The FEBS journal最新文献

A splicing-competent intron has been found to affect the expression of a gene at almost every step between transcription and translation. In this investigation, we performed a comprehensive analysis of the role of an intron from two yeast genes, ASC1 and APE2, on cellular fitness and at different steps of gene expression. Mutants containing intronless versions of either gene grew 30-60% slower than their intron-containing counterparts, and their growth was compromised on ethanol and glycerol medium. Furthermore, there was an enhanced R-loop signal in the coding region of both genes in the absence of the intron. Steady-state RNA levels of ASC1 and APE2 decreased by about 30-fold and 5-fold, respectively, in the absence of the intron. Nascent transcription analysis revealed a drop in transcription of both ASC1 and APE2 by 4-10 fold and 2-5 fold, respectively, in the intronless state. The half-life of mRNA of both genes registered a 2- to 3-fold decline in the absence of an intron. A fluorescence in situ hybridization approach detected an increase in nuclear retention of mRNA in the absence of the intron for both genes. Measurement of protein level by western blot found no detectable signal for either protein in the absence of an intron. These results suggest that the introns of both genes affect the expression of their genes at the level of transcription, mRNA stability, and nucleocytoplasmic transport of mRNA. Furthermore, both ASC1 and APE2 introns affect the fitness of cells in terms of growth rate and the ability to grow on different carbon sources.

Modern insulin still depends on phenol and zinc to keep the hormone stable in vials and pumps, yet both additives slow absorption and raise safety concerns. We therefore asked a simple, clinically driven question: Can we stabilize the fast-acting T-state of insulin without phenol/zinc by exploiting pH-dependent water and anion binding? Using high-resolution synchrotron crystallography (1.4-1.76 Å), we solved novel designer and acid-stable cubic insulin structures from pH 2 to 6 in citrate-sulfate buffers and mapped solvent/anion contacts onto computational analyses. Across the acidic range, we uncovered a conserved 'water-anion clamp' centered on the Phe¹ᴮ-Asn³ᴮ pocket that locks insulin in its bioactive T-conformation while neutralizing the protein's positive charge. This clamp: (i) removes the need for phenolic ligands, and (ii) keeps monomers soluble at high concentration. The structural blueprint we provide can guide formulation of phenol- and zinc-free, ultra-rapid insulin for subcutaneous pumps and high-strength cartridges, addressing unmet needs in intensive diabetes management. By clarifying how simple buffer anions and structured water can replace traditional preservatives, our work may link atomic-level detail to a practical therapeutic goal: faster, safer insulin delivery.

Mitochondrial structural and functional changes accompany psoriasis, yet the mitochondrial response to psoriatic inflammation in keratinocytes and fibroblasts remains unexplored. In this study, we investigated the effect of psoriasis-like inflammation (PLI) induced by a cytokine cocktail (interleukin (IL)-17A, IL-22 and tumour necrosis factor (TNF)-α) on mitochondrial network morphology and function in cultured keratinocytes (HaCaT) and fibroblasts (BJ-5ta). In both cell types, PLI triggered the expression of psoriasis-related Elafin and high amounts of cytokines (IL-1, IL-6), interferons (IFN-α, IFN-β, IFN-γ), and chemokines (C-C motif chemokine 5 (CCL5) and IL-8), accompanied by increased mitochondrial membrane potential, reactive oxygen species (ROS) production, respiration suppression, network fragmentation, swelling and cristae disassembly. Stimulated emission depletion (STED) nanoscopy revealed the disappearance of mitochondrial cristae in response to PLI, with the process starting more quickly and being more pronounced in keratinocytes than in fibroblasts. These findings highlight cell-specific mitochondrial responses to psoriatic inflammation, guiding future investigations towards new pharmacological targets for managing psoriasis.

Platelets (PLTs) have a significant impact on tumor development and progression, particularly in breast cancer, and contribute to cancer-associated thrombosis (CAT). Transforming growth factor beta (TGFβ), which is abundantly secreted by PLTs, is known to promote cancer aggressiveness. Nevertheless, the role of TGFβ in the PLT-cancer-cell interaction is largely unexplored. This study investigates how TGFβ stimulation of MCF7 breast cancer cells affects their capacity to interact with PLTs and induce PLT aggregation. MCF7, pre-treated with TGFβ and then exposed to PLTs, exhibited enhanced epithelial-mesenchymal transition (EMT) and a significantly increased ability to bind PLTs in suspension, as well as to stimulate PLT activation and aggregation. Gene expression and surface protein analyses revealed that TGFβ induced the upregulation of MCF7 adhesion molecules such as integrin-αv/CD51 and galectin-3. Intriguingly, these effects were abolished when cells were plated at high density, suggesting that TGFβ signaling may be influenced by cell junction regulation. Furthermore, we selected specific inhibitors of integrin-αv (cilengitide) and galectin-3 (GB1107) that did not interfere with PLT aggregation itself. Cilengitide, but not GB1107, effectively reduced the increased PLT-MCF7 interaction induced by TGFβ. Both inhibitors, however, significantly diminished PLT aggregation triggered by TGFβ-treated MCF7 cells. Complementary analyses of proteomic datasets from breast cancer tissues demonstrated a significant positive correlation between TGFβ1 and the platelet marker integrin alpha-IIb (ITGA2B; also known as CD41), particularly in luminal A subtypes and in cancers with lymph node involvement. These findings suggest that TGFβ stimulation enhances PLT-breast-cancer cell interactions and promotes PLT aggregation through the upregulation of specific adhesion proteins, thereby potentially contributing to CAT and metastatic progression.

Selective inhibitors are essential for targeted therapeutics and for probing enzyme functions in various biological systems. The two main challenges in identifying such protein-based inhibitors lie in the extensive experimental effort required, including the generation of large libraries, and in tailoring the selectivity of inhibitors to enzymes with homologous structures. To address these challenges, machine learning (ML) is being used to improve protein design by training on targeted libraries and identifying key interface mutations that enhance affinity and specificity. However, such ML-based methods are limited by inaccurate energy calculations and difficulties in predicting the structural impacts of multiple mutations. Here, we present an ML-based method that leverages HTS data to streamline the design of selective protease inhibitors. To demonstrate its utility, we applied our new method to find inhibitors of matrix metalloproteinases (MMPs), a family of homologous proteases involved in both physiological and pathological processes. By training ML models on binding data for three MMPs (MMP-1, MMP-3, and MMP-9), we successfully designed a novel N-TIMP2 variant with a differential specificity profile, namely, high affinity for MMP-9, moderate affinity for MMP-3, and low affinity for MMP-1. Our experimental validation showed that this novel variant exhibited a significant specificity shift and enhanced selectivity compared with wild-type N-TIMP2. Through molecular modeling and energy minimization, we obtained structural insights into the variant's enhanced selectivity. Our findings highlight the power of ML-based methods to reduce experimental workloads, facilitate the rational design of selective inhibitors, and advance the understanding of specific inhibitor-enzyme interactions in homologous enzyme systems.

Ferroptosis, an iron-dependent, non-apoptotic form of cell death, is characterised by pathogenic lipid reactive oxygen species accumulation, dysregulated iron homeostasis, and oxidative stress-induced membrane damage. Numerous studies have demonstrated that ferroptosis plays a critical role in the pathogenesis of metabolic disorders and related cancers, particularly metabolic dysfunction-associated steatotic liver disease (MASLD) and hepatocellular carcinoma. Epigenetic modifications, notably aberrant expression of histone lysine demethylases (KDMs), have been strongly associated with both ferroptosis and MASLD. This review systematically summarises how KDM family members regulate ferroptosis-related gene transcription through epigenetic mechanisms, along with their specific roles in MASLD progression. Additionally, current progress and challenges in the potential application of KDM inhibitors in regulating ferroptosis and MASLD treatment are discussed, with the aim of providing a scientific foundation for the translational development of therapeutic strategies.

The strategies adopted by viruses to counteract the potential antiviral effects of ribosomal quality control (RQC) that regulates the fidelity of protein translation, ribosome recycling, and the activation of ribosomal and integrated stress responses are poorly understood. Here, we investigated the capacity of the viral ubiquitin deconjugase (vDUB) encoded in the large tegument protein of human pathogenic herpesviruses to interfere with the triggering of RQC upon the induction of translational stress in cytosolic and endoplasmic reticulum (ER)-associated ribosomes. We found that the vDUBs encoded by Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), and Kaposi sarcoma virus (KSHV) share the capacity to counteract the ubiquitination of RPS10, RPS20, and RPS3, and the UFMylation of RPL26 in cells treated with the translation elongation inhibitor anisomycin (ANS), which resulted in the rescue of model RQC and ER-RQC substrates from proteasome- and lysosome-dependent degradation, readthrough of stall-inducing mRNAs, and inhibition of ER-phagy. In contrast, while inhibiting the ubiquitination of RPS10, RPS20, and RPS3, and rescuing RQC substrates almost as efficiently as the homologs, the herpes simplex virus-1 (HSV1) encoded vDUB failed to counteract RPL26 UFMylation. Furthermore, it was unable to rescue the ER-RQC substrate or inhibit ER-phagy, nor did it promote ZAKα phosphorylation or activate the ISR. Our findings pinpoint important differences in the strategies adopted by these human viruses for regulating translational stress responses.

Arginase converts arginine (Arg) to ornithine (Orn), regulating their availability for the metabolic pathways that utilize these amino acids. The roles of arginase isoenzymes, ARG1 and ARG2, vary by cell type, tissue, and physiological state. In the brain, ARG2 is the predominant isoenzyme, particularly enriched in the striatum, where it localizes to a striatum-specific neuronal population-medium spiny neurons (MSNs). While the precise role of ARG2 in MSNs remains unclear, its loss alters the striatal metabolomic profile, highlighting its metabolic significance. Here, to investigate the basis of these complex metabolic changes, we examined Arg metabolism in Arg2 knockout (Arg2^-/-) mice. Targeted analysis of Arg-related metabolites and selected proteins regulating Arg metabolic pathways revealed that Arg2 loss significantly increased Arg levels but did not affect Orn, likely due to compensatory synthesis of Orn from Arg (via arginine:glycine amidinotransferase) and/or proline (via ornithine aminotransferase). Additionally, markers of nitric oxide (NO) production remained unchanged, suggesting that striatal ARG2 is not involved in the regulation of this pathway, a role commonly attributed to arginase. Most notably, Arg2 loss disrupted polyamine homeostasis, shifting the balance toward higher polyamines at the expense of lower ones and altering the expression of polyamine-regulating proteins. These findings highlight ARG2's crucial role in striatal metabolism and its potential relevance to striatum-related disorders. Given that striatal ARG2 impairment has been reported in Huntington's disease, a neurodegenerative disorder specifically affecting MSNs, understanding its function may provide insights into the pathology.

Molecular chaperones ensure that proteins attain their mature state by assisting in proper folding, preventing aggregation, refolding misfolded proteins, and targeting irreparably misfolded proteins for degradation. This comprehensive role is vital for maintaining cellular homeostasis and responding to stress conditions. In this review, I focus on the multifaceted roles of chaperones in regulating protein production, spanning from ribosome biogenesis to controlling translation rate and translation fidelity through the folding of essential translation factors in eukaryotes. I discuss the function of ribosome- and nascent chain-bound molecular chaperones for the translation machinery and protein synthesis. Finally, I highlight findings on the interdependence of the two pillars of protein homeostasis when cells experience cellular stress and organisms face pathophysiological conditions.