Calpains dimerize via penta-EF-hand (PEF) domains, but their mechanical stability and kinetics remain incompletely characterized. Here, we used single-molecule force spectroscopy on an EGFP-tagged calpain small subunit P...Calpains dimerize via penta-EF-hand (PEF) domains, but their mechanical stability and kinetics remain incompletely characterized. Here, we used single-molecule force spectroscopy on an EGFP-tagged calpain small subunit PEF (CAPNS1-PEF) homodimer to quantify dissociation mechanics. Across retraction speeds of 0.2-10 μm/s, homodimer rupture required large forces (> 300 pN). Rupture forces and force-induced unfolding did not change when Ca was removed, suggesting Ca-independent mechanics, consistent with crystal structures showing minimal Ca-induced conformational changes in the PEF domain. Together, these results indicate that EF-hands in the CAPNS1 homodimer function primarily as structural elements within a mechanically robust, Ca-insensitive dimerization scaffold.
The ESX-5 secretion system in Mycobacterium tuberculosis exports PE/PPE virulence factors, with EccA5, an AAA+ ATPase, playing a pivotal role. We solved the crystal structure of EccA5's N-terminal TPR domain (EccA5NT) at...The ESX-5 secretion system in Mycobacterium tuberculosis exports PE/PPE virulence factors, with EccA5, an AAA+ ATPase, playing a pivotal role. We solved the crystal structure of EccA5's N-terminal TPR domain (EccA5NT) at 2.15 Å, revealing a monomeric fold with six TPR motifs and a variable β-finger. Biophysical studies, including SAXS and size exclusion chromatography, confirm its monomeric state. A flexible loop (residues 137-148) suggests dynamic substrate interactions. SPR, SAXS and in silico docking show moderate binding (K = 3.43 μm) between EccA5NT's β-finger and EspG5's β2-β3 loop, indicating a role in PE/PPE-EspG5 complex disassembly. These findings elucidate the role of EccA5 in ESX-5-mediated secretion.
Adrenergic-driven thermogenic activation of brown adipose tissue requires high amounts of nutrients including iron to support mitochondrial biogenesis. This is governed by rapid gene expression changes in ex vivo differe...Adrenergic-driven thermogenic activation of brown adipose tissue requires high amounts of nutrients including iron to support mitochondrial biogenesis. This is governed by rapid gene expression changes in ex vivo differentiated human cervical-derived brown adipocytes. Transferrin receptor 1 (TFRC) is upregulated in response to dibutyryl-cAMP. We aim to investigate the mechanism of facilitated iron uptake when thermogenesis is activated. Pharmacological inhibition and siRNA-mediated knock-down of TFRC during stimulation decrease intracellular iron content and prevent elevation of oxygen consumption and induction of thermogenic markers. Deferoxamine-mediated iron chelation also shows comparable effects. Contrarily, the expression of ferroportin exporter is suppressed during activation; however, its inhibition does not increase thermogenesis. Brown adipocytes constitutively express and secrete high amounts of transferrin, while melanotransferrin expression and release are upregulated only in activated adipocytes. In silico analysis suggests that melanotransferrin interacts with the helical domain of TFRC. Our findings support that iron is critical in stimulating adipocyte thermogenesis.
Centrosome abnormalities can lead to erroneous chromosome segregation during cell division, resulting in genomic instability. We identified a cancer-associated heterozygous missense mutation (S76L) in the centrosome prot...Centrosome abnormalities can lead to erroneous chromosome segregation during cell division, resulting in genomic instability. We identified a cancer-associated heterozygous missense mutation (S76L) in the centrosome protein STIL that promotes centrosome amplification and DNA damage. STIL was found to interact with BRCA1 regulating its stability; this, however, is disrupted by the S76L mutation. Mimicking the heterozygous state by overexpressing STIL-S76L redistributed BRCA1 from the nucleus to centrosomes and elevated centrosomal Aurora-A and PLK1 kinases that are responsible for centrosome amplification. Decreased nuclear BRCA1 in the mutant state induced DNA damage, which was rescued by co-expression of wild-type but not nuclear localization-deficient BRCA1. Despite amplified centrosomes, mutant cells maintain pseudo-bipolar spindle organization via kinesin HSET (KIFC1)-dependent clustering, a known cancer survival mechanism and potential therapeutic target. Together, our findings uncover a previously unrecognized STIL-BRCA1 regulatory axis that safeguards centrosome homeostasis and genome integrity. Impact statement Our study reveals a cancer-associated STIL mutation that disrupts its interaction with BRCA1, thereby destabilizing BRCA1 and leading to centrosome amplification and DNA damage.
Myeloid-derived suppressor cells (MDSCs) differentiate and proliferate in the pathological context of cancer, suppress T-cell responses, and promote tumor progression and therapeutic resistance. These cells express high...Myeloid-derived suppressor cells (MDSCs) differentiate and proliferate in the pathological context of cancer, suppress T-cell responses, and promote tumor progression and therapeutic resistance. These cells express high levels of TNF receptor type 2 (TNFR2), but the ligand TNF-α also activates TNFR1, masking TNFR2-specific function. We analyzed TNFR2 signaling using GM-CSF-induced MDSCs from TNFR2-knockout mice and scR2agoTNF-Fc, a TNFR2-selective agonist. Stimulation with scR2agoTNF-Fc maintained a highly suppressive monocytic subset. TNFR2 deficiency reduced MDSC-mediated T-cell suppression. TNFR2 activation also increased the expression of immunosuppressive effector molecules such as inducible nitric-oxide synthase and interleukin-10. These results indicate that TNFR2 is a promising therapeutic target for modulating the differentiation and immunosuppressive functions of MDSC subsets.
Liver cancer stands as the sixth leading cause of cancer-related deaths globally, with hepatocellular carcinoma (HCC) being the most frequently diagnosed subtype. The hepatic tumor microenvironment (TME) comprises a comp...Liver cancer stands as the sixth leading cause of cancer-related deaths globally, with hepatocellular carcinoma (HCC) being the most frequently diagnosed subtype. The hepatic tumor microenvironment (TME) comprises a complex array of cellular and non-cellular components, including activated hepatic stellate cells (HSCs), tumor-associated macrophages (TAM), endothelial cells, immune cells, and non-cellular elements such as growth factors, proteolytic enzymes, inhibitors, and extracellular matrix (ECM) proteins. The initiation and progression of HCC involve intricate interactions among hepatocytes, tumor cells, and non-tumor cells, including liver-resident non-parenchymal cells (NPCs). The Hippo-YAP pathway plays a crucial role in tumor development and initiation. YAP/TAZ, as primary effectors of the Hippo pathway, intricately connect with other signaling pathways relevant to tumors. YAP promotes the growth of cancer stem cells, the development of malignant phenotypes, and drug resistance, contributing significantly to cancer growth. This review focuses on the role of YAP in stromal cells as a mediator of HCC. We aim to present a comprehensive overview, not only consolidating existing knowledge but also paving the way for innovative exploration in pursuing effective therapeutic strategies against HCC.
Abnormal accumulation of misfolded proteins is a hallmark of neurodegenerative diseases. Amyloid aggregation of α-synuclein (α-Syn) and TAR DNA-binding protein 43 (TDP-43) contributes to Parkinson's disease and frontotem...Abnormal accumulation of misfolded proteins is a hallmark of neurodegenerative diseases. Amyloid aggregation of α-synuclein (α-Syn) and TAR DNA-binding protein 43 (TDP-43) contributes to Parkinson's disease and frontotemporal dementia, respectively. The heterotypic aggregates are increasingly recognized as highly cytotoxic. Given the frequent co-occurrence of α-Syn, TDP-43, and tau pathologies, we examined whether the first prion-like domain (PRD1) of CPEB3 modulates α-Syn and TDP-43 aggregation. Nuclear magnetic resonance (NMR) relaxation experiments revealed a direct interaction between PRD1 and the amyloid core of α-Syn, suppressing its aggregation, while phase separation assays showed delayed liquid-liquid phase separation (LLPS) -mediated α-Syn aggregation. In contrast, no interaction was detected with the C-terminal domain of TDP-43 (TDP-43), indicating selective inhibition of α-Syn aggregation by PRD1.
Proietti L, Beltrao P, Bruna A
… +13 more, Cortés-Ciriano I, Garnett MJ, Gonçalves E, Herranz-Ors C, Kaulich M, Lord CJ, Petsalaki E, Rad R, Ryan CJ, Savino A, Sesia D, Wessels L, Iorio F
Large-scale perturbational approaches have transformed cancer research, enabling systematic identification of tumour-specific dependencies and therapeutic vulnerabilities. However, many clinically relevant vulnerabilitie...Large-scale perturbational approaches have transformed cancer research, enabling systematic identification of tumour-specific dependencies and therapeutic vulnerabilities. However, many clinically relevant vulnerabilities arise from genetic interactions, including synthetic lethal and buffering relationships, and are shaped by cellular state, lineage and treatment history. Interpreting complex dependency landscapes increasingly relies on advanced computational and AI-based approaches integrating molecular, phenotypic and contextual information. In this rapidly evolving setting, dedicated forums are needed to connect experimental and computational perspectives. Following the success of the inaugural European Cancer Dependency Map Symposium, the 2nd EuroDepMap was held on 20 November 2025 at Human Technopole in Milan, bringing together leading scientists in functional genomics, genome-editing screens, disease models and AI-driven analysis, marking a pivotal moment for the field.
MiRNA export is a tightly regulated process crucial for maintaining balanced miRNA and target gene expression levels in metazoan cells. RNA-interacting proteins such as HuR play a key role in the selectivity and specific...MiRNA export is a tightly regulated process crucial for maintaining balanced miRNA and target gene expression levels in metazoan cells. RNA-interacting proteins such as HuR play a key role in the selectivity and specificity of miRNA export, thereby enabling context-dependent release of gene-repressing miRNAs from mammalian cells. Our results demonstrate that activated macrophages cooperatively export miRNAs, where hepatic miR-122 significantly enhances the export of miR-146a and other miRNAs. We also observe that this cooperative export causes a synchronized increase in the expression of pro-inflammatory target genes in activated macrophages. In investigating the molecular mechanisms, we found that the miRNA-binding protein HuR cooperatively binds to miRNAs and promotes their entry into endosomes, thereby facilitating their cooperative export. This highlights the selective and cooperative nature of endosome targeting happening in activated macrophages as a prerequisite for extracellular vesicle-mediated miRNA export. Impact statement This study uncovers a new mechanism for miRNA export regulation in mammalian cells, driven by the RNA-binding protein HuR. In macrophages, HuR cooperatively binds with miRNAs to export both low- and high-affinity substrates, thereby influencing macrophage activation. This cooperative export method is a common strategy for coordinated miRNA release across different cells.
The substantial increase in food allergy prevalence during the last decades has made it a significant public health concern, affecting around 10% of the global population, especially children. Despite significant progres...The substantial increase in food allergy prevalence during the last decades has made it a significant public health concern, affecting around 10% of the global population, especially children. Despite significant progress in understanding the general mechanisms of allergic sensitization, the development of oral tolerance remains a major challenge in advancing food allergy research and treatment. Additionally, each allergenic food source has a distinct immunological profile and tolerance trajectory, further complicating research efforts. Currently, oral allergen-specific immunotherapy is the only treatment that can help build tolerance to certain food allergens over time-although treatment outcomes vary. While B cells have been described and studied for their pathogenic role in food allergy, recent evidence suggests that they also modulate allergic responses through various effector and humoral functions. Notably, despite their low frequency, recent knowledge on the molecular and functional characteristics of food allergen-specific memory B cells has revealed important functions during both disease progression and therapeutic intervention. This review summarizes the current knowledge of IgE-mediated food allergy, highlighting the role of B cells, especially allergen-specific ones, in both disease and immune tolerance.
Aggregated amyloid beta peptide (Aβ) contributes to Alzheimer's disease through neurotoxic effects and a prion-like mode of transmission. We report that protein disulfide isomerase (PDI) exhibits disaggregase activity ag...Aggregated amyloid beta peptide (Aβ) contributes to Alzheimer's disease through neurotoxic effects and a prion-like mode of transmission. We report that protein disulfide isomerase (PDI) exhibits disaggregase activity against oligomeric but not fibrillar forms of Aβ. PDI did not bind monomeric Aβ, indicating its highly effective inhibition of fibril formation occurs through reversal of early-stage oligomers rather than prevention of the initial aggregate. Cells exposed to both PDI and oligomeric Aβ were protected from Aβ-induced toxicity. An S-nitrosylated form of PDI that is associated with neurodegeneration could not bind to oligomeric Aβ, thereby eliminating its neuroprotective disaggregase activity. Our observations suggest PDI could be used both physiologically and therapeutically to dissolve the oligomeric forms of Aβ.
Mitochondrial ribosomal proteins are involved in many cellular processes and not only protein synthesis from mitochondrial DNA. We previously showed that zebrafish mitochondrial ribosomal protein L4 (Mrpl4) is highly exp...Mitochondrial ribosomal proteins are involved in many cellular processes and not only protein synthesis from mitochondrial DNA. We previously showed that zebrafish mitochondrial ribosomal protein L4 (Mrpl4) is highly expressed in larval intestine. However, the physiological significance of this expression pattern remains unclear. Here, we observed significant defects in intestinal growth and maturation in mrpl4 knockout fish; this was accompanied by disruption of intestinal epithelial integrity leading to inflammatory responses, demonstrating that Mrpl4 plays an essential role in regulating zebrafish intestinal development. Moreover, we found that Notch signaling was downregulated in these mutants, and reactivation of Notch signaling can partially rescue their intestinal defects, suggesting involvement of Notch signaling in the effects of Mrpl4 on intestinal development.
Hepatic stellate cell (HSC) activation is a central mechanism in liver fibrosis, with histone acetyltransferase p300 acting as a pivotal transcriptional cofactor. To define upstream regulators of p300 stability during HS...Hepatic stellate cell (HSC) activation is a central mechanism in liver fibrosis, with histone acetyltransferase p300 acting as a pivotal transcriptional cofactor. To define upstream regulators of p300 stability during HSC activation, we performed a deubiquitinase inhibitor screen in activated HSCs and identified ubiquitin carboxyl-terminal hydrolase 2 (USP2) as a p300 deubiquitinase. Single-cell RNA sequencing of fibrotic human liver tissues revealed USP2 as the most specifically expressed USP family member in stromal populations, including HSCs, with marked upregulation in chronic liver disease and advanced metabolic dysfunction-associated steatotic liver disease (MASLD). Moreover, we demonstrated that USP2 stabilizes p300 and promotes HSC activation, whereas USP2 knockdown or pharmacological inhibition suppresses p300 accumulation and fibrogenic responses. These findings identify USP2 as a key regulator of p300 stability and HSC activation in liver fibrosis. Impact statement Our study identifies USP2 as a novel regulator of p300 stability and hepatic stellate cell activation, revealing a previously unrecognized mechanism driving liver fibrosis. These findings provide new insight into fibrogenesis and highlight USP2 as a potential therapeutic target, impacting both fundamental biology and translational fibrosis research.
Spinal muscular atrophy (SMA) is caused by a deficiency in survival motor neuron (SMN) protein; redox imbalance and oxidative stress are also implicated. Protein S-glutathionylation (PSSG) is a reversible redox modificat...Spinal muscular atrophy (SMA) is caused by a deficiency in survival motor neuron (SMN) protein; redox imbalance and oxidative stress are also implicated. Protein S-glutathionylation (PSSG) is a reversible redox modification that protects cysteines from irreversible oxidation and regulates protein function. Here, we report stage- and tissue-dependent defects in PSSG levels, accompanied by tissue-specific alterations in the expression of glutathione-related enzymes in Taiwanese SMA mice at early and late symptomatic stages. Importantly, we also provide evidence linking glutathione homeostasis defects with ferroptosis. Finally, partial restoration of SMN by antisense oligonucleotides selectively modulates these abnormalities in a tissue-dependent manner. Our findings suggest S-glutathionylation dysregulation as a novel SMA hallmark and highlight persistent redox imbalance as a therapeutic target beyond SMN restoration. Impact statement This study provides a multi-organ analysis of redox imbalance in spinal muscular atrophy, revealing systemic loss of protein S-glutathionylation in a stage- and tissue-dependent manner. By identifying the heart as particularly redox-vulnerable, this work refines understanding of oxidative stress beyond motor neurons and informs tissue-aware therapeutic evaluation.
Despite evolving independently in diverse organisms, circadian clocks ubiquitously employ period-ARNT-single minded (PAS) and cryptochrome (CRY) proteins as key regulators coupling environmental variables into circadian...Despite evolving independently in diverse organisms, circadian clocks ubiquitously employ period-ARNT-single minded (PAS) and cryptochrome (CRY) proteins as key regulators coupling environmental variables into circadian regulation. In these systems, we often observe complex gene duplication events and evolution of specialized function despite retaining high-sequence identity. These specialized functions often have evolved from ancestral photoactive proteins (LOV/CRY) where upon the ancestral photoactive ligand-binding pockets have been co-opted as protein-protein interaction motifs and targets for drug discovery. In this review, we dissect structural, biochemical, and computational studies of the PAS and CRY superfamilies within circadian clocks to highlight their molecular mechanisms and factors that position them as drug targets for diverse disease phenotypes. Particular focus is placed on discussing how photoactive members of the protein families can inform on allosteric mechanisms that couple cofactor-binding sites to regulation of flexible signaling motifs relevant to circadian regulation and drug discovery.
Circadian clocks are conserved timekeeping systems present across the tree of life. Despite sequence divergence, their negative elements share biophysical traits such as intrinsic disorder, phosphorylation clusters, and...Circadian clocks are conserved timekeeping systems present across the tree of life. Despite sequence divergence, their negative elements share biophysical traits such as intrinsic disorder, phosphorylation clusters, and charged low-complexity regions, suggesting a shared functional logic beyond primary sequence. Using the fungal protein FREQUENCY (FRQ) as a proxy, we review how intrinsic disorder enables temporal regulation. Using computational analysis, we explore the hypothesis that multisite phosphorylation may incrementally reshape FRQ conformational ensemble, with potential consequences for partner engagement over the circadian day. Thus, in this review we consider how, despite poor sequence conservation, circadian negative elements maintain a conserved mechanism rooted in the physical principles of disorder and post-translational modulation, positioning these properties at the very heart of the molecular chronosome.
Ferritin is a ubiquitous and evolutionarily conserved iron-storage protein that plays a fundamental role in cellular iron homeostasis. By catalyzing the oxidation of ferrous iron and sequestering it as a ferric mineral w...Ferritin is a ubiquitous and evolutionarily conserved iron-storage protein that plays a fundamental role in cellular iron homeostasis. By catalyzing the oxidation of ferrous iron and sequestering it as a ferric mineral within a protein nanocage, ferritin prevents toxic accumulation of labile iron and reactive oxygen species that damage proteins, lipids, and DNA. In humans, ferritin assembles into a 24-subunit nearly spherical shell enclosing a central cavity that safely stores thousands of iron atoms. This organized architecture enables ferritin to act as both an efficient iron detoxification system and a dynamic intracellular iron reservoir. Recent advances in cryo-electron microscopy (cryo-EM) have transformed ferritin research by revealing its structural organization, molecular interactions, and functional states at high resolution. Additionally, beyond protein-protein interactions, cryo-EM now enables direct visualization of ferritin-mediated biomineralization, allowing in situ observation of iron nucleation, mineral growth, and core organization within intact nanocages. Together, these advances establish cryo-EM as a transformative tool for elucidating ferritin structure, dynamics, and function - reshaping our understanding of iron metabolism and guiding the rational design of ferritin-based nanomaterials for biomedical applications.
This study characterizes a translin-like protein from Chlamydomonas reinhardtii, a unicellular alga. The efficient binding of the Crtranslin protein to both single-stranded DNA and RNA aligns it with the nucleic acid-bin...This study characterizes a translin-like protein from Chlamydomonas reinhardtii, a unicellular alga. The efficient binding of the Crtranslin protein to both single-stranded DNA and RNA aligns it with the nucleic acid-binding properties of the translin protein family, known for its roles in DNA repair, RNA metabolism, and mRNA transport. We report for the first time the presence of a translin-like protein that forms octameric rings, is more closely related to rice translin, and is localized to an organelle not yet known to harbor such a family of proteins, viz., in the basal body and flagella of C. reinhardtii. This study lays the groundwork for future investigations into the molecular functions of Crtranslin and its potential regulatory roles in flagellar dynamics. Impact statement This is the first report of the presence of a nucleic acid-binding protein, Translin, in the basal body and cilia.
Understanding how environmental changes affect the health of organisms and ecosystems is complex, but recent interdisciplinary advances and the recognition of immune function as a dynamic mediator offer exciting progress...Understanding how environmental changes affect the health of organisms and ecosystems is complex, but recent interdisciplinary advances and the recognition of immune function as a dynamic mediator offer exciting progress. Environmental immunotoxicology in teleost fishes is evolving beyond cataloguing stressors towards a mechanistic, integrative framework that leverages omics, in vivo tracking and cross-disciplinary modelling. However, knowledge gaps in immune mechanisms, toxicokinetics and multi-stressor interactions remain. The present work highlights these gaps, advocating for immune function as both a mechanistic lens and an integrative health indicator. Such a framework can improve predictive risk assessments, management strategies and our understanding of contaminant effects on resilience, disease susceptibility and population viability. While challenges remain, the field is poised for significant growth through collaborative innovation and advancing technology.
AlphaFold models provide static structural predictions, limiting their use in interpreting flexible regions in low-resolution cryo-EM maps. Here, we assess whether AlphaFold-generated distograms can instead reveal confor...AlphaFold models provide static structural predictions, limiting their use in interpreting flexible regions in low-resolution cryo-EM maps. Here, we assess whether AlphaFold-generated distograms can instead reveal conformational flexibility, focusing on binding-induced hinge motions. For this, we examined the key metabolic AK2/AIFM1 complex, where molecular dynamics and cryo-EM confirm a hinge motion in AK2 upon binding. Notably, this motion is captured in the AlphaFold2/3 distograms of apo AK2, even though it is absent in the predicted structures. By extending our analysis to other systems, we demonstrate that distograms may offer a valuable, model-independent method for interpreting ambiguous hinge regions in cryo-EM maps. Impact statement We reveal that AlphaFold distograms can successfully predict binding-induced hinge motions. This establishes distograms as a valuable, structure-free metric for identifying alternative conformational states, aiding the interpretation of ambiguous densities in cryo-EM maps.