The FEBS journal最新文献

Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein composed of a pyrin domain (PYD) and a caspase activation and recruitment domain (CARD). ASC plays a key role in the inflammasome complex by forming a supramolecular structure called the ASC speck, which promotes inflammation and pyroptosis. The assembly of ASC-dependent inflammasomes is mediated by homotypic interactions between receptor, adaptor, and effector proteins, with PYD-PYD and CARD-CARD interactions categorized into three major types (type I, II, and III). These interactions orchestrate the homo-oligomerization of inflammasome components, serving as a platform for caspase-1 activation. Through maintaining interaction homeostasis, ASC regulates innate immune responses and functions as a tumor suppressor. Dysregulation of ASC due to genetic mutations is implicated in various cancers and autoimmune diseases. However, the mechanisms driving ASC speck formation remain unclear, leaving questions on its domain-specific interactions. To address this, we used a cell line model to investigate the roles of single and double mutations within the PYD and CARD domains of ASC. We separately fused wild-type (wt)PYD and wtCARD domains to GFP and mCherry to assess the effects of these mutations on interaction dynamics using fluorescence microscopy and Förster resonance energy transfer (FRET) systems. Our results reveal previously unknown cooperative mechanisms in which specific PYD and CARD residues function as enhancers or disruptors of homo-oligomerization, highlighting the importance of cumulative interaction effects. Our study provides new insights into the molecular basis of ASC domain polymerizations.

Ustilago maydis causes smut disease in maize. In this study, we investigated the molecular basis of the contribution of small heat shock protein Hsp20 to U. maydis pathogenicity. Through biochemical studies, we demonstrate environment-dependent oligomeric plasticity and phase separation properties associated with purified Hsp20. Using genetic deletion mutants of hsp20, we evaluated the involvement of the protein in major cellular processes leading to the pathogenic development of the fungus. Hsp20 was found to form higher order oligomers in vitro that assemble into liquid condensates. Within cells, Hsp20 formed distinct punctate structures that we believe play a pivotal role in its function. These punctate structures were demonstrated to sequester proteins such as actin and septin within them. The absence of Hsp20 was found to significantly affect key cellular processes, such as endocytosis, budding, cell polarity determination and mating, in U. maydis cells. The deletion mutant failed to sporulate and complete the pathogenic life cycle. Further studies are needed to obtain mechanistic insights into the regulation of these cellular processes by Hsp20.

Developing techniques to visualize intracellular structures, which influence the spatiotemporal functionality of biomolecules, is essential for elucidating mechanisms governing cellular behavior. In this study, we demonstrate that label-free external apodized phase-contrast (ExAPC) microscopy serves as a valuable tool for the simultaneous observation of various intracellular structures with high spatiotemporal resolution, while successfully mitigating halo artifacts. Additionally, through quantitative analysis of images obtained by combining ExAPC microscopy with fluorescence microscopy, we identified distinct heterogeneities in biomolecular condensates, lipid droplets, and mitochondria. Our findings highlight the potential of ExAPC microscopy to provide detailed insights into alterations in intracellular structures associated with diverse cellular processes, corroborating the existing knowledge and potentially contributing to the discovery of previously unknown cellular mechanisms.

Photorhabdus bacteria live in mutualistic relationships with Heterorhabditis nematodes, and together, they act as effective insect pathogens. These bacteria produce a diverse array of lectins, sugar-binding proteins that are believed to play crucial roles in the complex tripartite interaction among Photorhabdus, nematodes, and their insect hosts. One such lectin, Photorhabdus laumondii tumor necrosis factor (TNF)-like lectin (PLTL), identified in Photorhabdus laumondii subsp. laumondii TTO1, exhibits notable sequence similarity to the N-terminal domain of the BC2L-C lectin (BC2L-CN), a TNF-like lectin recognized for its specificity toward fucosylated glycans associated with human embryonic stem cells and certain cancers. Through glycan array analysis and surface plasmon resonance, we identified PLTL's binding preference for branched histo-blood group oligosaccharides. The crystallographic structure of PLTL in complex with the BLe^b pentasaccharide reveals a network of direct and water-mediated hydrogen bonds simultaneously stabilizing the Fucα1-2 and Galα1-3 moieties, which define its narrow glycan specificity. A combination of mass spectrometry, protein crystallography, and analytical ultracentrifugation showed a unique hexameric PLTL architecture stabilized by intermolecular disulfide bridges. Our data suggest that PLTL may contribute to the mutualistic relationship between Photorhabdus and its nematode symbiont, Heterorhabditis bacteriophora, rather than playing a role in the interaction with the insect host. This study provides a structural and functional characterization of PLTL, a newly identified member of the TNF-like lectin family. Comparative analysis with BC2L-CN highlights both conserved and distinct structural features, suggesting potential applications in glycan recognition-based diagnostics or biotechnological tools beyond its biological role. Our findings underscore its complex glycan specificity and offer insights into its potential role in Photorhabdus-nematode symbiosis.

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.