The FEBS journal最新文献

Angiotensin I-converting enzyme (ACE) is a zinc-dependent dipeptidyl carboxypeptidase involved in blood pressure regulation through proteolysis of angiotensin I (Ang-I) into the potent vasoconstrictor, angiotensin II (Ang-II). Inhibition of ACE is therefore used for the treatment of hypertension, heart failure, myocardial infarction, stroke and chronic kidney disease. Current ACE inhibitors (ACEi) bind both the N- and C-catalytic domains of ACE (referred to as nACE and cACE), and this has been linked to the occurrence of side effects due to the wide substrate specificity of ACE. The development of domain selective ACEi with reduced side effects is therefore key for improved therapeutic intervention. Understanding how current ACEi bind nACE and cACE, and their differences in domain selectivity should aid structure-based development of more selective ACEi by identifying different chemical groups that increase or decrease selectivity. We present the kinetic and structural characterisation of nACE and cACE with three thiolate ACEi, captopril (K_i, nACE = 2.53 nm and cACE = 2.04 nm), rentiapril (monomer K_i, nACE = 2.22 nm and cACE = 6.77 nm) and zofenoprilat (K_i, nACE = 2.86 nm and cACE = 0.61 nm). Detailed structural analysis indicated that the S2' subsite likely contributes to the variation in domain selectivity observed for rentiapril and zofenoprilat due to differences in hydrophobicity and displacement of water molecules that contribute to ACE's hydration shell. Interestingly, in the cACE crystal structure, rentiapril bound as a dimer, and kinetic data revealed that both the monomeric and dimeric (dimer K_i, nACE = 15.11 nm and cACE = 36.38 nm) forms of rentiapril inhibit ACE with nanomolar affinity.

Recent advances highlight extracellular vesicles (EVs) as key mediators of intercellular communication, carrying a complex cargo that includes extracellular matrix (ECM) components and associated modulators. Among them, ADAMTS proteases are emerging as pivotal regulators due to their ability to orchestrate precise ECM remodeling events and influence cellular behavior in pathological contexts such as cancer, vascular diseases, and tissue regeneration. Notably, the identification of specific ADAMTS family members within EV populations suggests that EVs may serve as vehicles for paracrine delivery and localized proteolytic activity, enabling spatially and temporally restricted ECM modulation. This review synthesizes current knowledge on the association between EVs and ADAMTS proteases, including their known substrates, and highlights their converging roles in shaping the extracellular landscape. We also discuss key knowledge gaps, especially concerning the diversity of ADAMTS-EV interactions, their functional impact in different physiological and pathological settings, and some reflections regarding their potential translational opportunities.

The shikimate pathway in Saccharomyces cerevisiae is pivotal for the biosynthesis of aromatic amino acids, which are essential for protein synthesis and cellular homeostasis. Here, we investigated the regulatory mechanisms within this pathway, focusing on the two key enzymes: phospho-2-dehydro-3-deoxyheptonate aldolase, tyrosine-inhibited (Aro4) and phospho-2-dehydro-3-deoxyheptonate aldolase, phenylalanine-inhibited (Aro3). Deletion of either the ARO4 or ARO3 genes led to significant intracellular accumulation of phenylalanine or tyrosine, respectively, upon feeding with the corresponding metabolite. Remarkably, we discovered that this metabolite accumulation promotes the formation of amyloid-like assemblies within cells, as evidenced by amyloid-specific staining and antibody recognition towards phenylalanine and tyrosine assemblies. These assemblies strongly correlated with reduced cell viability. Treatment with common amyloid inhibitors significantly mitigated the formation of these toxic assemblies, improving cell growth, and viability in the mutant strains. Additionally, although overexpression of ARO3 in the aro4Δ background partially rescued the growth phenotype, combining ARO3 overexpression with trimethylamine N-oxide (TMAO) treatment provided an additive growth rescue effect. Our findings highlight the role of cellular self-assembly in regulating the shikimate pathway and reveal a previously unknown link between metabolic dysregulation and the formation of toxic amyloid-like structures. This discovery offers new insights into inherited metabolic disorders and potential therapeutic approaches.

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.

Keratins assemble into mechanically resilient polymers that physically stabilize epithelial cells. When epithelial cells divide, keratin polymers must be severed to allow cell separation during cytokinesis. Phosphorylation has been implicated in this process, but how keratins are regulated during cell division is not understood. Aurora B kinase, which is part of the chromosome passenger complex (CPC), accumulates at the cell center during cytokinesis and has been implicated in regulating intermediate filaments. We mapped six Aurora B kinase sites in Keratin 8. Phosphorylation of Keratin 8 at S34 occurred specifically at the cleavage furrow and persisted at the midzone until the completion of cytokinesis. Inhibition of Aurora B or expression of a nonphosphorylatable Keratin 8 mutant impaired keratin disassembly at the cleavage furrow. We propose that Aurora B-mediated phosphorylation promotes localized keratin filament disassembly at the cleavage furrow, allowing spatially regulated disassembly during cytokinesis. Aurora B binds to keratin filaments, and its localization to midzones was reduced in Keratin 8 knockout cells, showing that Keratin 8 facilitates Aurora B targeting during cytokinesis. This suggests a positive feedback cycle whereby Keratin 8 promotes midzone localization of Aurora B and, in turn, is locally disassembled by its kinase activity. This cycle is required for successful furrow ingression and completion of cell division in cancer cells of epithelial origin and might provide a target for solid tumor treatment.

Glucocorticoid receptor (GR) is a critical regulator of erythroid progenitor proliferation, while Nemo-like kinase (NLK) is reported to be hyperactivated in Diamond-Blackfan anemia (DBA), suggesting a possible cross talk. Here, we demonstrate that NLK directly interacts with multiple domains of GR and promotes its ubiquitin-mediated proteasomal degradation. Co-immunoprecipitation assays confirmed endogenous NLK-GR interaction in K562 cells, enhanced by proteasomal inhibition. NLK overexpression downregulated levels of GR in a kinase-dependent manner across HEK293T, K562, and MEL cells, an effect reversed by MG132 or a ubiquitination-defective mutant. NLK directly phosphorylated GR at Ser226, as shown by in vitro kinase assays and site-specific immunoblotting. Conversely, NLK depletion reduced basal GR phosphorylation while increasing total GR. We identified OTS167 as a direct NLK inhibitor through cellular thermal shift and kinase assays. OTS167 suppressed NLK autophosphorylation and decreased Ser226 phosphorylation of GR, stabilizing GR protein. Functionally, among all the inhibitors tested, OTS167 maximally inhibited proliferation of K562 and MEL cells by 40-90%. OTS167 also induced erythroid differentiation in K562 and MEL by increasing CD71/TER119 expression and benzidine-positive cells by 60-80%, while NLK overexpression inhibited hemin-induced benzidine staining by 25%. In primary human CD34⁺ cells, NLK and GR exhibited inverse temporal expression during erythropoiesis. OTS167 or dexamethasone expanded CD71⁺ and CFU-E populations and enhanced proliferation (Ki67⁺) across BFU-E, CFU-E, and proerythroblast stages. Conversely, dexamethasone upregulated NLK, suppressing GR and suggesting a feedback loop. Thus, NLK-mediated GR downregulation constrains erythropoiesis, and its inhibition by OTS167 promotes erythroid expansion, revealing a targetable pathway in erythroid disorders.

Acute kidney injury (AKI) is a common and often fatal condition characterized by tubular epithelial cell necrosis and immune cell infiltration. The macrophage (MФ) plays multiple roles in kidney injury and repair after AKI. However, the classification and function of MФ subsets involved in AKI remain poorly understood. In this study, kidney bulk-sequencing showed the injury pattern (day 0 to day 3) to repair pattern (day 4 to day 7) after cisplatin-induced AKI (Cis-AKI). Single-cell RNA sequencing of Cis-AKI mouse kidneys dissected the transcriptome heterogeneity of renal MФs, and depletion and adoptive transfer experiments were used to explore the functional distinction of MФ subsets. The single-cell atlas identified four MФ subsets with distinct transcriptomic profiles (Mo-MФ, Mki67⁺MФ, Cd74⁺MФ, Cx3cr1⁺MФ). The dynamic proportion change of MФ subsets from progression to regression analyses revealed that Mo-MФ, defined as recruit-MФ in this study, was primarily distributed in the progression stage, whereas the other MФ subsets, defined as resident-MФ, were dominant in the non-pathological condition and regression stage. By depletion and adoptive transfer experiments, our results confirmed that the Cx3cr1⁺MФ subset plays a crucial role in the protection against Cis-AKI. Further cell coculture experiments revealed that the tyrosine-protein kinase receptor UFO (AXL)-growth arrest-specific protein 6 (GAS6) ligand-receptor pair is involved in regulating the efferocytosis of apoptotic renal tubular epithelial cells by Cx3cr1⁺MФs, representing one of the molecular mechanisms in promoting kidney repair. In conclusion, our study dissected the heterogeneity of renal macrophage subsets during Cis-AKI, and the discovery of the protective Cx3cr1⁺MФ subset may provide new therapeutic targets for AKI intervention.

Metacaspases are cysteine proteases found in fungi, protozoa, and plants, where they regulate critical cellular processes such as programmed cell death (PCD), cell cycle progression, and protein homeostasis. Although structurally related to caspases, metacaspases differ in mechanism of activation, substrate specificity, and biological roles. Unlike caspases, metacaspases are monomeric calcium-dependent enzymes that cleave substrates after basic residues such as arginine or lysine. This review provides a comprehensive overview of the structural classification, biochemical regulation, and physiological functions of metacaspases in model eukaryotes. We discuss their roles in stress adaptation, cell death, and proteostasis in organisms such as Saccharomyces cerevisiae, Candida albicans, Trypanosoma brucei, and Trypanosoma cruzi. We highlight recent advances in understanding their activation via calcium binding and autocatalytic processing, and explore their functional diversity across species. In addition, we examine the therapeutic potential of metacaspases as drug targets due to their absence in mammals and essential roles in pathogenic microbes. Challenges in substrate identification, enzymatic characterization, and inhibitor design are also addressed, along with emerging tools that may accelerate metacaspase research. Altogether, this review underscores the growing importance of metacaspases in eukaryotic biology and their promising applications in antifungal and antiparasitic drug development.

Although macrophages (MФs) are vital regulators of acute kidney injury (AKI), their diverse roles in renal injury and repair remain elusive. Li et al. leveraged single-cell RNA sequencing to dissect MФ dynamics at different stages of cisplatin-induced AKI. They identified four distinct renal MФ subsets, in which monocyte-derived MФs (Mo-MФs) drive major renal inflammation during AKI progression, whereas renal resident Cx3cr1⁺ MФs promote renal repair via AXL-GAS6 signaling-mediated efferocytosis during AKI regression. This work advances our understanding of innate immune responses in AKI and provides insights into the heterogeneity of MФs in renal injury and repair.

Erylysin A (EryA), an aegerolysin protein produced by the edible king oyster mushroom (Pleurotus eryngii), interacts strongly with an invertebrate-specific membrane sphingolipid ceramide phosphoethanolamine. Recently, a fluorescently fused variant of EryA was shown to bind to artificial and bacterial lipid membranes containing cardiolipin (CL). This tetra-acylated glycerophospholipid, present in bacteria and in inner mitochondrial membranes of eukaryotic cells, was shown to be externalized to the plasma membrane surface during the process of apoptosis. In this work, we evaluated the interaction of EryA-mCherry with CL-containing artificial lipid vesicles and with mammalian cells undergoing apoptosis and compared its binding affinity and specificity to that of the well-established apoptosis marker, annexin V-FITC. Our results show that, in contrast to annexin V-FITC, which binds several negatively charged glycerophospholipids, EryA-mCherry specifically recognizes and binds CL in artificial membrane systems. However, this binding of EryA-mCherry to CL-supplemented membranes is less effective (K_D = 4.7 ± 1.6 μm) than that of annexin V-FITC, whose binding is observed at nanomolar concentrations. Experiments using mammalian cells showed the ability of EryA-mCherry to selectively label the membranes of apoptotic cells, binding to the same membrane regions as anti-CL antibodies and annexin V-FITC. Our data suggest that EryA-mCherry might be used as a marker of early apoptosis, as well as a marker of CL in biological and artificial lipid membranes.