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Science Advances Oct 2023Interleukins are secreted proteins that regulate immune responses. Among these, the interleukin 12 (IL-12) family holds a central position in inflammatory and infectious...
Interleukins are secreted proteins that regulate immune responses. Among these, the interleukin 12 (IL-12) family holds a central position in inflammatory and infectious diseases. Each family member consists of an α and a β subunit that together form a composite cytokine. Within the IL-12 family, IL-35 remains particularly ill-characterized on a molecular level despite its key role in autoimmune diseases and cancer. Here we show that both IL-35 subunits, IL-12α and EBI3, mutually promote their secretion from cells but are not necessarily secreted as a heterodimer. Our data demonstrate that IL-12α and EBI3 are stable proteins in isolation that act as anti-inflammatory molecules. Both reduce secretion of proinflammatory cytokines and induce the development of regulatory T cells. Together, our study reveals IL-12α and EBI3, the subunits of IL-35, to be functionally active anti-inflammatory immune molecules on their own. This extends our understanding of the human cytokine repertoire as a basis for immunotherapeutic approaches.
Topics: Humans; Cytokines; Interleukin-12; Interleukins; Minor Histocompatibility Antigens; T-Lymphocytes, Regulatory
PubMed: 37878703
DOI: 10.1126/sciadv.adg6874 -
The EMBO Journal Sep 2023Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby...
Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby exposing to the cytosol intraluminal, otherwise shielded danger signals such as glycans and sphingomyelin. The detection of glycans by galectin-8 triggers anti-bacterial autophagy, but how cells sense and respond to cytosolically exposed sphingomyelin remains unknown. Here, we identify TECPR1 (tectonin beta-propeller repeat containing 1) as a receptor for cytosolically exposed sphingomyelin, which recruits ATG5 into an E3 ligase complex that mediates lipid conjugation of LC3 independently of ATG16L1. TECPR1 binds sphingomyelin through its N-terminal DysF domain (N'DysF), a feature not shared by other mammalian DysF domains. Solving the crystal structure of N'DysF, we identified key residues required for the interaction, including a solvent-exposed tryptophan (W154) essential for binding to sphingomyelin-positive membranes and the conjugation of LC3 to lipids. Specificity of the ATG5/ATG12-E3 ligase responsible for the conjugation of LC3 is therefore conferred by interchangeable receptor subunits, that is, the canonical ATG16L1 and the sphingomyelin-specific TECPR1, in an arrangement reminiscent of certain multi-subunit ubiquitin E3 ligases.
Topics: Animals; Sphingomyelins; Microtubule-Associated Proteins; Autophagy-Related Proteins; Carrier Proteins; Autophagy; Ubiquitin-Protein Ligases; Autophagy-Related Protein 5; Mammals
PubMed: 37409490
DOI: 10.15252/embj.2022113012 -
PLoS Pathogens Dec 2023Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the...
Protein phosphatases are post-translational regulators of Toxoplasma gondii proliferation, tachyzoite-bradyzoite differentiation and pathogenesis. Here, we identify the putative protein phosphatase 6 (TgPP6) subunits of T. gondii and elucidate their role in the parasite lytic cycle. The putative catalytic subunit TgPP6C and regulatory subunit TgPP6R likely form a complex whereas the predicted structural subunit TgPP6S, with low homology to the human PP6 structural subunit, does not coassemble with TgPP6C and TgPP6R. Functional studies showed that TgPP6C and TgPP6R are essential for parasite growth and replication. The ablation of TgPP6C significantly reduced the synchronous division of the parasite's daughter cells during endodyogeny, resulting in disordered rosettes. Moreover, the six conserved motifs of TgPP6C were required for efficient endodyogeny. Phosphoproteomic analysis revealed that ablation of TgPP6C predominately altered the phosphorylation status of proteins involved in the regulation of the parasite cell cycle. Deletion of TgPP6C significantly attenuated the parasite virulence in mice. Immunization of mice with TgPP6C-deficient type I RH strain induced protective immunity against challenge with a lethal dose of RH or PYS tachyzoites and Pru cysts. Taken together, the results show that TgPP6C contributes to the cell division, replication and pathogenicity in T. gondii.
Topics: Animals; Humans; Mice; Catalytic Domain; Cell Cycle; Cell Division; Parasites; Protozoan Proteins; Toxoplasma; Virulence; Phosphoprotein Phosphatases
PubMed: 38091362
DOI: 10.1371/journal.ppat.1011831 -
Nature Communications Aug 2023Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for...
Assembly of ribosomes in bacteria is highly efficient, taking ~2-3 min, but this makes the abundance of assembly intermediates very low, which is a challenge for mechanistic understanding. Genetic perturbations of the assembly process create bottlenecks where intermediates accumulate, facilitating structural characterization. We use cryo-electron microscopy, with iterative subclassification to identify intermediates in the assembly of the 50S ribosomal subunit from E. coli. The analysis of the ensemble of intermediates that spans the entire biogenesis pathway for the 50 S subunit was facilitated by a dimensionality reduction and cluster picking approach using PCA-UMAP-HDBSCAN. The identity of the cooperative folding units in the RNA with associated proteins is revealed, and the hierarchy of these units reveals a complete assembly map for all RNA and protein components. The assembly generally proceeds co-transcriptionally, with some flexibility in the landscape to ensure efficiency for this central cellular process under a variety of growth conditions.
Topics: Cryoelectron Microscopy; Escherichia coli; Ribosome Subunits, Large, Bacterial; Bacteria; RNA
PubMed: 37633970
DOI: 10.1038/s41467-023-40859-w -
BioRxiv : the Preprint Server For... Sep 2023Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic (C) subunit of protein kinase A (PKA). Not only this...
Allosteric cooperativity between ATP and substrates is a prominent characteristic of the cAMP-dependent catalytic (C) subunit of protein kinase A (PKA). Not only this long-range synergistic action is involved in substrate recognition and fidelity, but it is likely to regulate PKA association with regulatory subunits and other binding partners. To date, a complete understanding of the molecular determinants for this intramolecular mechanism is still lacking. Here, we used an integrated NMR-restrained molecular dynamics simulations and a Markov Model to characterize the free energy landscape and conformational transitions of the catalytic subunit of protein kinase A (PKA-C). We found that the apo-enzyme populates a broad free energy basin featuring a conformational ensemble of the active state of PKA-C (ground state) and other basins with lower populations (excited states). The first excited state corresponds to a previously characterized inactive state of PKA-C with the αC helix swinging outward. The second excited state displays a disrupted hydrophobic packing around the regulatory (R) spine, with a flipped configuration of the F100 and F102 residues at the tip of the αC-β4 loop. To experimentally validate the second excited state, we mutated F100 into alanine and used NMR spectroscopy to characterize the binding thermodynamics and structural response of ATP and a prototypical peptide substrate. While the activity of PKA-C toward a prototypical peptide substrate is unaltered and the enzyme retains its affinity for ATP and substrate, this mutation rearranges the αC-β4 loop conformation interrupting the allosteric coupling between nucleotide and substrate. The highly conserved αC-β4 loop emerges as a pivotal element able to modulate the synergistic binding between nucleotide and substrate and may affect PKA signalosome. These results may explain how insertion mutations within this motif affect drug sensitivity in other homologous kinases.
PubMed: 37745542
DOI: 10.1101/2023.09.12.557419 -
Frontiers in Cellular and Infection... 2023The pilus is an extracellular structural part that can be detected in some () isolates (type I pili are found in approximately 30% of strains, while type II pili are... (Review)
Review
The pilus is an extracellular structural part that can be detected in some () isolates (type I pili are found in approximately 30% of strains, while type II pili are found in approximately 20%). It is anchored to the cell wall by LPXTG-like motifs on the peptidoglycan. Two kinds of pili have been discovered, namely, pilus-1 and pilus-2. The former is encoded by pilus islet 1 (PI-1) and is a polymer formed by the protein subunits RrgA, RrgB and RrgC. The latter is encoded by pilus islet 2 (PI-2) and is a polymer composed mainly of the structural protein PitB. Although pili are not necessary for the survival of , they serve as the structural basis and as virulence factors that mediate the adhesion of bacteria to host cells and play a direct role in promoting the adhesion, colonization and pathogenesis of . In addition, as candidate antigens for protein vaccines, pili have promising potential for use in vaccines with combined immunization strategies. Given the current understanding of the pili of regarding the genes, proteins, structure, biological function and epidemiological relationship with serotypes, combined with the immunoprotective efficacy of pilins as protein candidates for vaccines, we here systematically describe the research status and prospects of pili and provide new ideas for subsequent vaccine research and development.
Topics: Bacterial Proteins; Streptococcus pneumoniae; Fimbriae, Bacterial; Fimbriae Proteins; Vaccines; Polymers
PubMed: 37799336
DOI: 10.3389/fcimb.2023.1270848 -
Biochemistry Oct 2023Protein production by ribosomes is fundamental to life, and proper assembly of the ribosome is required for protein production. The RNA, which is post-transcriptionally...
Protein production by ribosomes is fundamental to life, and proper assembly of the ribosome is required for protein production. The RNA, which is post-transcriptionally modified, provides the platform for ribosome assembly. Thus, a complete understanding of ribosome assembly requires the determination of the RNA post-transcriptional modifications in all of the ribosome assembly intermediates and on each pathway. There are 26 RNA post-transcriptional modifications in 23S RNA of the mature () large ribosomal subunit. The levels of these modifications have been investigated extensively only for a small number of large subunit intermediates and under a limited number of cellular and environmental conditions. In this study, we determined the level of incorporations of 2-methyl adenosine, 3-methyl pseudouridine, 5-hydroxycytosine, and seven pseudouridines in an early-stage large-subunit assembly intermediate with a sedimentation coefficient of 27S. The 27S intermediate is one of three large subunit intermediates accumulated in cells lacking the DEAD-box RNA helicase DbpA and expressing the helicase inactive R331A DbpA construct. The majority of the investigated modifications are incorporated into the 27S large subunit intermediate to similar levels to those in the mature 50S large subunit, indicating that these early modifications or the enzymes that incorporate them play important roles in the initial events of large subunit ribosome assembly.
Topics: RNA Processing, Post-Transcriptional; Escherichia coli; RNA, Bacterial; Escherichia coli Proteins; RNA, Ribosomal, 23S; Ribosome Subunits, Large, Bacterial; DEAD-box RNA Helicases; Pseudouridine; Ribosomes
PubMed: 37751522
DOI: 10.1021/acs.biochem.3c00291 -
International Journal of Molecular... Jul 2023The changes in epigenetic modifications during early embryonic development significantly impact mammalian embryonic genome activation (EGA) and are species-conserved to...
The changes in epigenetic modifications during early embryonic development significantly impact mammalian embryonic genome activation (EGA) and are species-conserved to some degree. Here, we reanalyzed the published RNA-Seq of human, mouse, and goat early embryos and found that (zinc finger protein 296) expression was higher at the EGA stage than at the oocyte stage in all three species (adjusted -value < 0.05 |log2(foldchange)| ≥ 1). Subsequently, we found that was conserved across human, mouse, goat, sheep, pig, and bovine embryos. In addition, we identified that ZFP296 interacts with the epigenetic regulators KDM5B, SMARCA4, DNMT1, DNMT3B, HP1β, and UHRF1. The Cys-His(C2H2) zinc finger domain TYPE2 TYPE3 domains of ZFP296 co-regulated the modification level of the trimethylation of lysine 9 on the histone H3 protein subunit (H3K9me3). According to ChIP-seq analysis, ZFP296 was also enriched in , , , , and in the mESC genome. Then, knockdown of the expression of at the late zygote of the mouse led to the early developmental arrest of the mouse embryos and failure resulting from a decrease in H3K9me3. Together, our results reveal that is an H3K9me3 modulator which is essential to the embryonic genome activation of mouse embryos.
Topics: Animals; Cattle; Humans; Mice; CCAAT-Enhancer-Binding Proteins; DNA Helicases; Embryonic Development; Histones; Mouse Embryonic Stem Cells; Nuclear Proteins; Sheep; Swine; Transcription Factors; Ubiquitin-Protein Ligases; Zygote; Embryo, Mammalian
PubMed: 37511136
DOI: 10.3390/ijms241411377 -
Cell Chemical Biology Dec 2023An emerging strategy for the therapeutic targeting of protein phosphatases involves the use of compounds that interfere with the binding of regulatory subunits or...
An emerging strategy for the therapeutic targeting of protein phosphatases involves the use of compounds that interfere with the binding of regulatory subunits or substrates. However, high-throughput screening strategies for such interfering molecules are scarce. Here, we report on the conversion of the NanoBiT split-luciferase system into a robust assay for the quantification of phosphatase subunit and substrate interactions in cell lysates. The assay is suitable to screen small-molecule libraries for interfering compounds. We designed and validated split-luciferase sensors for a broad range of PP1 and PP2A holoenzymes, including sensors that selectively report on weak interaction sites. To facilitate efficient hit triaging in large-scale screening campaigns, deselection procedures were developed to eliminate assay-interfering molecules with high fidelity. As a proof-of-principle, we successfully applied the split-luciferase screening tool to identify small-molecule disruptors of the interaction between the C-terminus of PP1β and the ankyrin-repeat domain of the myosin-phosphatase targeting subunit MYPT1.
Topics: Protein Phosphatase 1; Protein Binding; Myosin-Light-Chain Phosphatase; Phosphorylation
PubMed: 37625414
DOI: 10.1016/j.chembiol.2023.07.018 -
Vaccines Sep 2023Developing recombinant proteins as nasal vaccines for inducing systemic and mucosal immunity against respiratory viruses is promising. However, additional adjuvants are...
Developing recombinant proteins as nasal vaccines for inducing systemic and mucosal immunity against respiratory viruses is promising. However, additional adjuvants are required to overcome the low immunogenicity of protein antigens. Here, a self-adjuvanted protein-RNA ribonucleoprotein vaccine was developed and found to be an effective nasal vaccine in mice and the SARS-CoV-2 infection model. The vaccine consisted of spike RBD (as an antigen), nucleoprotein (as an adaptor), and ssRNA (as an adjuvant and RNA scaffold). This combination robustly induced mucosal IgA, neutralizing antibodies and activated multifunctional T-cells, while also providing sterilizing immunity against live virus challenge. In addition, high-resolution scRNA-seq analysis highlighted airway-resident immune cells profile during prime-boost immunization. The vaccine also possesses modularity (antigen/adaptor/RNA scaffold) and can be made to target other viruses. This protein-RNA ribonucleoprotein vaccine is a novel and promising approach for developing safe and potent nasal vaccines to combat respiratory virus infections.
PubMed: 37896953
DOI: 10.3390/vaccines11101550