Ewing sarcoma (ES) is a highly aggressive pediatric tumor driven by the EWS/FLi1 transcription factor, influencing epithelial–mesenchymal transition (EMT). EMT stabilizes a hybrid cell state, boosting metastatic potential and drug resistance. Nevertheless, the mechanisms underlying this ES phenotype remain elusive. Through a computational model, we predicted ZEB2, miR-200, and miR-145 circuits responsible for maintaining hybrid states in ES.

Ewing sarcoma (ES) is a highly aggressive pediatric tumor driven by the RNA-binding protein EWS (EWS)/friend leukemia integration 1 transcription factor (FLI1) chimeric transcription factor, which is involved in epithelial–mesenchymal transition (EMT). EMT stabilizes a hybrid cell state, boosting metastatic potential and drug resistance. Nevertheless, the mechanisms underlying the maintenance of this hybrid phenotype in ES remain elusive. Our study proposes a logical EMT model for ES, highlighting zinc finger E-box-binding homeobox 2 (ZEB2), miR-145, and miR-200 circuits that maintain hybrid states. The model aligns with experimental findings and reveals a previously unknown circuit supporting the mesenchymal phenotype. These insights emphasize the role of ZEB2 in the maintenance of the hybrid state in ES.

Grimontia hollisae collagenase (Ghcol) consists of 767 amino acid residues with a single catalytic domain containing the zinc-binding motif H⁴⁹²EYVH⁴⁹⁶. The crystal structure of Ghcol in complex with its substrate (Gly-Pro-hydroxyproline-Gly-Pro-hydroxyproline) and site-directed mutagenesis of active-site Tyr residues revealed the catalytic mechanism: Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state.

Grimontia hollisae collagenase (Ghcol) exhibits high collagen-degrading activity. To explore its catalytic mechanism, its substrate (Gly-Pro-Hyp-Gly-Pro-Hyp, GPOGPO)-complexed crystal structure was determined at 2.0 Å resolution. A water molecule was observed near the active-site zinc ion. Since this water was not observed in the product (GPO)-complexed Ghcol, it was hypothesized that the GPOGPO-complexed Ghcol structure reflects a Michaelis complex, providing a structural basis for understanding the catalytic mechanism. Analyses of the active-site geometry and site-directed mutagenesis of the active-site tyrosine residues revealed that Glu493 and Tyr564 were essential for catalysis, suggesting that Glu493 functions as an acid and base catalyst while Tyr564 stabilizes the tetrahedral complex in the transition state. These results shed light on the catalytic mechanism of bacterial collagenase.

In this study, the role of STAP-2 in B cell functions was analyzed. BCR-mediated B cell activation was enhanced in STAP-2 KO mice by inhibiting recruitment of Csk to Lyn. In accordance with the results, antibody production was significantly higher in STAP-2 KO mice compared with WT mice. Therefore, STAP-2 is important for the regulation of BCR signaling in B cells.

Although signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN.

This work rectifies the previous view that eukaryotic inositol lipids may derive from archaea that used such lipids together with membrane traffic proteins to engulf an aerobic bacterium, which became the mitochondrion. Eukaryotic biosynthesis of inositol lipids is of bacterial origin, as exemplified by the phylogeny of D-myo-inositol 3-phosphate synthase (MIPS), the initial enzyme of the biosynthetic pathway.

The merger of two very different microbes, an anaerobic archaeon and an aerobic bacterium, led to the birth of eukaryotic cells. Current models hypothesize that an archaeon engulfed bacteria through external protrusions that then fused together forming the membrane organelles of eukaryotic cells, including mitochondria. Images of cultivated Lokiarchaea sustain this concept, first proposed in the inside-out model which assumes that the membrane traffic system of archaea drove the merging with bacterial cells through membrane expansions containing inositol lipids, considered to have evolved first in archaea. This assumption has been evaluated here in detail. The data indicate that inositol lipids first emerged in bacteria, not in archaea. The implications of this finding for the models of eukaryogenesis are discussed.

Hybrid RNA–DNA triplexes belong to two different motifs: Parallel pyrimidine or antiparallel purine. Their formation at engineered Escherichia coli promoters produced transcription modulation with enhancing or inhibiting effects, depending on triplex geometry. These modular synthetic biology units were used to build molecular gates XOR and XNOR and a threshold gate.

In recent years, increasing numbers of noncoding RNA molecules were identified as possible components of endogenous DNA–RNA hybrid triplexes involved in gene regulation. Triplexes are potentially involved in complex molecular signaling networks that, if understood, would allow the engineering of biological computing components. Here, by making use of the enhancing and inhibiting effects of such triplexes, we demonstrate in vitro the construction of triplex-based molecular gates: ‘exclusive OR’ (XOR), ‘exclusive NOT-OR’ (XNOR), and a threshold gate, via transcription of a fluorogenic RNA aptamer. Precise modulation was displayed by the biomolecular-integrated systems over a wide interval of transcriptional outputs, ranging from drastic inhibition to significant enhancement. The present contribution represents a first example of molecular gates developed using DNA–RNA triplex nanostructures.

Iron is a key nutrient for the growth of almost all bacteria. The pathogen Pseudomonas aeruginosa is able to express at least 15 different iron acquisition pathways, each involving a specific outer membrane transporter. Most of these iron-uptake pathways rely on small iron chelators (siderophores) produced by other microorganisms. We identified the outer membrane transporters involved in the uptake of iron via two α-carboxylate siderophores and three mixed α-carboxylate/hydroxamate siderophores.

Iron is an essential nutrient for the survival and virulence of Pseudomonas aeruginosa. The pathogen expresses at least 15 different iron-uptake pathways, the majority involving small iron chelators called siderophores. P. aeruginosa produces two siderophores, but can also use many produced by other microorganisms. This implies that the bacterium expresses appropriate TonB-dependent transporters (TBDTs) at the outer membrane to import the ferric form of each of the siderophores used. Here, we show that the two α-carboxylate-type siderophores rhizoferrin-Fe and staphyloferrin A-Fe are transported into P. aeruginosa cells by the TBDT ActA. Among the mixed α-carboxylate/hydroxamate-type siderophores, we found aerobactin-Fe to be transported by ChtA and schizokinen-Fe and arthrobactin-Fe by ChtA and another unidentified TBDT. Our findings enhance the understanding of the adaptability of P. aeruginosa and hold significant implications for developing novel strategies to combat antibiotic resistance.

Human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoenzyme associated with a variety of human diseases, possesses high plasticity in the catalytic site. We report the crystal structure of NQO1 with phenylmethylsulfonyl fluoride (PMSF) covalently bound to the Tyr128 residue. We show that, unexpectedly, the catalytic activity of the enzyme was not abolished, indicating that the PMSF molecule does not limit the dynamics of this residue.

A large conformational heterogeneity of human NAD(P)H:quinone oxidoreductase 1 (NQO1), a flavoprotein associated with various human diseases, has been observed to occur in the catalytic site of the enzyme. Here, we report the X-ray structure of NQO1 with phenylmethylsulfonyl fluoride (PMSF) at 1.6 Å resolution. Activity assays confirmed that, despite being covalently bound to the Tyr128 residue at the catalytic site, PMSF did not abolish NQO1 activity. This may indicate that the PMSF molecule does not reduce the high flexibility of Tyr128, thus allowing NADH and DCPIP substrates to bind to the enzyme. Our results show that targeting Tyr128, a key residue in NQO1 function, with small covalently bound molecules could possibly not be a good drug discovery strategy to inhibit this enzyme.

In this study, we demonstrated that sphingomyelin (SM) synthase 1 (SMS1) displayed phosphatidylcholine (PC)-specific phospholipase C (PC-PLC), phosphatidylethanolamine (PE)-PLC, and ceramide phosphoethanolamine (CPE) synthase (CPES) activities, in addition to SMS activity. Moreover, SMS1 exhibited a substrate specificity for saturated fatty acid (SFA)- or monounsaturated fatty acid (MUFA)-containing PC molecular species, but not polyunsaturated fatty acid-containing PC. It is possible that SMS1 preferably produces SFA-containing diacylglycerol (SFA-DG) independently of ceramide.

Sphingomyelin (SM) synthase 1 (SMS1), which is involved in lipodystrophy, deafness, and thrombasthenia, generates diacylglycerol (DG) and SM using phosphatidylcholine (PC) and ceramide as substrates. Here, we found that SMS1 possesses DG-generating activities via hydrolysis of PC and phosphatidylethanolamine (PE) in the absence of ceramide and ceramide phosphoethanolamine synthase (CPES) activity. In the presence of the same concentration (4.7 mol%) of PC and ceramide, the amounts of DG produced by SMS and PC-phospholipase C (PLC) activities of SMS1 were approximately 65% and 35% of total DG production, respectively. PC-PLC activity showed substrate selectivity for saturated and/or monounsaturated fatty acid-containing PC species. A PC-PLC/SMS inhibitor, D609, inhibited only SMS activity. Mn²⁺ inhibited only PC-PLC activity. Intriguingly, DG attenuated SMS/CPES activities. Our study indicates that SMS1 is a unique enzyme with PC-PLC/PE-PLC/SMS/CPES activities.