https://hdl.handle.net/11000/5252 2026-09-08T09:43:55Z 2026-09-08T09:43:55Z Valls-Chiva, Angeles Hornos, Felipe Hueso, Jose L. Martín-Pardillos, Ana Santamaria, Jesus https://hdl.handle.net/11000/40348 2026-09-03T01:03:33Z 2026-09-02T15:40:10Z

Título : Immobilization of glucose oxidase and catalase on magnetite nanoparticles as functional catalyst supports Autor : Valls-Chiva, Angeles; Hornos, Felipe; Hueso, Jose L.; Martín-Pardillos, Ana; Santamaria, Jesus Resumen : In this work, we have prepared magnetite nanoparticles (MNPs) coated with polyethylenimine (PEI) to obtain a positively charged surface and showed their suitability as functional supports to successfully immobilize glucose oxidase (GOx) and catalase (CAT) enzymes. We have compared two immobilization strategies, namely electrostatic binding and covalent attachment mediated by glutaraldehyde cross-linking. To quantify the amount of immobilized enzyme (qmax), we have developed a methodological approach that minimizes the dependence of the qmax value on the equilibrium constant of the adsorption process (K). Catalytic studies showed high retention of enzymatic activity (100% for covalent binding and 75% for electrostatic binding), indicating that the immobilization protocol preserves the native conformation of the enzyme. Furthermore, the covalent strategy demonstrated stable binding even when the nanohybrid was subjected to extreme conditions (pH 3), retaining 91% of the enzyme on the surface, compared to 6% when immobilized electrostatically. These results highlight the importance of surface engineering in the design of magnetic biocatalysts for potential application in starvation or oxygen generation therapies.

2026-09-02T15:40:10Z Álvarez-Rodríguez, Maria Gabriela Vega, Sonia Hornos, Felipe Velazquez-Campoy, Adrian Rizzuti, Bruno Neira, José L. https://hdl.handle.net/11000/40347 2026-09-03T01:03:32Z 2026-09-02T15:39:16Z

Título : Unfolding Behavior and Conformational Changes Under Different Denaturing Conditions of MAPK 1 (MEK1) Autor : Álvarez-Rodríguez, Maria Gabriela; Vega, Sonia; Hornos, Felipe; Velazquez-Campoy, Adrian; Rizzuti, Bruno; Neira, José L. Resumen : 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.

2026-09-02T15:39:16Z Álvarez-Rodríguez, María Gabriela Vega, Sonia Hornos, Felipe Fienco-Bacusoy, Augusto Abian, Olga Velazquez-Campoy, Adrian Rizzuti, Bruno Neira, José L. https://hdl.handle.net/11000/40346 2026-09-03T01:03:31Z 2026-09-02T15:38:12Z

Título : Citrullination at the Nuclear Localization Signal of the Inhibitor of Growth 4 (ING4) Interferes With its Binding to Importin a3 Autor : Álvarez-Rodríguez, María Gabriela; Vega, Sonia; Hornos, Felipe; Fienco-Bacusoy, Augusto; Abian, Olga; Velazquez-Campoy, Adrian; Rizzuti, Bruno; Neira, José L. Resumen : 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.

2026-09-02T15:38:12Z Encinas-Gimenez, Miguel Garcia-Peiro, Jose I. Hueso, Jose L. Caleiras, Eduardo Notario, Laura Hornos, Felipe Martin-Duque, Pilar Dela Vieja, Antonio Santamaria, Jesus https://hdl.handle.net/11000/40345 2026-09-03T01:03:30Z 2026-09-02T15:36:53Z

Título : Ultrasmall Platinum Nanoparticles for Radiation-Enhanced Cancer Therapy Autor : Encinas-Gimenez, Miguel; Garcia-Peiro, Jose I.; Hueso, Jose L.; Caleiras, Eduardo; Notario, Laura; Hornos, Felipe; Martin-Duque, Pilar; Dela Vieja, Antonio; Santamaria, Jesus Resumen : Despite decades of intense research, cancer remains a major global health challenge, with a clear need for new and more efficient therapies. Among emerging approaches, high atomic number (Z) nanomaterials capable of modulating the interaction between radiation and matter have attracted growing interest as radiotherapy enhancers. In this work, ultra-small Platinum-based nanoparticles with a metallic core diameter of 2–3 nm were synthesized from H2PtCl6 using polyvinylpyrrolidone as stabilizer. This nanomaterial exhibits low acute toxicity, catalase-like activity and enhances DNA damage under pre-clinical X-ray irradiation, increasing the antitumoral effect of X-ray alone. In vitro and in vivo studies, including intratumoral and intravenous administration, demonstrate improved tumor control and prolonged survival without detectable systemic toxicity under the tested conditions, establishing ultra-small Pt-nanoparticles as efficient materials for radiotherapy enhancement.

2026-09-02T15:36:53Z Guerrero-López, Paula Garcia-Peiro, Jose I. Hornos, Felipe Hueso, Jose L. Santamaria, Jesus Garcia-Aznar, J.Manuel https://hdl.handle.net/11000/40344 2026-09-03T01:03:29Z 2026-09-02T15:35:42Z

Título : Copper–Collagen Interactions Regulate the Mechanical and Invasive Properties of Tumor Spheroids Autor : Guerrero-López, Paula; Garcia-Peiro, Jose I.; Hornos, Felipe; Hueso, Jose L.; Santamaria, Jesus; Garcia-Aznar, J.Manuel Resumen : Copper is a key trace element with established cytotoxic properties, yet its interactions with the extracellular matrix and their implications for the evolution of 3D culture models remain poorly understood. Here, we study how copper entrapped in collagen can regulate the structural and invasive properties of 3D tumor spheroids. Our findings reveal that copper influences cytoskeletal organization, protrusion dynamics, and epithelial–mesenchymal transitions. Interestingly, we also highlight its dual capacity to either suppress or enhance invasive behavior depending on the context of exposure. These results position copper as a contextdependent modulator of tumor progression and underscore the importance of dose and timing in determining therapeutic outcomes. By integrating a physiologically relevant 3D model, this study provides new insights into copper–ECM interactions and identifies potential strategies to exploit them for cancer therapy.

2026-09-02T15:35:42Z Álvarez-Rodríguez, María Gabriela Estaras, Matías Hornos, Felipe Rizzuti, Bruno Santofimia-Castaño, Patricia Iovanna, Juan L. Neira, José L. https://hdl.handle.net/11000/40343 2026-09-03T01:03:29Z 2026-09-02T15:34:32Z

Título : Evaluating the therapeutic role of salvianolic acid A on pancreatic cancer cells through interaction with the intrinsically disordered protein NUPR1 Autor : Álvarez-Rodríguez, María Gabriela; Estaras, Matías; Hornos, Felipe; Rizzuti, Bruno; Santofimia-Castaño, Patricia; Iovanna, Juan L.; Neira, José L. Resumen : The nuclear protein 1 (NUPR1) is an intrinsically disordered protein (IDP) involved in stress processes in the cell. We have been developing, by using organic chemistry, several small molecules which hamper in vitro and in cell- based assays NUPR1 biomolecular interactions by targeting its two hot-spots around Ala33 and Thr68. In this work, we used a natural compound, salvianolic acid A (SAA), to target NUPR1. SAA has anti-cancer and anti- inflammatory properties. Binding in vitro was monitored by using fluorescence, isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR); the affinity was in the low micromolar range, as shown by fluorescence and ITC. The NMR spectra of NUPR1, in the absence and in the presence of SAA, indicated that binding involved several polypeptide patches of NUPR1, as well as its hot-spots; however, the binding did not alter the disordered nature of the protein. Moreover, molecular docking simulations provided a model of the binding at the atomic level. Results of proliferation cellular assays with MIA PaCa-2 cells indicated that the IC was ~20 μ 50 M, confirming the affinity values found by fluorescence and ITC. SAA was also capable of inhibiting the formation of stress granules (SGs) triggered by NUPR1. Our work shows that a plant-derived molecule can target an IDP involved in cancer, with affinities in the low micromolar range. Our model compound can hamper the pathological state (SG formation) triggered by this IDP, and indicates that the search for potential inhibitors of disordered proteins could be further extended to bioactive natural compounds.

2026-09-02T15:34:32Z Garcia-Peiro, Jose I. Sancho-Albero, María Miguel, Silvia Mosseri, Andrea Hornos, Felipe Contreras-Montoya, Rafael Hueso, Jose L. Santamaria, Jesus https://hdl.handle.net/11000/40342 2026-09-03T01:03:27Z 2026-09-02T15:33:27Z

Título : Evolving Platinum-Copper Nanostructures for Enhanced Photothermal Therapy and Controlled Copper Release in Cancer Therapy Autor : Garcia-Peiro, Jose I.; Sancho-Albero, María; Miguel, Silvia; Mosseri, Andrea; Hornos, Felipe; Contreras-Montoya, Rafael; Hueso, Jose L.; Santamaria, Jesus Resumen : Nanotechnology provides a panoply of new tools in cancer therapy, enabling advanced treatments such as chemodyamic therapy (CDT), radiation therapy (RT), photothermal therapy (PTT) or photodynamic therapy (PDT). The therapeutic effect can be increased by combining one or more of these actions and this is usually considered already from the design of the nanoparticles (NPs). Bimetallic NPs are obvious candidates in this respect, being able to trigger a multifunctional response associated to each metal and achieving a synergistic action in combined therapies. In this work, we designed platinum-copper bimetallic NPs (PtCu NPs) capable to evolve during treatment and fulfill a dual role as chemotherapeutic agents, (controlled release of ionic Cu), and as tumor ablation agents under near infrared (NIR) irradiation. Tuning the synthesis conditions demonstrated the influence of metal composition and alloying degree on the Cu release pattern when exposed to physiological media. The Cu ions released disrupt the redox balance in cells by generating reactive hydrogen species (likely hydroxyl radicals •OH through reaction with H2O2 overexpressed in the tumor microenvironment) while simultaneously oxidizing glutathione (GSH). After Cu release, the remaining Pt-rich structures present enhanced photothermal (PT) response. The efficacy of the combined therapy enabled by these NPs has been evaluated in vitro and in vivo.

2026-09-02T15:33:27Z Carpio, Laureano E. Álvarez-Rodríguez, María Gabriela Recio-Moreno, David Hornos, Felipe Georgieva-Nikolova, Izabel Camara-Artigas, Ana Rejas, Virginia Vidal, Miguel Palomino-Schätzlein, Martina Romero, Camino de Juan Neira, José L. https://hdl.handle.net/11000/40341 2026-09-03T01:03:28Z 2026-09-02T15:33:00Z

Título : The binding of the salvianolic acid A to the citrullinating enzyme PADI4 as a potential treatment for cancer Autor : Carpio, Laureano E.; Álvarez-Rodríguez, María Gabriela; Recio-Moreno, David; Hornos, Felipe; Georgieva-Nikolova, Izabel; Camara-Artigas, Ana; Rejas, Virginia; Vidal, Miguel; Palomino-Schätzlein, Martina; Romero, Camino de Juan; Neira, José L. Resumen : Cancer cells must maintain molecular mechanisms relying on an energy trade-off between resistance and key functions to survive. PADI4 (peptidyl arginine deiminase 4) is an enzyme implicated in the conversion of arginine to citrulline (citrullination), that has been related to the development of several types of cancers and in numerous inflammatory routes. Salvianolic acid A (SAA) is a stilbenoid, obtained from the radix of Salvia miltiorrhiza, with several anti-cancer and anti-inflammatory features. In this work, we report and characterize the interaction between PADI4 and SAA. The binding reaction was followed by using several biophysical probes, namely fluorescence, isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR); the affinity constant was ∼600 nM (ITC). The enzyme binding to the C-terminal region of RING1B (E3 ubiquitin-protein ligase really interesting new gene 1 B), which is a substrate of PADI4, was altered in the presence of SAA, as shown by NMR. Colorimetric assays indicated that SAA was an activator of the citrullinating activity of PADI4. Moreover, the results in silico suggested that binding of SAA to the dimeric PADI4 occurred at the dimerization interface, and not at its active site, potentially stabilizing the dimeric, active form of the protein. The effect of SAA was shown to be cell-line-dependent, and its presence in glioblastoma cells hampered the binding between intact RING1B and the enzyme. Altogether, this work shows that PADI4 was capable of binding to SAA, at a novel site, and opens the venue to develop its use in modulating in vitro and in cellular assays the deimination processes.

2026-09-02T15:33:00Z Hornos, Felipe Rizzuti, Bruno Neira, José L. https://hdl.handle.net/11000/40340 2026-09-03T01:03:26Z 2026-09-02T15:29:04Z

Título : Importin α3 Is Tolerant to Nuclear Localization Signal Chirality Autor : Hornos, Felipe; Rizzuti, Bruno; Neira, José L. Resumen : Several carrier proteins are involved in nuclear translocation from the cytoplasm to the nucleus in eukaryotic cells. We have previously demonstrated the binding of several intact folded and disordered proteins to the human isoform importin α3 (Impα3); furthermore, disordered peptides, corresponding to their nuclear localization signals (NLSs), also interact with Impα3. These proteins and their isolated NLSs also bind to the truncated importin species ∆Impα3, which does not contain the N-terminal disordered importin binding domain (IBB). In this work, we added a further ‘layer’ of conformational disorder to our studies, testing whether the isolated D-enantiomers of NLSs of selected proteins, either folded or unfolded, were capable of binding to both Impα3 and ∆Impα3. The D-enantiomers, like their L-form counterparts, were monomeric and disordered in isolation, as shown by nuclear magnetic resonance (NMR). We measured the ability of such D-enantiomeric NLSs to interact with both importin species by using fluorescence, biolayer interferometry (BLI), isothermal titration calorimetry (ITC), and molecular simulations. In all cases, the binding affinities were within the same range as those measured for their L-isomer counterparts for either Impα3 or ∆Impα3, and the binding locations corresponded to the major NLS binding site of the protein. Thus, the stereoisomeric nature is not important in defining the binding of proteins to the main component of classical cellular translocation machinery, although the primary structure of the hot-spot site for NLS binding of importin is well defined.

2026-09-02T15:29:04Z Pavia, Natalia Potenza, Alberto Hornos, Felipe Poveda, José A. Gonorazky, Gabriela Neira, José L. Giudici, Ana M. Beligni, MaríaVerónica https://hdl.handle.net/11000/40339 2026-09-03T01:03:25Z 2026-09-02T15:28:11Z

Título : The Diacylglycerol Acyltransferase 3 of Chlamydomonas reinhardtii Is a Disordered Protein Capable of Binding to Lipids Derived from Chloroplasts Autor : Pavia, Natalia; Potenza, Alberto; Hornos, Felipe; Poveda, José A.; Gonorazky, Gabriela; Neira, José L.; Giudici, Ana M.; Beligni, MaríaVerónica Resumen : Understanding triacylglycerol (TAG) metabolism is crucial for developing algae as a source of biodiesel. TAGs are the main reservoir of energy in most eukaryotes. The final, rate-limiting step in the formation of TAGs is catalyzed by 1,2-diacylglycerol acyltransferases (DGATs). In the green alga Chlamydomonas reinhardtii, DGAT3 is phylogenetically related to plant DGAT3 but unrelated to other DGATs from eukaryotes, such as DGAT1 and DGAT2. In this study, we described the conformational preferences and the lipid-binding features of the DGAT3 from C. reinhardtii. To characterize its conformational stability and structural features, we used several biophysical probes, namely, fluorescence, circular dichroism (CD), and differential scanning calorimetry (DSC). Our results showed that the protein was mainly disordered, containing a small population of folded conformations in a narrow pH range (pH 8 to 10). The conformational stability of the folded structure of DGAT3 was very low, as shown by urea or guanidinium denaturations. Thermal denaturation, followed by fluorescence or CD, as well as calorimetric denaturation, followed by DSC, did not yield any transition in the pH range where DGAT3 acquired a “native-like” conformation. Furthermore, we used two approaches to demonstrate the interaction of DGAT3 with lipid membranes at the pH at which it had acquired a “native-like” conformation. The first involved the measurement of anisotropy and fluorescence quenching of the protein. The second approach focused on examining possible modifications of the biophysical properties of lipids due to their interaction with DGAT3, through anisotropy measurements and leakage assays. Both methods produced consistent results, suggesting that DGAT3 preferentially interacted with negatively charged membranes. These results will allow the design of a more efficient and stable DGAT3, as well as an in-depth understanding of how the metabolism of TAGs is accomplished in C. reinhardtii.

2026-09-02T15:28:11Z