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Ubiquitously expressed aminoacyl-tRNA synthetases play essential roles in decoding genetic information required for protein synthesis in every living species. Growing evidence suggests that they also function as crossover mediators of multiple biological processes required for homeostasis. In humans, eight cytoplasmic tRNA synthetases form a central machinery called the multi-tRNA synthetase complex (MSC). The formation of MSCs appears to be essential for life, although the role of MSCs remains unclear. Glutamyl-prolyl-tRNA synthetase 1 (EPRS1) is the most evolutionarily derived component within the MSC that plays a critical role in immunity and metabolism (beyond its catalytic role in translation) via stimulus-dependent phosphorylation events. This review focuses on the role of EPRS1 signaling in inflammation resolution and metabolic modulation. The involvement of EPRS1 in diseases such as cancer is also discussed. EPRS1 signaling: a key player in immunity and metabolism EPRS1, a vital enzyme for protein synthesis, has been found to play a significant role in various biological processes beyond translation. Researchers have discovered that EPRS1 can act as a molecular switch, regulating inflammation, metabolism, and disease. The enzyme’s phosphorylation at specific sites allows it to dissociate from the multi-tRNA synthetase complex (MSC) and interact with different proteins to perform various functions. For example, EPRS1 regulates inflammation resolution in response to LPS and bacteria, adipocyte lipid metabolism in response to insulin, and antiviral immune function in response to RNA viruses. These findings highlight the potential of EPRS1 as a therapeutic target for treating inflammatory diseases, obesity, aging, and viral infections. Further research is needed to better understand the mechanisms underlying EPRS1’s diverse functions and develop targeted therapies.

A fundamental question in biology is how vertebrates evolved and differ from invertebrates, and little is known about differences in the regulation of translation in the two systems. Herein, we identify a threonyl-tRNA synthetase (TRS)-mediated translation initiation machinery that specifically interacts with eIF4E homologous protein, and forms machinery that is structurally analogous to the eIF4F-mediated translation initiation machinery via the recruitment of other translation initiation components. Biochemical and RNA immunoprecipitation analyses coupled to sequencing suggest that this machinery emerged as a gain-of-function event in the vertebrate lineage, and it positively regulates the translation of mRNAs required for vertebrate development. Collectively, our findings demonstrate that TRS evolved to regulate vertebrate translation initiation via its dual role as a scaffold for the assembly of initiation components and as a selector of target mRNAs. This work highlights the functional significance of aminoacyl-tRNA synthetases in the emergence and control of higher order organisms. The initiation of translation is a highly regulated process that contributes to specific gene expression programs. Here the authors find that, in vertebrate, threonyl-tRNA synthetase (TRS) can act as a scaffold for the initiation machinery to stimulate the translation of a specific set of mRNAs.

The redox-dependent inhibition of thioredoxin (TRX) by thioredoxin-interacting protein (TXNIP) plays a pivotal role in various cancers and metabolic syndromes. However, the molecular mechanism of this regulation is largely unknown. Here, we present the crystal structure of the TRX–TXNIP complex and demonstrate that the inhibition of TRX by TXNIP is mediated by an intermolecular disulphide interaction resulting from a novel disulphide bond-switching mechanism. Upon binding to TRX, TXNIP undergoes a structural rearrangement that involves switching of a head-to-tail interprotomer Cys63-Cys247 disulphide between TXNIP molecules to an interdomain Cys63-Cys190 disulphide, and the formation of a de novo intermolecular TXNIP Cys247-TRX Cys32 disulphide. This disulphide-switching event unexpectedly results in a domain arrangement of TXNIP that is entirely different from those of other arrestin family proteins. We further show that the intermolecular disulphide bond between TRX and TXNIP dissociates in the presence of high concentrations of reactive oxygen species. This study provides insight into TRX and TXNIP-dependent cellular regulation. The protein thioredoxin regulates the activity of many signalling molecules. Here, Hwang et al. report structural and biochemical evidence that the interaction between thioredoxin and its inhibitor thioredoxin-interacting protein is regulated by a redox-dependent disulphide bond-switching mechanism.

The AKT signaling pathway plays critical roles in the resolution of inflammation. However, the underlying mechanisms of anti-inflammatory regulation and signal coordination remain unclear. Here, we report that anti-inflammatory AKT signaling is coordinated by glutamyl-prolyl-tRNA synthetase 1 (EPRS1). Upon inflammatory activation, AKT specifically phosphorylates Ser999 of EPRS1 in the cytoplasmic multi-tRNA synthetase complex, inducing release of EPRS1. EPRS1 compartmentalizes AKT to early endosomes via selective binding to the endosomal membrane lipid phosphatidylinositol 3-phosphate and assembles an AKT signaling complex specific for anti-inflammatory activity. These events promote AKT activation-mediated GSK3β phosphorylation, which increase anti-inflammatory cytokine production. EPRS1-deficient macrophages do not assemble the early endosomal complex and consequently exacerbate inflammation, decreasing the survival of EPRS1-deficient mice undergoing septic shock and ulcerative colitis. Collectively, our findings show that the housekeeping protein EPRS1 acts as a mediator of inflammatory homeostasis by coordinating compartment-specific AKT signaling. The PI3K/AKT signaling pathway is carefully regulated in specific cellular compartments. Lee and colleagues show that the housekeeping gene glutamyl-prolyl-tRNA synthetase 1 coordinates early endosome-specific AKT signaling necessary for inflammation resolution.

Multiple bacterial genera take advantage of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin to invade host cells. Secretion of the MARTX toxin by Vibrio vulnificus , a deadly opportunistic pathogen that causes primary septicemia, the precursor of sepsis, is a major driver of infection; however, the molecular mechanism via which the toxin contributes to septicemia remains unclear. Here, we report the crystal and cryo-electron microscopy (EM) structures of a toxin effector duet comprising the domain of unknown function in the first position (DUF1)/Rho inactivation domain (RID) complexed with human targets. These structures reveal how the duet is used by bacteria as a potent weapon. The data show that DUF1 acts as a RID-dependent transforming NADase domain (RDTND) that disrupts NAD + homeostasis by hijacking calmodulin. The cryo-EM structure of the RDTND-RID duet complexed with calmodulin and Rac1, together with immunological analyses in vitro and in mice, provide mechanistic insight into how V. vulnificus uses the duet to suppress ROS generation by depleting NAD(P) + and modifying Rac1 in a mutually-reinforcing manner that ultimately paralyzes first line immune responses, promotes dissemination of invaders, and induces sepsis. These data may allow development of tools or strategies to combat MARTX toxin-related human diseases. The MARTX toxins secreted by clinical V. vulnificus strains release the DUF1-RID effector duet in infected host cells. Here, the authors show that the duet hijacks calmodulin and Rac1, respectively, and transforms into a potent weapon to promote sepsis.

Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.

Pathogenic Vibrio species cause diseases in diverse marine animals reared in aquaculture. Since their pathogenesis, persistence, and survival in marine environments are regulated by quorum sensing (QS), QS interference has attracted attention as a means to control these bacteria in aquatic settings. A few QS inhibitors of Vibrio species have been reported, but detailed molecular mechanisms are lacking. Here, we identified a novel, potent, and selective Vibrio QS inhibitor, named QStatin [1-(5-bromothiophene-2-sulfonyl)-1H-pyrazole], which affects Vibrio harveyi LuxR homologues, the well-conserved master transcriptional regulators for QS in Vibrio species. Crystallographic and biochemical analyses showed that QStatin binds tightly to a putative ligand-binding pocket in SmcR, the LuxR homologue in V. vulnificus , and changes the flexibility of the protein, thereby altering its transcription regulatory activity. Transcriptome analysis revealed that QStatin results in SmcR dysfunction, affecting the expression of SmcR regulon required for virulence, motility/chemotaxis, and biofilm dynamics. Notably, QStatin attenuated representative QS-regulated phenotypes in various Vibrio species, including virulence against the brine shrimp ( Artemia franciscana ). Together, these results provide molecular insights into the mechanism of action of an effective, sustainable QS inhibitor that is less susceptible to resistance than other antimicrobial agents and useful in controlling the virulence of Vibrio species in aquacultures. IMPORTANCE Yields of aquaculture, such as penaeid shrimp hatcheries, are greatly affected by vibriosis, a disease caused by pathogenic Vibrio infections. Since bacterial cell-to-cell communication, known as quorum sensing (QS), regulates pathogenesis of Vibrio species in marine environments, QS inhibitors have attracted attention as alternatives to conventional antibiotics in aquatic settings. Here, we used target-based high-throughput screening to identify QStatin, a potent and selective inhibitor of V. harveyi LuxR homologues, which are well-conserved master QS regulators in Vibrio species. Structural and biochemical analyses revealed that QStatin binds tightly to a putative ligand-binding pocket on SmcR, the LuxR homologue in V. vulnificus , and affects expression of QS-regulated genes. Remarkably, QStatin attenuated diverse QS-regulated phenotypes in various Vibrio species, including pathogenesis against brine shrimp, with no impact on bacterial viability. Taken together, the results suggest that QStatin may be a sustainable antivibriosis agent useful in aquacultures. Yields of aquaculture, such as penaeid shrimp hatcheries, are greatly affected by vibriosis, a disease caused by pathogenic Vibrio infections. Since bacterial cell-to-cell communication, known as quorum sensing (QS), regulates pathogenesis of Vibrio species in marine environments, QS inhibitors have attracted attention as alternatives to conventional antibiotics in aquatic settings. Here, we used target-based high-throughput screening to identify QStatin, a potent and selective inhibitor of V. harveyi LuxR homologues, which are well-conserved master QS regulators in Vibrio species. Structural and biochemical analyses revealed that QStatin binds tightly to a putative ligand-binding pocket on SmcR, the LuxR homologue in V. vulnificus , and affects expression of QS-regulated genes. Remarkably, QStatin attenuated diverse QS-regulated phenotypes in various Vibrio species, including pathogenesis against brine shrimp, with no impact on bacterial viability. Taken together, the results suggest that QStatin may be a sustainable antivibriosis agent useful in aquacultures.

Restoration of p53 activity is a promising chemotherapeutic approach, and because of the high binding affinity between HAUSP, MDM2 and p53, blocking HAUSP activity should have the net effect of robust p53 stabilization. HAUSP is inhibited by belt-like binding of vIRF4 from Kaposi's sarcoma–associated herpesvirus. Two peptides derived from vIRF4 can additively inhibit HAUSP, leading to p53-dependent cell cycle arrest and xenograft tumor regression. Herpesvirus-associated ubiquitin-specific protease (HAUSP) regulates the stability of p53 and the p53-binding protein MDM2, implicating HAUSP as a therapeutic target for tuning p53-mediated antitumor activity. Here we report the structural analysis of HAUSP with Kaposi's sarcoma–associated herpesvirus viral interferon (IFN) regulatory factor 4 (vIRF4) and the discovery of two vIRF4-derived peptides, vif1 and vif2, as potent and selective HAUSP antagonists. This analysis reveals a bilateral belt-type interaction that results in inhibition of HAUSP. The vif1 peptide binds the HAUSP TRAF domain, competitively blocking substrate binding, whereas the vif2 peptide binds both the HAUSP TRAF and catalytic domains, robustly suppressing its deubiquitination activity. Peptide treatments comprehensively blocked HAUSP, leading to p53-dependent cell-cycle arrest and apoptosis in culture and to tumor regression in xenograft mouse model. Thus, the virus has developed a unique strategy to target the HAUSP–MDM2–p53 pathway, and these virus-derived short peptides represent biologically active HAUSP antagonists.

During the 2021/2022 winter season, we isolated highly pathogenic avian influenza (HPAI) H5N1 viruses harbouring an amino acid substitution from Asparagine(N) to Aspartic acid (D) at residue 193 of the hemagglutinin (HA) receptor binding domain (RBD) from migratory birds in South Korea. Herein, we investigated the characteristics of the N193D HA-RBD substitution in the A/CommonTeal/Korea/W811/2021[CT/W811] virus by using recombinant viruses engineered via reverse genetics (RG). A receptor affinity assay revealed that the N193D HA-RBD substitution in CT/W811 increases α2,6 sialic acid receptor binding affinity. The rCT/W811-HA virus caused rapid lethality with high virus titres in chickens compared with the rCT/W811-HA virus, while the rCT/W811-HA virus exhibited enhanced virulence in mammalian hosts with multiple tissue tropism. Surprisingly, a ferret-to-ferret transmission assay revealed that rCT/W811-HA virus replicates well in the respiratory tract, at a rate about 10 times higher than that of rCT/W811-HA , and all rCT/W811-HA direct contact ferrets were seroconverted at 10 days post-contact. Further, competition transmission assay of the two viruses revealed that rCT/W811-HA has enhanced growth kinetics compared with the rCT/W811-HA , eventually becoming the dominant strain in nasal turbinates. Further, rCT/W811-HA exhibits high infectivity in primary human bronchial epithelial (HBE) cells, suggesting the potential for human infection. Taken together, the HA-193D containing HPAI H5N1 virus from migratory birds showed enhanced virulence in mammalian hosts, but not in avian hosts, with multi-organ replication and ferret-to-ferret transmission. Thus, this suggests that HA-193D change increases the probability of HPAI H5N1 infection and transmission in humans.

Pathogenic and commensal bacteria that experience limited nutrient availability in their host have evolved sophisticated systems to catabolize the mucin sugar N -acetylneuraminic acid, thereby facilitating their survival and colonization. The correct function of the associated catabolic machinery is particularly crucial for the pathogenesis of enteropathogenic bacteria during infection, although the molecular mechanisms involved with the regulation of the catabolic machinery are unknown. This study reports the complex structure of NanR, a repressor of the N -acetylneuraminate (nan) genes responsible for N -acetylneuraminic acid catabolism, and its regulatory ligand, N -acetylmannosamine 6-phosphate (ManNAc-6P), in the human pathogenic bacterium Vibrio vulnificus . Structural studies combined with electron microscopic, biochemical, and in vivo analysis demonstrated that NanR forms a dimer in which the two monomers create an arched tunnel-like DNA-binding space, which contains positively charged residues that interact with the nan promoter. The interaction between the NanR dimer and DNA is alleviated by the ManNAc-6P–mediated relocation of residues in the ligand-binding domain of NanR, which subsequently relieves the repressive effect of NanR and induces the transcription of the nan genes. Survival studies in which mice were challenged with a ManNAc-6P–binding-defective mutant strain of V. vulnificus demonstrated that this relocation of NanR residues is critical for V. vulnificus pathogenesis. In summary, this study presents a model of the mechanism that regulates sialic acid catabolism via NanR in V. vulnificus.