest production of neutralizing antibodies against both RSV A and B subtypes, G-specific IgG and IFN-γ production in splenocytes, and interleukin-2 and interferon-γ expression in CD4 T cells. Significant humoral and cellular immune responses were observed in mice immunized with G combined with AddaS03™ or cyclosporin A adjuvants. The vaccine containing the AddaS03™ adjuvant showed significantly high expression of interleukin-4 in CD4 T cells. Cross-protection against a challenge with either RSV A or B subtypes was observed in the G plus adjuvant groups, resulting in a significant decrease in viral load and reduced pathological damage in the mouse lungs. These findings offer valuable insights into the development and application of recombinant RSV G-subunit vaccines with adjuvants.
PubMed: 38932414
DOI: 10.3390/vaccines12060686 -
Vaccines Jun 2024spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal-oral route, causing significant morbidity and mortality, especially among...
spp. are responsible for bacillary dysentery or shigellosis transmitted via the fecal-oral route, causing significant morbidity and mortality, especially among vulnerable populations. There are currently no licensed vaccines. spp. use a type III secretion system (T3SS) to invade host cells. We have shown that L-DBF, a recombinant fusion of the T3SS needle tip (IpaD) and translocator (IpaB) proteins with the LTA1 subunit of enterotoxigenic labile toxin, is broadly protective against spp. challenge in a mouse lethal pulmonary model. Here, we assessed the effect of LDBF, formulated with a unique TLR4 agonist called BECC470 in an oil-in-water emulsion (ME), on the murine immune response in a high-risk population (young and elderly) in response to challenge. Dual RNA Sequencing captured the transcriptome during infection in vaccinated and unvaccinated mice. Both age groups were protected by the L-DBF formulation, while younger vaccinated mice exhibited more adaptive immune response gene patterns. This preliminary study provides a step toward identifying the gene expression patterns and regulatory pathways responsible for a protective immune response against . Furthermore, this study provides a measure of the challenges that need to be addressed when immunizing an aging population.
PubMed: 38932347
DOI: 10.3390/vaccines12060618 -
Viruses Jun 2024Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant threat to the global swine industry. The development of highly effective subunit...
Porcine reproductive and respiratory syndrome virus (PRRSV) presents a significant threat to the global swine industry. The development of highly effective subunit nanovaccines is a promising strategy for preventing PRRSV variant infections. In this study, two different types of ferritin (Ft) nanovaccines targeting the major glycoprotein GP5, named GP5m-Ft and (Bp-IVp)-Ft, were constructed and evaluated as vaccine candidates for PRRSV. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) demonstrated that both purified GP5m-Ft and (Bp-IVp)-Ft proteins could self-assemble into nanospheres. A comparison of the immunogenicity of GP5m-Ft and (Bp-IVp)-Ft with an inactivated PRRSV vaccine in BALB/c mice revealed that mice immunized with GP5m-Ft exhibited the highest ELISA antibody levels, neutralizing antibody titers, the lymphocyte proliferation index, and IFN-γ levels. Furthermore, vaccination with the GP5m-Ft nanoparticle effectively protected piglets against a highly pathogenic PRRSV challenge. These findings suggest that GP5m-Ft is a promising vaccine candidate for controlling PRRS.
Topics: Animals; Porcine respiratory and reproductive syndrome virus; Ferritins; Mice, Inbred BALB C; Swine; Mice; Antibodies, Viral; Antibodies, Neutralizing; Nanoparticles; Porcine Reproductive and Respiratory Syndrome; Viral Vaccines; Viral Envelope Proteins; Female; Interferon-gamma; Nanovaccines
PubMed: 38932282
DOI: 10.3390/v16060991 -
Viruses Jun 2024Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed,... (Review)
Review
Vaccines are one of the most effective medical interventions, playing a pivotal role in treating infectious diseases. Although traditional vaccines comprise killed, inactivated, or live-attenuated pathogens that have resulted in protective immune responses, the negative consequences of their administration have been well appreciated. Modern vaccines have evolved to contain purified antigenic subunits, epitopes, or antigen-encoding mRNAs, rendering them relatively safe. However, reduced humoral and cellular responses pose major challenges to these subunit vaccines. Protein nanoparticle (PNP)-based vaccines have garnered substantial interest in recent years for their ability to present a repetitive array of antigens for improving immunogenicity and enhancing protective responses. Discovery and characterisation of naturally occurring PNPs from various living organisms such as bacteria, archaea, viruses, insects, and eukaryotes, as well as computationally designed structures and approaches to link antigens to the PNPs, have paved the way for unprecedented advances in the field of vaccine technology. In this review, we focus on some of the widely used naturally occurring and optimally designed PNPs for their suitability as promising vaccine platforms for displaying native-like antigens from human viral pathogens for protective immune responses. Such platforms hold great promise in combating emerging and re-emerging infectious viral diseases and enhancing vaccine efficacy and safety.
Topics: Humans; Nanoparticles; Animals; Viral Vaccines; Virus Diseases; Viruses; Antigens, Viral; Vaccines, Subunit
PubMed: 38932228
DOI: 10.3390/v16060936