FEBS Letters

UDP‐glucuronic acid 4‐epimerase (UGAepi) catalyzes the NAD ⁺ ‐dependent interconversion of UDP‐glucuronic acid (UDP‐GlcA) and UDP‐galacturonic acid (UDP‐GalA) through a mechanism involving C4‐oxidation, 4‐keto‐intermediate rotation, and subsequent reduction. Here, the functional significance of the substrate's carboxylate group in the epimerization process was investigated using UDP‐4‐keto‐pentose, an analogous intermediate that lacks a carboxylate moiety. Site‐directed mutations were introduced into UGAepi from Bacillus cereus (BcUGAepi) to increase substrate binding pocket flexibility, enabling the variant enzymes to accommodate UDP‐4‐keto‐pentose more efficiently than the wild‐type does. Although these BcUGAepi variants partially maintained nonstereospecific C4‐epimerization activity with UDP‐GlcA, they demonstrated fully stereospecific reduction of UDP‐4‐keto‐pentose to UDP‐xylose. These findings highlight the critical role of the carboxylate moiety as an essential element for epimerization in BcUGAepi, and elucidate the structural determinants of substrate specificity in UGAepis.

During cortical development, neural stem/precursor cells (NS/PCs) sequentially produce neurons, astrocytes, and oligodendrocytes. Before producing these cells, human (h) NS/PCs undergo prolonged self‐renewal to form a larger cortex than other mammals, although the mechanisms are mostly unknown. Here, we performed a gene knockout screen using the CRISPR/Cas9 system to search for genes involved in hNS/PC self‐renewal. We identified RMND5A , encoding an E3 ubiquitin ligase, among the candidate genes. We further demonstrated that knockdown of RMND5A decreased proliferation and promoted neuronal differentiation of hNS/PCs through the activation and suppression of the Wnt and mTOR signaling pathways, respectively. Taken together, our findings suggest that RMND5A participates in the maintenance of hNS/PC self‐renewal by modulating the Wnt and mTOR signaling pathways. Impact statement During cortical development, human neural stem/precursor cells (hNS/PCs) undergo prolonged self‐renewal to form a larger cortex than other mammals, although the mechanisms are mostly unknown. We identified RMND5A, an E3 ubiquitin ligase, as essential for maintaining self‐renewal of hNS/PCs, providing valuable insights into the evolutionary expansion of the human brain.

Gene gp52 of bacteriophage Phi11 encodes a putative holin (“GeneID:1258070”). Holins are bacteriophage proteins that control host cell lysis and determine the timing of the phage's infectious cycle. This study assessed the effect of overexpressing Gp52 and its mutants upon the growth rate and morphology of Escherichia coli . Gp52 caused aggressive host cell lysis, while two of the deletion mutants caused a decline in lytic potency. Lysis was completely abolished by the third mutant, which lacked the N‐terminal domain and the two putative transmembrane domains. This is a first‐hand study reporting the domain‐dependent antibacterial activity of Gp52 and could contribute to the development of novel therapeutic interventions targeting bacterial membrane integrity, especially in the context of rising antimicrobial resistance. Impact statement We studied the functional domains of Phi11 holin and their impact on host lysis. The identification of the smallest region of holin which can lyse bacterial cells will open doors for novel phage‐based therapies, thereby circumventing traditional antibiotics and benefiting both the scientific community and society's fight against antimicrobial resistance.

αB‐crystallin, a small heat shock protein, is crucial for maintaining lenticular transparency and prevents protein aggregation as a molecular chaperone in various tissues. Mutations in αB‐crystallin can lead to diseases such as cataracts, cardiomyopathy, and neurodegenerative disorders. This study explores the effects of the p.R157C mutation in the C‐terminal domain, near the IXI motif, which is associated with cardiomyopathy. The mutant protein was generated through site‐directed mutagenesis, expressed in bacterial systems, and purified by ion‐exchange chromatography. Biophysical and computational techniques revealed significant alterations in secondary structure, oligomerization, and conformational stability. The mutation also enhanced chaperone activity and promoted amyloid fibril formation. These alterations may disrupt the interactions of the p.R157C mutant αB‐crystallin with cardiac proteins such as desmin and calcineurin, potentially contributing to cardiomyopathy. These findings offer mechanistic insights into αB‐crystallin‐related cardiomyopathy, shedding light on its pathological role and potential therapeutic targets.

Chikungunya (CHIKV) and dengue (DENV) viruses pose a public health risk and lack antiviral treatments. Structure‐based molecular docking of a natural MTase substrates library identified herbacetin (HC) and caffeic acid phenethyl ester (CAPE) as potential CHIKV nsP1 and DENV NS5 MTase inhibitors. Binding affinities and MTase inhibition were confirmed using purified proteins. The crystal structure of DENV 3 NS5 MTase and CAPE complex revealed CAPE binding at viral RNA capping sites. Interestingly, HC and CAPE depleted polyamines crucial for RNA virus replication and decreased viral titer with IC 50 values of ~ 13.44 and ~ 0.57 μ m against CHIKV, and ~ 7.24 and ~ 1.01 μ m against DENV 3, respectively. Polyamine addition did not reverse the antiviral effects, suggesting a dual inhibition mechanism. Impact statement This study reveals the antiviral potential of natural small molecules, Herbacetin (HC) and Caffeic acid phenethyl ester (CAPE) against Dengue and Chikungunya viruses. The molecules deplete polyamine levels and directly inhibit viral methyltransferases. This study opens new avenues for developing antiviral strategies that target both host factors and viral components.

Mutations in polycystin‐1 (PC1) or polycystin‐2 (PC2) cause autosomal‐dominant polycystic kidney disease (ADPKD). Structural data suggest that one PC1 and three PC2 form heterotetrameric ion channels with an ion permeation pathway blocked by PC1 (R4100, R4107, and H4111) and PC2 (L677, N681) residues. Here, we demonstrate that replacing these residues with alanines results in a gain‐of‐function (GOF) PC2/PC1 construct with distinct selectivity properties compared to PC2 homomers. We also show preferential formation of PC2/PC1 heteromeric complexes over PC2 homomers. Re‐interpretation of published PC2/PC1 cryo‐electron microscopy data, combined with cysteine modification experiments, suggests that the pore‐forming domain of PC1 adopts a canonical TRP channel‐like conformation. This novel PC2/PC1 GOF construct offers the opportunity to investigate the functional impact of ADPKD mutations.

The ubiquitin‐like interferon (IFN)‐stimulated gene 15 (ISG15) is a unique molecular effector that functions both intra‐ and extracellularly. Central to its pleiotropic nature is the ability to coordinate cellular responses following its conjugation to target proteins via ISGylation or in its free form. The activity of ISG15 is highly context‐dependent: in the case of viral infections, ISG15 can serve as a pro‐ or antiviral factor. While ISG15 has been studied extensively, several gaps persist in our understanding of its role in dysregulated immune homeostasis. In particular, the role of ISG15 in the central nervous system (CNS), which has traditionally been considered an immune‐privileged site, remains ill‐defined. Interestingly, elevated ISG15 expression is observed in the CNS following instances of brain injury, autoimmunity, neurodegeneration, and viral infection. In this review, we seek to provide a comprehensive analysis of these studies as they pertain to ISG15 and its potential roles in the CNS. Furthermore, we discuss questions and challenges in the field while highlighting ISG15 as a potential diagnostic biomarker or therapeutic target. Impact statement While ISG15 has been studied extensively, several gaps remain in our understanding of its role in dysregulated immune homeostasis and its impact within the central nervous system (CNS). In this review, we provide a comprehensive analysis of the emerging roles of ISG15 in brain injury, autoimmunity, neurodegeneration, and viral infection within the CNS.

Building on experimental evidence and replicator theories, I propose a 3‐stage scenario for a transition from autocatalysis into template‐based replication of RNA, providing a pathway for the origin of life. In stage 1, self‐reproduction occurs via autocatalysis using oligomer substrates, replicator viability relies on substrate‐specificity, and heritable variations are mediated by structural interactions. In stage 2, autocatalysis coexists with the templated ligation of external substrates. This dual mode of reproduction combined with limited diffusion avoids the error catastrophe. In stage 3, template‐based replication takes over and uses substrates of decreasing size, made possible by enhanced catalytic properties and compartmentalization. Structural complexity, catalytic efficiency, metabolic efficiency, and cellularization all evolve gradually and interdependently, ultimately leading to evolutionary processes similar to extant biology. Impact statement This perspective proposes a testable stepwise scenario for the emergence of evolution in an RNA origin of life. It shows how evolution could appear in a gradual manner, thanks to catalytic feedback among random mixtures of molecules. It highlights possible couplings between the different facets of molecular self‐organization, which could bootstrap life.

Autophagy is a catabolic process by which cells maintain cellular homeostasis through the degradation of dysfunctional cytoplasmic components, such as toxic misfolded proteins and damaged organelles, within the lysosome. It is a multistep process that is tightly regulated by nutrient, energy, and stress‐sensing mechanisms. Autophagy plays a pivotal role in various biological processes, including protein and organelle quality control, defense against pathogen infections, cell metabolism, and immune surveillance. As a result, autophagy dysfunction is linked to a variety of pathological conditions. The role of autophagy in cancer is complex and dynamic. Depending on the context, autophagy can have both tumor‐suppressive and pro‐tumorigenic effects. In contrast, its role is more clearly defined in protein conformational disorders, where autophagy serves as a mechanism to reduce toxic protein aggregation, thereby improving cellular homeostasis. Because autophagy‐based therapies hold promising potential for the treatment of cancer and protein conformational disorders, this review will highlight the latest findings and advancements in these areas.

Short‐chain fatty acids (SCFAs), produced by dietary fiber fermentation in the colon, play essential roles in cellular metabolism, with butyrate notably modulating immune responses and epigenetic regulation. Their production contributes to an acidic colonic environment where protonated SCFAs permeate membranes, leading to intracellular acidification and SCFA accumulation. Using our method to measure intracellular pH, we investigated how extracellular pH influences butyrate‐induced acidification and immunomodulatory effects in human macrophages. Our data show that butyrate accumulates and acidifies cells at acidic extracellular pH due to the permeability of its protonated form. While inflammatory cytokine production was mildly influenced by extracellular pH, butyrate‐induced histone acetylation exhibited a pH dependence, underscoring the importance of considering extracellular pH when assessing the SCFA's functions.

Choroideremia (CHM) is a rare X‐linked recessive form of inherited retinal degeneration caused by the deficiency of the Rab escort protein 1 (REP1)‐encoding CHM gene. REP1 is essential for the post‐translational prenylation of the key players in intracellular membrane trafficking, the Rab GTPases. In this study, we aimed to analyze the mechanisms of retinal degeneration caused by Rep deficiency using the Drosophila retina as a model system. Rab GTPases lost their membrane association ability and diffused into the cytoplasm, and the accumulation of unprenylated Rab6 and Rab7 was observed in Rep‐deficient photoreceptors. Notably, Rep‐deficient photoreceptors underwent progressive cell death via cell swelling and rupture rather than apoptosis. These findings provide new insight to seek a therapeutic approach to CHM. Impact statement Choroideremia is an inherited retinal degeneration caused by a deficiency of Rab escort protein 1 (Rep‐1). We used the Drosophila retina as a model to study the mechanism of retinal degeneration in Rep‐deficiency and found that Rep‐deficient photoreceptors undergo progressive cell death via cell swelling and rupture rather than apoptosis.

Spot 14 (S14), encoded by Thrsp , is a thyroid hormone‐responsive transcriptional activator that regulates lipogenesis, though its mechanisms remain unclear. We aimed to study the role of S14 on gene expression in adipocytes. We analyzed Thrsp and its paralog Mid1ip1 in brown (EB5), beige (EB7), and white (F442A) adipocytes. Thrsp expression was higher in EB5 and EB7 than in F442A and increased with thyroid hormone T3 in EB5 and EB7 but decreased in F442A. Mid1ip1 expression rose moderately in EB5 and EB7, influencing lipid metabolism genes. Silencing Thrsp upregulated Mid1ip1 in EB7 and reduced thermogenic gene expression in EB5 and EB7. These findings underscore the roles of Thrsp and Mid1ip1 in metabolic and thermogenic pathways, highlighting the responsiveness of S14 to thyroid hormones and nutrient signals. Impact statement This study reveals that Thyroid Hormone‐Induced Protein 8 (THRSP), also known as Spot‐14, and its paralog Spot‐14R, regulate metabolic and thermogenic gene expression differently in brown and beige adipocytes. These findings provide insights into adipocyte metabolism, offering potential targets for obesity and metabolic disorder treatments.

Heart failure with preserved ejection fraction (HFpEF) accounts for half of heart failure cases and is characterised by reduced pericyte coverage. While the contributions of other cardiac cell types to HFpEF are well‐studied, the role of pericytes remains less understood. Using murine single‐nucleus RNA‐sequencing to study cardiac pericytes in HFpEF, we identified reduced STAT3 expression as a hallmark of HFpEF pericytes. Mechanistic studies in vitro revealed that STAT3 deletion induces cellular senescence and impairs pericyte adhesion, recapitulating HFpEF‐like characteristics. These findings suggest that STAT3 is crucial for maintaining pericyte homeostasis and highlight its reduction as a potential driver of pericyte loss, a defining feature of HFpEF.

The mouse 3D8 anti‐DNA antibody can enter cells and localize in the cytoplasm, primarily facilitated by the complementarity‐determining region 1 of the variable light chain (CDR L1) domain. In this study, we grafted the CDR L1 loop from 3D8 onto non‐cell‐penetrating IgG antibodies to investigate whether these IgGs could acquire cytoplasmic localization ability while retaining antigen‐binding activity. One of three IgGs was successfully delivered into the cytoplasm while maintaining antigen‐binding activity. In silico protein modeling suggests that this capability is linked to structural similarity between CDR L1 in the grafted Ab and that in 3D8. This study proposes a strategy to confer cell‐penetrating capability by incorporating a specific CDR loop into an antibody backbone while retaining affinity.

We present a transcription‐coupled Flp‐nick system enabling a stable protein‐bound nick mimicking a topoisomerase I–DNA cleavage complex. The nick is introduced at a single site within a controllable LacZ gene inserted into the Saccharomyces cerevisiae genome. This system allows unique single‐site studies of a frequently occurring damage within a transcription unit in vivo . As proof of principle, we demonstrate RNA polymerase II accumulation at the damage site when MG132 inhibits the proteasome. Similarly, accumulation occurs when polymerase ubiquitination is abolished by deletion of the ubiquitinase ELC1 gene. This indicates that a topoisomerase I–DNA mimicking cleavage complex per se induces RNA polymerase II ubiquitination and degradation. These findings advance understanding of cellular responses to topoisomerase I‐targeting drugs used in cancer chemotherapy.

Extracellular vesicles (EVs) are critical in cell communication, transfer of biomolecules, and host‐pathogen interaction. A newly identified subset, “interaction vesicles” (iEVs), forms through host‐pathogen contact, merging membrane elements from both. These iEVs may arise through multiple mechanisms, including direct cell–cell contact, membrane contact sites, uptake and repackaging of foreign EVs, and post‐release fusion of EVs. These hybrid vesicles enable pathogens to modify host environments, aiding immune evasion and infection persistence. However, iEVs may also act in favor of the host, contributing to pathogen recognition and elimination. Advanced techniques, including proteomics and high‐resolution microscopy, are beginning to clarify their composition and fusion. Yet, isolating these hybrid EVs remains challenging. Overcoming these barriers could enhance understanding of infection mechanisms and support diagnostic and therapeutic innovation.

Phycocyanin (PC), a pigment–protein complex with diverse biotechnological applications, plays a key role in light energy transfer for photosynthesis in cyanobacteria. PC (O‐PC) from a thermotolerant cyanobacteria Oscillatoria sp. N09DM exhibits remarkable stability compared to its mesophilic counterparts, making it highly valuable for industrial and medical applications. To understand the basis of its stability, the crystal structure of O‐PC is solved and analysed. Structural analysis reveals a key molecular interaction, including hydrogen bonds, salt bridges and hydrophobic interactions, along with amino acid substitutions that provide the thermal stability. Additionally, structural results provide insights into chromophore‐protein interactions for understanding O‐PC's role in the efficient transfer of light energy.

The neuromuscular junction (NMJ) performs the crucial function of controlling skeletal muscle contraction. NMJ formation depends on the Agrin/Lrp4/MuSK/Dok‐7 signaling pathway. However, signaling downstream of Dok‐7 remains incompletely understood. Here we used the phosphorylated iTRAQ technique to identify downstream molecules of Dok‐7 in muscle cells. We found 16 Agrin/Dok‐7‐mediated serine/threonine phosphorylated proteins, and we validated the role of one phosphorylated protein, JPH2, in regulating AChR clustering. Our phosphoproteomics analysis sheds light on the underappreciated signaling network downstream of Agrin/Dok‐7, thus providing new clues for understanding pathogenesis and developing treatment methods for neuromuscular diseases.

MicroRNAs (miRNAs) control organogenesis in mammals by inhibiting translation of mRNA. Skin is an excellent model to study the role of miRNAs in epidermis and the mesenchyme. Previous research demonstrated miRNA‐29 family functions in skin; however, the mRNA targets and the downstream mechanisms of miRNA‐29‐mediated regulation are missing. We used the miRNA crosslinking and immunoprecipitation method to find direct targets of miRNA‐29 in keratinocytes and fibroblasts from human skin. miRNA‐29 inhibition using modified antisense oligonucleotides in 2D and 3D cultures of keratinocytes and fibroblasts enhanced cell‐to‐matrix adhesion through autocrine and paracrine mechanisms of miRNA‐29‐dependent tissue growth. We reveal a full transcriptome of human keratinocytes with enhanced adhesion to the matrix, which supports regeneration of the epidermis and is regulated by miRNA‐29. Impact statement The functions of small, therapeutically targetable microRNA molecules identified in our study can provide a new approach to improve wound healing by restoring and enhancing the inner molecular mechanisms of a cell and its surrounding matrix. We also provide a plethora of new mRNA targets for follow‐up studies of cell adhesion and extracellular matrix formation in humans.

Since stress can be transmitted to congeners via social metabolites, it is paramount to understand how the social context of abiotic stress influences aquatic organisms' responses to global changes. Here, we integrated the transcriptomic and phenotypic responses of zebrafish embryos to a UV damage/repair assay following scenarios of heat stress, its social context and their combination. Heat stress preceding UV exposure had a hormetic effect through the cellular stress response and DNA repair, rescuing and/or protecting embryos from UV damage. However, experiencing heat stress within a social context negated this molecular hormetic effect and lowered larval fitness. We discuss the molecular basis of interindividual chemical transmission within animal groups as another layer of complexity to organisms' responses to environmental stressors.

The main mediators for the amino acid uptake in Saccharomyces cerevisiae are the permeases belonging to the yeast amino acid transporter family. Recently, we discovered that members of this family support growth on more amino acids than previously described. Here we study the substrate spectrum of Lyp1, the main transporter responsible for the uptake of lysine in yeast. We show that overexpressed Lyp1 supports growth on alanine, asparagine, leucine, methionine, phenylalanine, serine, and valine when these are provided as the sole source of nitrogen to a strain severely deficient for the uptake of amino acids. We show that alanine and serine compete with lysine for the common transport system, albeit with much lower affinity. Thus, Lyp1 has a much broader substrate spectrum than previously thought, which may be true for many amino acid transporters.

B cells migrate within lymphoid organs during maturation and activation, processes orchestrated by the interplay between B cell receptor (BCR) signaling and microenvironmental cues. Integrins act as mechanoreceptors, linking BCR activation to cytoskeletal remodeling, facilitating immune synapse formation, antigen recognition, and extraction. BCR activation models describe receptor clustering and mechanical changes within the antigen–BCR complex. Upon activation, immune synapses form, enabling antigen extraction and downstream signaling. Integrins stabilize these synapses, amplify BCR signaling, and modulate BCR positioning via actin reorganization. In chronic lymphocytic leukemia (CLL), aberrant BCR signaling and integrins are major players in leukemic cell homing, prognosis, and therapy resistance. In this review, we summarize the current understanding of the interplay of BCR mechanics and B cell localization, with a particular focus on communication between BCR signaling and integrin‐mediated processes via actin dynamics. We give insights into normal B cell biology and then outline aspects typical to CLL.

The evolutionarily conserved E‐Twenty‐Six (ETS) family of transcription factors acts downstream of major signal transduction pathways and plays a pivotal role in tissue development and maintenance. Importantly, their function is frequently corrupted in a substantial proportion of tumour types, and they are also indispensable for angiogenic sprouting, a hallmark of cancer, which is essential for fuelling tumour enlargement and dissemination. Consequently, targeting aberrant ETS activity could potentially represent a precise and effective means by which to block tumour growth. Here, we present proof‐of‐principle high‐throughput screens and an initial characterization of candidate hits, as a methodological and conceptual framework for the identification of novel ETS transcription factor inhibitors, which may ultimately lead to new therapeutic avenues for treating cancer.

Endolysins—enzymes produced by tailed bacteriophages to degrade bacterial cell walls—have traditionally been classified as canonical or signal‐anchor‐release (SAR) endolysins. However, analysis of expanding viral (meta)genomic data has revealed a third class, which we designate as C‐terminal anchor (CTA) endolysins. These enzymes feature an N‐terminal enzymatic domain, a C‐terminal transmembrane domain, and typically lack signal sequences, distinguishing them from SAR endolysins. CTA endolysins span all known enzymatic activities and exhibit diverse architectures, though most have a single transmembrane helix and an N‐out, C‐in topology, consistent with periplasmic activity. While their functional mechanisms remain to be elucidated, our findings suggest that CTA endolysins are nearly as prevalent as SAR endolysins and represent a distinct, previously unrecognized branch of the endolysin world.

MicroRNAs (miRNAs) are a prominent class of small non‐coding RNAs that control gene expression. This comprehensive review explores the intricate roles of miRNAs in metabolism and immunity, as well as the emerging field of immunometabolism. The core of this work delves into the functional and regulatory capabilities of miRNAs, examining their complex influence on glucose and lipid metabolism, as well as their pivotal roles in shaping T‐cell development and function. Specifically, this review addresses how miRNAs orchestrate the complex interaction between cellular metabolic processes and immune responses, underscoring the essential nature of these small regulatory molecules in maintaining homeostasis. Finally, we examine the emerging role of Artificial Intelligence (AI) in miRNA research, focusing on how machine learning techniques are revolutionizing the identification and validation of potential miRNA biomarkers. By integrating these diverse aspects, this review underscores the multifaceted roles of miRNAs in biological processes and their significant potential in advancing biomedical research and clinical applications.