The inhibitor of growth 4 (ING4) acts as a tumor suppressor regulating chromatin structure. Due to this nuclear function, ING4 has a nuclear localization signal (NLS), which is recognized by the cellular translocation machinery ‒ mainly formed by proteins named importins, which include the isoform importin α3 (Impα3) ‒ to allow movement through the nuclear membrane. Peptidyl arginine iminohydrolases (PADIs) are enzymes involved in the posttranslational modification of arginine to citrulline. PADI4, one of the five isoforms of PADI in humans, citrullinates ING4 at the NLS region. We studied in vitro and in silico how the different degrees of citrullination affected binding of the NLS of ING4 to PADI4, Impα3 and its truncated species (ΔImpα3), lacking the importin binding domain, by using several biophysical techniques and molecular simulations. To that end, we synthesized eight peptides encompassing the NLS of ING4 (residues 130-152), with single, double and triple citrulline replacements at Arg132, Arg142 and Arg144. The peptides were monomeric and disordered, as tested by DOSY, 1D- and 2D-1H NMR experiments. All the peptides were capable of binding to PADI4 with low micromolar affinities, but their affinity decreased as the fraction of citrullination increased. Moreover, all peptides could bind to both importin species with affinities in the low micromolar range, and their affinities were also dependent on the citrullination degree. The peptides targeted the canonical NLS binding site for cargo proteins of both importin species. These findings suggest that: (i) citrullination at the NLS might interfere with ING4 nuclear translocation; and (ii) successive citrullination at the NLS affected binding to PADI4.

IntroductionThe STimulator of Interferon Genes (STING) is a key adaptor protein in the innate immune response to cytosolic DNA, making it a promising therapeutic target. Identifying novel STING ligands could provide new opportunities for immune modulation.MethodsWe employed high-throughput virtual screening to identify potential STING ligands and selected Teniposide, an anticancer drug primarily used for infant leukemia. Direct binding of Teniposide to STING’s cytosolic domain was confirmed via isothermal titration calorimetry (ITC) and validated using a double mutant STING variant unable to bind Teniposide. Computational docking and molecular dynamics simulations were performed to characterize the binding mode.ResultsTeniposide activated the IFN-β signaling pathway in a STING-dependent manner, independent of dsDNA sensors cyclic GMP-AMP synthase (cGAS) and Interferon Gamma Inducible Protein 16 (IFI16). ITC confirmed direct interaction, and the STING double mutant abolished binding. Computational analyses revealed a symmetrical binding mode involving two Teniposide molecules interacting with STING.DiscussionThese findings suggest that Teniposide activates STING through a previously unrecognized, cGAS-independent mechanism, while retaining potential for canonical cGAS-STING stimulation. Our combined computational and experimental evidence supports repurposing Teniposide as a STING agonist, highlighting new therapeutic possibilities for innate immune stimulation.

Protein kinases have key roles in cells as they regulate diverse signal transduction pathways. Mitogen-activated protein kinase (MAPK) signaling route modulates several processes, such as cell proliferation, cell programming, metabolic changes and stress responses. Within the group of proteins participating in this pathway, the MAPK kinase (MEK1) is a dimeric, 393-residue-long, dual-specificity protein kinase that phosphorylates both tyrosine and threonine residues. In this study, we explored the conformational changes occurring during the unfolding of MEK1, by using orthogonal biophysical techniques. Intrinsic fluorescence, extrinsic 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, dynamic light scattering (DLS), and far-ultraviolet (UV) circular dichroism (CD) showed that the protein acquired a native-like conformation within a narrow pH range (8.0 to 9.0). Urea and guanidinium hydrochloride (GdmCl) denaturations followed by intrinsic and ANS fluorescence and far-UV CD, at pH 8.1, where the protein acquired a native-like conformation, showed that: (i) the apparent conformational stability of isolated MEK1 was low; and (ii) the unfolding occurred through the presence of intermediates. The presence of several unfolding intermediates was also evidenced through: (i) differential scanning calorimetry (DSC) in the absence of the ligand ATP; and (ii) unfolding simulations with the help of computational techniques based on constraint network analysis (CNA). We propose that the apparent low stability of this protein was related to its flexibility and modulates its ability to interact with diverse molecular partners.

The Polycomb repressive complexes can be considered a paradigm of chromatin-based gene regulation in animals. Two of the proteins involved in such complexes are the Ring finger protein 1 (RING1) and the intrinsically disordered protein RING1A and YY1 binding protein (RYBP), which binds to RING1 in some of the Polycomb complexes. Peptidyl arginine iminohydrolases (PADIs) are enzymes involved in the post-translational modification of arginine to citrulline. PADI4, one of the five isoforms of PADI in humans, citrullinates RING1B and RYBP at specific arginines at their C-terminal (Arg231) and N-terminal (Arg53) regions, respectively. In this work, we studied in vitro and in silico whether isolated peptides from each protein, encompassing those wild-type arginines and their citrulline-modified counterparts, were capable of binding to PADI4. The isolated peptides, either in wild-type or modified form, were monomeric and disordered as indicated by far-UV CD and NMR. The affinity constants of peptides for PADI4, as measured by fluorescence, biolayer interferometry (BLI) and isothermal titration calorimetry (ITC), were in the low micromolar range (dissociation constants around 10-20 μM), and the affinities were smaller for the citrullinated variants. Molecular docking simulations showed that the isolated peptides from each protein, either in wild-type form or modified variants, targeted the active site of PADI4, corresponding to the catalytic residue Cys645. These findings suggest that the isolated peptides were capable of binding to the enzyme with affinities similar to those of intact RING1B or RYBP, and that citrullination of the arginines diminished the affinity for the enzyme.

Nucleophosmin (NPM1) is a nucleolar protein commonly mutated in ~30% of newly diagnosed acute myeloid leukemia (AML) cases. These mutations occur in the terminal exon of the NPM1 gene, affecting the C-terminal DNA-binding domain of the protein and causing its delocalization to the cytoplasm-a hallmark of NPM1-mutated AML. NPM1 shuttling to the nucleoplasm is tightly regulated by posttranslational modifications, such as phosphorylation of Ser254, Ser260, and Tyr271 of the DNA-binding domain. However, the structural mechanisms underlying this process remain unclear. In this work, we show that Ser-to-Asp (S254D-S260D) and Tyr-to-pCMF (para-carboxymethyl phenylalanine) (Y271pCMF) phosphomimetic mutations induce significant structural and dynamical rearrangements, as well as drastic modifications in electrostatic surface potential. These changes compromise recognition of a G-quadruplex sequence from the c-MYC promoter by reducing DNA-binding affinity, reshape histone capturing dynamics, and fade charge segregation in the histone-binding domain. Combination of such substitutions in a triple phosphomimetic variant (S254D-S260D-Y271pCMF) further destabilizes the domain's structure and triggers protein aggregation. Altogether, these findings suggest that phosphorylation of Ser254, Ser260, and Tyr271 of the C-end DNA-binding domain weakens both DNA affinity and charge block-driven liquid-liquid phase separation, offering a molecular explanation for the delocalization of NPM1 outside of the nucleolus.

Bacteroides fragilis is a key component of the human gut microbiota, although enterotoxigenic strains (ETBF), which produce B. fragilis toxin (BFT), can act as opportunistic pathogens. BFT disrupts intestinal epithelial integrity and contributes to conditions such as inflammatory bowel disease and colorectal cancer. This study aimed to characterize three allosteric inhibitors of BFT-3 (isoform 3 of BFT), previously identified by our group through high-throughput screening of US Food and Drug Administration approved drugs. We evaluated their activities in vitro and in vivo. Using Galleria mellonella larvae as a novel infection model for B. fragilis, we assessed the antimicrobial and antivirulence potential of these compounds. Among the three tested compounds, MOA4 demonstrated superior efficacy, enhanced bacterial clearance in vivo, and increased larval survival in a dose-dependent manner, with minimal toxicity. Synergy studies have revealed the potential combinatory effects of MOA4 and conventional antibiotics. These findings establish G. mellonella as a valuable alternative model for studying B. fragilis infections and highlight MOA4 as a promising candidate to be repurposed for the treatment of B. fragilis-mediated diseases while preserving commensal microbiota.

Phenylketonuria (PKU) is an inherited metabolic disorder caused by pathogenic variants in phenylalanine hydroxylase (PAH), leading to toxic phenylalanine accumulation and severe neurological complications if untreated. Current pharmacological treatment relies on tetrahydrobiopterin (BH4), which benefits only a subset of patients, highlighting a major unmet need for alternative therapies. Here, we combined high-throughput screening, computational modelling, and drug repurposing to identify pharmacological chaperones capable of rescuing PAH function. We evaluated 26 structurally diverse small molecules in HEK293T cells expressing wild-type PAH or one of eight PKU-associated variants spanning phenotypes from mild to classical disease. Chaperoning efficacy was strongly variant-dependent, and for every variant tested at least one compound produced a greater activity increase than BH4 under identical assay conditions. Notably, belinostat, a clinically approved histone deacetylase inhibitor, emerged as the most effective compound for several clinically severe variants. Mechanistically, functional rescue consistently correlated with an increased population of tetrameric, catalytically competent PAH, as quantified by mass photometry. The crystal structure of the PAH-belinostat complex (PDB ID: 9T1O), together with structural models for all compounds, provide a framework for rational optimization. These results establish a preclinical proof-of-concept for genotype-guided pharmacological chaperone therapy in PKU and support the feasibility of personalized, variant-specific treatment strategies.

Background Pancreatic cancer continues to be one of the most challenging malignancies to diagnose and treat, and it is often detected at an advanced stage owing to asymptomatic early phases and limitations in current diagnostic tools, highlighting the need for improved diagnostic methodologies. Methods We aimed to evaluate the potential for integrating information from thermal liquid biopsy (TLB) thermograms with clinical biomarkers to improve the diagnosis and prognosis of pancreatic cancer. Serum samples from 381 Danish patients with pancreatic cancer and 325 patients referred with non-organ specific signs and symptoms of cancer, but without confirmed cancer after clinical and computed tomography evaluation, were analyzed. TLB thermograms provided information on the partial excess heat capacity of the serum as a function of temperature. Three classification models were constructed using machine learning algorithms based on variable selection with penalization, applying cross-validation and resampling techniques: iClin Model (age, Eastern Cooperative Oncology Group Performance Status, carbohydrate antigen 19.9, and C-reactive protein), iTLB Model (discordant pairs of temperatures from thermograms), and iTLB + iClin Model (discordant pairs of temperatures and clinical data). Results The iClin Model achieved high diagnostic performance, with a validation area under the curve (AUC) of 0.95 ± 0.01 for differentiating symptomatic controls from patients with pancreatic ductal adenocarcinoma (PDAC), but showed no significant association with overall survival (OS). The iTLB Model showed limited diagnostic performance, with a validation AUC of 0.59 ± 0.03, but was associated with OS in patients with PDAC. The combined iTLB + iClin Model preserved high diagnostic performance, with a validation AUC of 0.95 ± 0.02, sensitivity of 96.20%, specificity of 91.20%, positive predictive value of 90.48%, and negative predictive value of 96.59%. For early-stage PDAC, the iTLB + iClin Model achieved an AUC of 0.93 (95% CI: 0.89–0.98), compared with 0.96 (95% CI: 0.95–0.98) for stages III–IV. Conclusion This study demonstrates the potential of combining specific clinical biomarker information with advanced techniques such as TLB, to improve the accuracy of pancreatic cancer diagnosis and prognosis.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease marked by progressive motor neuron loss, leading to muscle paralysis and respiratory failure. Genetic mutations, notably in the ANXA11 gene, have been implicated in both familial and sporadic ALS forms. ANXA11 functions as a cellular “tether,” orchestrating the transport of RNA-protein complexes and lysosomes through its N-terminal (Nt) and C-terminal (Ct) domains, respectively. This study uncovers a novel calcium-dependent regulatory mechanism governing the intramolecular interaction between these domains. Using biochemical, biophysical, and computational approaches, we suggest that in the absence of calcium, ANXA11 adopts a closed conformation with stable Nt-Ct interactions. Elevated calcium levels induce a conformational shift, disrupting this interaction and exposing binding sites for RNA and membranes. Crucially, we show that the ALS-associated D40G mutation in the Nt domain impairs this calcium-regulated interaction, favoring a persistent open conformation that predisposes to toxic protein aggregation. These findings reveal that calcium acts as a molecular switch modulating ANXA11 conformation and function, providing new insights into its role in ALS pathogenesis and potential therapeutic targets.

Mutations in the AIFM1 gene, encoding the apoptosis-inducing factor (AIF), have been associated with a spectrum of neurometabolic disorders. However, the mechanistic basis underlying their pathogenicity remains poorly understood. In this work, we identified and comprehensively characterized a novel hemizygous AIFM1 mutation c.1006G > A (E336K) in a male patient presenting with a progressive hereditary axonal sensorimotor polyneuropathy with childhood onset, inherited in an X-linked recessive pattern, associated with sensorineural hearing loss and without cognitive impairment. The clinical phenotype was consistent with Charcot-Marie-Tooth disease type 4 (CMTX4). Patient-derived fibroblasts exhibited reduced AIF protein stability despite preserved mRNA expression, impaired growth in OXPHOS-dependent conditions, decreased basal respiration, and altered assembly of mitochondrial respiratory supercomplexes. These defects were accompanied by reduced CHCHD4 protein levels and mitochondrial content. The purified E336K protein exhibited compromised FAD retention, decreased thermal stability, impaired NADH affinity, destabilization of the charge-transfer complex crucial for sustaining the AIF: CHCHD4 interaction, and a shift in coenzyme preference toward NADPH. Structurally, the substitution of Glu336 with Lys remodels the electrostatic environment of the NADH-binding cleft, thereby compromising redox function and weakening CHCHD4 binding. Despite these defects, the protein with the E336K mutation retained DNA binding, nuclease activity, and binding to nuclear partners, although parthanatos induction was attenuated in patient fibroblasts. Collectively, these molecular alterations converge on defective mitochondrial bioenergetics and dynamics, providing a direct mechanistic link to the patient’s clinical evolution. These findings provide a framework for understanding AIFM1-related disorders and pave the way for the development of future personalized molecular therapies.

Abstract Apoptosis-inducing factor is a mitochondrial flavoprotein that links redox metabolism to mitochondrial homeostasis through its interaction with the disulfide relay protein CHCHD4. Although NADH-dependent AIF dimerization has been proposed as the activated state mediating CHCHD4 engagement, whether it is strictly required for productive AIF–CHCHD4 function remains unclear. Here, combining cellular, biochemical and biophysical approaches, we show that disruption of the AIF dimer interface compromises oxidative phosphorylation, respiratory-chain organization and CHCHD4-dependent mitochondrial homeostasis, yet preserves partial AIF function. Our data reveal that the AIF–CHCHD4 system operates as a conformational dynamic redox module in which distinct AIF oligomeric and redox states sustain CHCHD4 activity with different efficiencies. Mechanistically, dimerization is coupled to NADH-dependent conformational changes that regulate coenzyme binding, charge-transfer complex stabilization and catalytic efficiency. In turn, CHCHD4 binding remodels AIF conformational and redox properties, partially compensating for defects in dimer stabilization or redox coupling. Consistently, a peptide derived from the CHCHD4 N-terminus partially restores redox function in a pathogenic AIF variant defective in dimer stabilization, supporting partner-assisted allosteric regulation as a potential therapeutic strategy.

Early detection of colorectal cancer is essential to improving survival, where yet current diagnostic tools show limited performance. This study aimed to enhance diagnostic accuracy by integrating clinical variables with thermogram profiles obtained through serum-based thermal liquid biopsy and analyzed using machine learning models. We evaluated 328 patients with colorectal cancer and 355 symptomatic individuals with non-organ-specific cancer signs but negative diagnostic evaluations, to reproduce clinically relevant decision settings. The combined model showed improved classification performance compared with the use of clinical variables alone, particularly in patients with early-stage disease. In addition, proteomic analysis of samples stratified bythermogram patterns identified proteins associated with survival, including fibrinogen-like protein 1, supporting the biological relevance of these thermodynamic profiles. Together, these findings indicate that integrating serum thermogram information with routine clinical data can modestly strengthen diagnostic assessment and help identify biologically meaningful patient subgroups, offering a promising non-invasive colorectal cancer evaluation.

Arginine iminohydrolases are a family of enzymes involved in the conversion of arginine to citrulline. There are five isoforms in humans (PADI1, 2, 3, 4, and 6). Some of them are observed experimentally in the cytoplasm and in the nucleus of the cell; for moving to the latter location, they must pass through the cell nuclear membrane by using the translocation machinery, mainly formed by the proteins named importins. We have previously described and characterized the isolated PADI4 nuclear localization sequences (NLSs) and their binding to importin α3 (Impα3). By using theoretical predictors, here we foretold the existence of several NLSs in isoforms PADI1, PADI2, and PADI3. These predicted polypeptide regions were chemically synthesized, and the soluble ones were conformationally characterized in isolation. We studied their ability to bind Impα3 and its truncated species (ΔImpα3) without the importin binding domain, by using several biophysical techniques and molecular simulations. The isolated peptides were disordered and monomeric in solution. Moreover, all of them were capable of binding to both importin species with affinities in the low micromolar range, and targeting the canonical NLS binding site for cargo proteins. These findings suggest that the predicted NLS regions could be the sites for the binding of the corresponding intact PADI protein to importin, and therefore, any of the PADI enzymes could be translocated into the nucleus.

Tuberculosis remains a critical global health challenge, which underscores the need for new therapeutic targets. A potential drug target is the rhodanese-like thiosulfate sulfurtransferase SseA, which plays a role in macrophage infection by Mycobacterium tuberculosis (Mtb) and its resistance to oxidative stress. In our research, we identified a protein (Rv3284), herein referred to as SufEMtb, that interacts with SseA and modulates its activity. Sequence analysis and molecular modeling revealed that SufEMtb enhances SseA enzymatic function by binding to its non-catalytic N-terminal domain and favoring an activating conformational change in a regulatory loop of SseA. This interaction appears crucial for effective enzyme activity and the maintenance of redox homeostasis in Mtb, making the SseA-SufEMtb complex a potential target for new therapies.

Vitamin B12 (cobalamin, Cbl) is a coordination compound of the cobalt, located at the center of a corrin ring composed of four pyrrolic-like groups. The cobalt ion can be bound to a variety of upper axial ligands, which vary among different cobalamin forms, including hydroxocobalamin (OHCbl), cyanocobalamin (CNCbl), methylcobalamin (MeCbl), and adenosylcobalamin (AdoCbl). MeCbl and AdoCbl are considered the biologically active forms, serving as cofactors in the metabolism of methylmalonic acid (MMA) and homocysteine (HCY). Impaired conversion of these metabolites leads to their pathological accumulation, resulting in severe cellular damage. This is precisely what occurs in cblC deficiency, a rare inborn disorder caused by mutations in the MMACHC protein, which plays a crucial role in binding and processing the various cobalamin forms. Mutations affecting MMACHC function impair its ability to correctly handle cobalamins, leading to the disease. In this study, we evaluated the impact of various cobalamin forms, specifically AdoCbl, MeCbl, and CNCbl, on the stability and oligomeric organization of the wild type MMACHC protein, using circular dichroism spectroscopy, native gel electrophoresis, and small-angle X-ray scattering. Moreover, isothermal titration calorimetry experiments provided insights into the thermodynamic parameters governing MMACHC binding to these cobalamins. In addition, we also assessed how the R161Q mutation in MMACHC alters the affinity of this protein for the different vitamin B12 forms, leading to decreased stability and impaired homodimerization, a process likely relevant to its functional role. Our findings provide molecular insights into cblC pathogenesis and advance our understanding of MMACHC structure–function relationships.

HDAC8 (histone deacetylase 8), a class I HDAC, is a promising target for different disorders: X-linked intellectual disability, fibrotic disease, cancer, and several neuropathological diseases. The structural and functional similarity between HDACs hinders the development of selective HDAC8 inhibitors. To date, no drugs based on competitive inhibition have been approved. In order to identify weaknesses in HDAC8 amenable to drug discovery, we report here a comprehensive thermodynamic characterization of the structural stability and its modulation by the interaction with its zinc (Zn2+) cofactor using a combination of experimental and computational techniques. HDAC8 represents an interesting example of an allosteric protein in the broad sense, in which its cofactor, the zinc ion, modulates its conformational equilibrium. The native, inactive, zinc-free state is physiologically relevant considering that the intracellular concentration of zinc is very low and may constitute a valid target for drug discovery.

The aggregation of transthyretin (TTR) results in life-threatening transthyretin amyloidosis. Familial forms of the disease arise from point mutations that destabilize the TTR tetramer, leading to its dissociation and/or monomer unfolding and subsequent formation of amyloid fibrils. Small molecules that kinetically stabilize the native tetramer effectively inhibit this aggregation. Although over 300 X-ray crystal structures of TTR have been determined, these data refer to a static structure and do not capture the conformational effects of mutations and ligand binding. Here, we demonstrate that hydrogen-deuterium exchange (HDX) and fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry (MS) offer critical insights into the conformational dynamics associated with TTR amyloidogenic mutations and the binding of kinetic stabilizers. The results indicate that the design of TTR binders should consider the specific conformational traits of each TTR pathogenic variant. We propose that incorporating MS-based techniques into TTR drug discovery will expedite the development of effective pathology-specific aggregation inhibitors.

Flavin ferredoxin-thioredoxin reductases (FFTRs) constitute a unique class of enzymes that transfer electrons from low-potential ferredoxins (Fdxs) to thioredoxins (Trxs) through a flavin cofactor. They are widely distributed across bacterial lineages, including cyanobacteria and anaerobes such as Clostridium, where they function either as the sole Trx reductase or in parallel with canonical NADPH-dependent or iron-sulfur systems. A distinguishing feature of cyanobacterial FFTRs is a C-terminal tail containing a conserved tryptophan (Trp) that engages in a π-stacking interaction with the flavin-a motif absent in clostridial orthologs. In this study, we use the cyanobacterial FFTR from Gloeobacter violaceus as a model to demonstrate that the C-terminal tail and its conserved aromatic residue modulate the FAD electronic environment, electron transfer efficiency, and Fdx donor interactions. Mutants lacking the tail or Trp exhibited increased flavin solvent exposure, a redox potential shift of over 200 mV toward less negative values, altered reduction kinetics, impaired electron flow to the redox-active disulfide, and reduced specificity for Fdx binding. These results reveal a dual role for the C-terminal tail: it establishes a productive donor-binding interface and shapes the FAD environment to meet the thermodynamic and kinetic requirements for efficient intramolecular electron transfer to the redox-active disulfide. Collectively, these findings provide mechanistic insight into how peripheral structural features, such as FAD-aromatic π-stacking interactions, govern flavin reactivity, donor specificity, and redox behavior in cyanobacterial FFTRs, underscoring their relevance and offering a framework for engineering redox-active biocatalysts for synthetic biology and metabolic applications.