The FEBS journal最新文献

Hepatocellular carcinoma (HCC) remains one of the most lethal malignancies worldwide, largely due to late diagnosis and limited therapeutic options. Tumor hypoxia is a hallmark of HCC and drives aggressive disease behavior, in part through activation of hypoxia-inducible factors (HIFs). While HIF-1α is classically understood as a hypoxia-responsive transcription factor, its role under normoxic conditions in cells is less clear. The study presented by Gkotinakou and colleagues in this issue provides important insight into this question by revealing a previously underappreciated dependence of HIF-1 signaling in controlling cell cycle progression in HCC, even in the presence of normal oxygen levels.

Cis-(+)-12-oxophytodienoic acid (OPDA) is a bioactive oxylipin and phytohormone participating in regulation of plant stress responses, growth, and development. Due to its α, β-unsaturated carbonyl moiety, OPDA covalently binds to free thiol groups by Michael addition. This binding, termed OPDAylation, alters the activity of target proteins, such as cyclophilin 20-3 (EC:5.2.1.8) and thioredoxins, that are essential components of the cellular redox regulatory network. To function as a reversible redox regulatory mechanism, OPDAylation should be complemented by a process of de-OPDAylation allowing for fine-tuning of OPDA-dependent regulation. This study explored OPDAylation and de-OPDAylation in vitro with emphasis on the involvement of glutathione. OPDA can be transferred from protein to glutathione (GSH) and vice versa. In a competition experiment, OPDAylation of thioredoxins (TRX) occurred rapidly in the presence of GSH, while over extended incubation times, de-OPDAylation of TRX occurred due to the stoichiometric excess of GSH. These results support the hypothesis that the initial TRX-based OPDAylation is proceeding under kinetic control due to the higher reactivity of the more nucleophilic cysteine moiety in TRX compared to the one of GSH, while the OPDAylation of GSH observed at prolonged incubation time is then the result of a thermodynamically controlled process. De-OPDAylation depends on the protein's sensitivity towards OPDA, the pH and the concentration of excess thiol groups. This likely allows for precise modulation of OPDA amounts, as the rapid modification of protein activity enables subsequent induction of OPDA signaling, whereas de-OPDAylation, triggered by increasing glutathione, increasing cellular reduction or presumably enzymatically, reverses this effect.

To maintain proteome integrity within distinct subcellular compartments, cells rely on tightly regulated proteostasis mechanisms, including protein synthesis, folding, trafficking, and degradation. Disruption of these processes leads to the accumulation of damaged proteins and structural changes that progressively compromise organelle function, contributing to aging and age-associated disorders, such as neurodegeneration, cancer, and metabolic dysfunction. Here, we discuss recent insights into how proteostasis influences the integrity and function of specific organelles, including the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes, as well as membraneless organelles, such as stress granules, processing bodies, the nucleolus, and nuclear speckles. We further discuss how dysfunction in these systems contributes to different hallmarks of aging and disease progression, highlighting potential therapeutic strategies aimed at maintaining organelle homeostasis to promote healthy aging.

Lysine acetylation within the tandem repeat region of cortactin (CTTN) regulates its actin-binding function and has been linked to cancer cell migration and neuronal development. While several lysine deacetylases (KDACs) have been implicated in modulating CTTN acetylation in cells, their site specificity and direct enzymatic roles remain poorly defined. Here, we use genetic code expansion to generate seven site-specifically acetylated CTTN variants and assess their deacetylation by human KDACs in a fully reconstituted in vitro system. Our results identify HDAC6 as the primary CTTN deacetylase, acting via its second catalytic domain (DD2), and demonstrate that SIRT1 and SIRT2 also directly deacetylate CTTN at overlapping sites in an NAD⁺-dependent manner. In contrast, other zinc-dependent HDACs, including HDAC8, displayed negligible or very weak activity on full-length CTTN. These findings provide new mechanistic insight into KDAC substrate preferences and highlight the value of biochemical reconstitution for dissecting complex acetylation networks.

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.