The FEBS journal最新文献

The FEBS Journal's Editorial Office is delighted to present our first Focus Issue of the year. In this Issue, we highlight some excellent research that advances the field of molecular microbiology. We welcome you to this celebration of all things bugs, drugs and 'biochem'. Read on to get a feel for the contents of this issue, then make sure to check out your favourites.

Understanding the molecular basis of regulated nitrogen (N₂) fixation is essential for engineering N₂-fixing bacteria that fulfill the demand of crop plants for fixed nitrogen, reducing our reliance on synthetic nitrogen fertilizers. In Azotobacter vinelandii and many other members of Proteobacteria, the two-component system comprising the anti-activator protein (NifL) and the Nif-specific transcriptional activator (NifA)controls the expression of nif genes, encoding the nitrogen fixation machinery. The NifL-NifA system evolved the ability to integrate several environmental cues, such as oxygen, nitrogen, and carbon availability. The nitrogen fixation machinery is thereby only activated under strictly favorable conditions, enabling diazotrophs to thrive in competitive environments. While genetic and biochemical studies have enlightened our understanding of how NifL represses NifA, the molecular basis of NifA sequestration by NifL depends on structural information on their interaction. Here, we present mechanistic insights into how nitrogen fixation is regulated by combining biochemical and genetic approaches with a low-resolution cryo-electron microscopy (cryo-EM) map of the oxidized NifL-NifA complex. Our findings define the interaction surface between NifL and NifA and reveal how this interaction can be manipulated to generate bacterial strains with increased nitrogen fixation rates able to secrete surplus nitrogen outside the cell, a crucial step in engineering improved nitrogen delivery to crop plants.

Nicotinamide/nicotinic acid mononucleotide adenylyltransferase 2 (NMNAT2) is a crucial enzyme for synthesizing nicotinamide adenine dinucleotide (NAD) and plays a vital role in neuronal health. NMNAT2 mRNA levels correlate positively with cognitive function in older adults but decline after injuries or proteinopathies. In this study, we used chromosome conformation capture followed by high-throughput sequencing (4C-seq) to unbiasedly identify NMNAT2 regulatory regions throughout the human genome. Using various bioinformatics analyses with these genomic regions, referred to as interactomes, we identified NMNAT2-associated genes and putative transcription factors (TFs). NMNAT2 transcription increases in SH-SY5Y cells when they differentiate into a neuron-like state. Excitingly, our 4C-seq data revealed distinct sets of interactomes interacting with the NMNAT2 promoter in undifferentiated versus neuron-like SH-SY5Y cells. Using the Religious Orders Study and the Rush Memory and Aging Project (ROSMAP) snRNA-seq data, we showed that the expression levels of many NMNAT2-associated genes are significantly correlated with NMNAT2 transcription in human neurons. Our biological validation studies confirmed the requirement of two specific genomic regions and four TFs, including cyclic AMP-dependent transcription factor ATF4, cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6), transcription factor SOX11, and heat shock factor protein 1 (HSF1), in NMNAT2 transcription. ATF4 has been identified as an injury-responsive TF, whereas HSF1 is modulated by protein stress. Together, our study identifies distinctive genomic loci containing NMNAT2 regulatory elements in undifferentiated versus neuron-like SH-SY5Y cells, NMNAT2-associated genes, and putative NMNAT2-TFs.

In this Editorial, Editorial Board member Kirsten C. Sadler responds to two recent opinion pieces discussing the effects of increased representation of women in science. This Editorial discusses her own experiences and views as a prominent academic in the biological sciences.

The CRISPR/Cas9 system has revolutionized molecular biology and gene editing, yet key aspects of its regulation, especially within eukaryotic environments, remain enigmatic. In this Viewpoint article, I will speculate on and explore the provocative hypothesis that Cas9 may possess previously unrecognized effector-like functions when expressed in host cells, potentially shaped by host-mediated post-translational modifications (PTMs). Of particular interest is SUMOylation at lysine 848, a key residue for DNA binding within the catalytic site, raising the possibility that this modification is not incidental, but functionally significant and precisely regulated. SUMOylation, a eukaryotic PTM, is increasingly recognized as a mechanism that also targets bacterial and viral effector proteins and virulence factors during infection, exerting context-dependent effects that may either enhance or hinder pathogen replication. Could Cas9, beyond its canonical role in bacterial CRISPR immunity, act as a host-modulating effector during infection, akin to known bacterial nucleomodulins such as transcription activator-like (TAL) effectors? If so, this would imply that certain pathogenic bacteria may have evolved Cas9 variants capable of exploiting host PTM machinery and targeting the host genome-an adaptation with potential implications for microbial virulence, host-pathogen interactions, and co-evolutionary dynamics. This perspective underscores the importance of systematically mapping Cas9 PTMs and examining their evolutionary conservation, functional significance, and pharmacological tunability, not only for basic biological insight and to deepen our understanding of microbial strategies, but also to refine the precision and safety of Cas9-based therapeutic platforms.

Perturbations in the metabolism of microtubule-associated protein tau (tau) underlie the pathology of a broad array of dementias, including chronic traumatic encephalopathy, amyotrophic lateral sclerosis (ALS) with cognitive impairment (ALSci) and approximately half of the dementias associated with frontotemporal lobar degeneration. We recently observed significantly increased hippocampal tau pathology in rats injected with pseudophosphorylated human tau (2N4R tau^T175D) co-expressing an ALS-associated TAR DNA-binding protein 43 (TDP-43) mutant (TDP-43^M337V) when compared to wild-type rats. To understand this mechanism, we examined whether the extracellular vesicles (EVs) derived from wild-type TDP-43 (wtTDP-43) or tau-expressing cells could transfer expression of these proteins to recipient cells, and whether co-localization of these proteins occurs. mCherry-wtTDP-43 or EGFP-tau constructs were expressed in HEK293 or SH-SY5Y cells. The secretome and EV fractions contained wtTDP-43 or 2N4R tau protein and RNA, and could transfer proteins into nontransfected cells. Co-localization was also detected in the cytosol of recipient cells. In silico modeling of tau and TDP-43 interactions suggests hydrogen bonding underlies this interaction. These studies further our understanding of the interaction between tau and TDP-43 by demonstrating their ability to co-aggregate and in providing a mechanism by which cell-cell transfer of either protein via extracellular vesicles can lead to these synergistic interactions.

Soluble IgMs, among the most potent activators of the classical pathway, are key mediators of complement-dependent cytotoxicity, which render them as promising drug candidates for the development of alternative drugs in treating autoimmune or inflammatory diseases. In the present study, we investigated the biochemical and in vitro functional properties of recombinant fragments from IgMs corresponding to the fragment crystallizable region (Fc)-core in their pentameric or hexameric forms. Biophysical experiments confirmed the crucial role of the IgM Joining (J) chain in favoring homogeneous pentamers, whereas its absence led to a heterogeneous population with a mixture of oligomeric forms. By combining size-exclusion chromatography with mass photometry, isolation of enriched samples with IgM hexamers or IgM pentamers without the J chain was possible. Biolayer interferometry demonstrated that both IgM-Fc forms bind C1q, and an ELISA showed that they induce the in vitro C4b deposition when in solid phase. Additionally, our data confirmed the higher efficacy of IgM hexamers compared to pentamers in activating the first component of the classical pathway. Finally, hemolytic assays demonstrate the ability of IgM-Fc constructs to inhibit Ig-induced complement-dependent cytotoxicity, which is likely made possible by the absence of fragment antigen binding region. These findings support a possible mechanism of C1 sequestration in plasma by IgM cores and consumption of the initial complement component C4. Our data thus provide important information for the development of IgM-based anti-inflammatory molecules that target specifically complement activation.

1,8-cineole synthase from Streptomyces clavuligerus (bCinS) is the only known bacterial terpene synthase that shows exclusive activity towards the monoterpene substrate geranyl diphosphate (GPP; C₁₀). Unlike most plant terpene synthases, bCinS is a high-fidelity enzyme producing 1,8-cineole as the predominant product (> 95%). A large number of bulky aromatic residues in the active site steer the carbocationic intermediates down a single path and restrict the conversion of larger prenyl-diphosphate substrates. Previously, we have shown that a single Phe-to-Ala mutation (F74A or F179A) allows bCinS to convert farnesyl diphosphate (FPP; C₁₅) into sesquiterpenoid products, including sesquicineole and germacrene A. Here, we made combinatorial mutations of aromatic active site residues to further expand the substrate scope of bCinS. The F74A-F179A double variant was not only more active than the wild type but showed increased activity towards FPP over GPP, with sesquicineole and cineole as the main products from these substrates, respectively. Computational active site volume analysis identified an additional residue, W58A, that unlocked activity towards the diterpene substrate geranylgeranyl diphosphate (GGPP; C₂₀), with the W58A-F74A-F179A triple variant showing the highest activity on this substrate. Remarkably, these key variants all appear to use the same 1,6 cyclisation cascade to form their main products from GPP, FPP, and GGPP. These results show that even high-fidelity terpene synthases such as bCinS can be engineered to accept different prenyl-pyrophosphate substrates without affecting the fundamental reaction cascade.

Multicellular animals arose along with complex gene programs controlling tissue integrity. A comprehensive understanding of the core mechanisms underlying tissue maintenance in metazoans requires studying early-branching animal groups. Here, we present whole-body regeneration (reaggregation from single cells) in sponges as a promising model for exploring conserved mechanisms maintaining tissue homeostasis in Metazoa. Using bulk RNA sequencing, we linked differential expression to major morphological changes in tissue and cell structure following the mechanical dissociation of intact tissues of a demosponge, Halisarca dujardinii. Reaggregation involves a pronounced shift in the expression of transcripts related to cell adhesion, cell polarity, proliferation, and stem cell maintenance. Cell cycle machinery is underexpressed during the reaggregation, whereas programs of stem cell maintenance and epithelial-mesenchymal transition (EMT) are upregulated. Altogether, the expression profiles render reaggregation a morphallactic reparative process driven primarily by cell plasticity rather than cell proliferation.

G protein-coupled receptors (GPCRs) are the largest superfamily of cell surface receptors. They regulate critical physiological events and serve as potential therapeutic targets. G protein-coupled receptor 35 (GPR35), a class A rhodopsin-like GPCR expressed in various tissues, including adipose tissue and the gastrointestinal tract, has roles in diverse functions, including antioxidant, anticarcinogenic, and anti-inflammatory effects. Although many endogenous and synthetic GPR35 agonists have been identified, the understanding of food-derived agonists is limited. In this study, we discovered pelargonidin as a newly identified food-derived GPR35 agonist through a systematic screening approach. We evaluated 28 dietary phytochemicals using a transforming growth factor α (TGFα) shedding assay to evaluate GPR35 activation, and found that cyanidin, a common 3-hydroxyanthocyanidin present in various red fruits and vegetables, induced GPR35 activation. Among a series of 3-hydroxyanthocyanidins tested, pelargonidin, characterized by its monohydroxylated B-ring, exhibited the most potent agonistic activity. Mutational studies demonstrated that the hydrogen bond between the 3-hydroxy group in the C-ring of pelargonidin and Asn169, as well as the hydrophobic interaction between the A-ring of pelargonidin and Phe163, is crucial for GPR35 activation. Furthermore, pelargonidin inhibited the production of interleukin-8, a pro-inflammatory cytokine, by activating endogenous GPR35 in Caco-2 cells. These findings suggest that GPR35 may serve as a potential receptor for dietary anthocyanidins, such as pelargonidin, and provide new insights into the molecular mechanisms underlying the potential chemopreventive effects of anthocyanidins.