https://hdl.handle.net/11000/30622 2026-04-03T16:49:39Z 2026-04-03T16:49:39Z Madruga, Enrique García-Rubia, Alfonso Sánchez-Núñez, Carlos Martínez-González, Loreto Fernández-Escamilla, Ana María Lastres-Becker, Isabel Gil, Carmen Martínez, Ana https://hdl.handle.net/11000/39585 2026-03-26T02:06:57Z 2026-03-25T13:47:05Z

Título : Brain Permeable SGK1 Inhibitors: A Promising Therapeutic Strategy for Neurodegenerative Diseases Autor : Madruga, Enrique; García-Rubia, Alfonso; Sánchez-Núñez, Carlos; Martínez-González, Loreto; Fernández-Escamilla, Ana María; Lastres-Becker, Isabel; Gil, Carmen; Martínez, Ana Resumen : A major challenge in modern medicine is developing new therapies for aging-related diseases such as neurodegenerative disorders, whose prevalence increases with longer life expectancy. Although kinase inhibitors have achieved clinical success, their development for central nervous system (CNS) disorders remains limited due to the complexity of kinase networks and poor blood− brain barrier (BBB) permeability. Serum/glucocorticoid-regulated kinase 1 (SGK1) participates in multiple signaling pathways but remains an underexplored target in neurodegeneration. Following a mixed ligand- and structure-based virtual screening, we have previously identified a brain-penetrant SGK1 inhibitor. A medicinal chemistry program based on hit expansion and optimization for BBB permeability reported here has generated a new family of SGK1 inhibitors as chemical probes that enable the investigation of SGK1’s role in neurological disorders and serve as promising starting points for drug development. These findings highlight SGK1 as a potential therapeutic target for neurodegenerative diseases, such as Alzheimer’s disease.

2026-03-25T13:47:05Z Cianferoni, Damiano Vizarraga, David Fernández-Escamilla, Ana Mª Fita, Ignacio Hamdani, Rahma Reche, Raul Delgado, Javier Serrano, Luis https://hdl.handle.net/11000/39409 2026-02-24T02:08:47Z 2026-02-23T17:35:32Z

Título : Artificial intelligence and first-principle methods in protein redesign: A marriage of convenience? Autor : Cianferoni, Damiano; Vizarraga, David; Fernández-Escamilla, Ana Mª; Fita, Ignacio; Hamdani, Rahma; Reche, Raul; Delgado, Javier; Serrano, Luis Resumen : Since AlphaFold2’s rise, many deep learning methods for protein design have emerged. Here, we validate widely used and recognized tools, compare them with first-principle methods, and explore their combinations, focusing on their effectiveness in protein redesign and potential for therapeutic repurposing. We address two challenges: evaluating tools and combinations ability to detect the effects of multiple concurrent mutations in protein variants, and leveraging large-scale datasets to compare modelingfree methods, namely force fields, which handle point mutations well with limited backbone rearrangement, and inverse folding tools, which excel at native sequence recovery but may struggle with non-natural proteins. Debuting TriCombine, a tool that identifies residue triangles in input structures, matches them to a structural database, and scores mutants based on substitution frequencies, we shortlisted candidates, modeled them with FoldX, and generated 16 SH3 mutants carrying up to 9 concurrent substitutions. The dataset was expanded to include 36 mutants and 11 crystal structures (7 newly solved), along with a parallel set of multiple non-concurrent mutants from three additional proteins. For broader validation, we analyzed 160,000 four-site GB1 mutants and 163,555 (single and double) variants across 179 natural and de novo domains. We show that combining AIbased modeling tools with force field scoring functions yields the most reliable results. Inverse folding tools perform very well but lose accuracy on less-represented proteins. First-principle force fields like FoldX remain the most accurate for point mutations. All methods perform worse when applied to unsolved de novo models, underscoring the need for hybrid strategies in robust protein design.

2026-02-23T17:35:32Z Espinosa-Urgel, Manuel Serrano, Luis Ramos, Juan Luis Fernández-Escamilla, Ana Mª https://hdl.handle.net/11000/39408 2026-02-24T02:08:46Z 2026-02-23T17:14:17Z

Título : Engineering Biological Approaches for Detection of Toxic Compounds: A New Microbial Biosensor Based on the Pseudomonas putida TtgR Repressor Autor : Espinosa-Urgel, Manuel; Serrano, Luis; Ramos, Juan Luis; Fernández-Escamilla, Ana Mª Resumen : Environmental contamination by toxic organic compounds and antimicrobials is one of the causes for the recent surge of multidrug-resistant pathogenic bacteria. Monitoring contamination is therefore the first step in containment of antimicrobial resistance and requires the development of simple, sensitive, and quantitative tools that detect a broad spectrum of toxic compounds. In this study, we have engineered a new microbial biosensor based on the ttgR-regulated promoter that controls expression of the TtgABC extrusion efflux pump of Pseudomonas putida, coupled to a gfp reporter. The system was introduced in P. putida DOT-T1E, a strain characterized by its ability to survive in the presence of high concentrations of diverse toxic organic compounds. This whole-cell biosensor is capable to detect a wide range of structurally diverse antibiotics, as well as compounds such as toluene or flavonoids.

2026-02-23T17:14:17Z Espinosa-Urgel, Manuel Serrano, Luis Ramos, Juan Luis Fernández-Escamilla, Ana Mª https://hdl.handle.net/11000/39333 2026-02-17T02:08:18Z 2026-02-16T17:03:17Z

Título : Engineering Biological Approaches for Detection of Toxic Compounds: A New Microbial Biosensor Based on the Pseudomonas putida TtgR Repressor Autor : Espinosa-Urgel, Manuel; Serrano, Luis; Ramos, Juan Luis; Fernández-Escamilla, Ana Mª Resumen : Environmental contamination by toxic organic compounds and antimicrobials is one of the causes for the recent surge of multidrug-resistant pathogenic bacteria. Monitoring contamination is therefore the first step in containment of antimicrobial resistance and requires the development of simple, sensitive, and quantitative tools that detect a broad spectrum of toxic compounds. In this study, we have engineered a new microbial biosensor based on the ttgR-regulated promoter that controls expression of the TtgABC extrusion efflux pump of Pseudomonas putida, coupled to a gfp reporter. The system was introduced in P. putida DOT-T1E, a strain characterized by its ability to survive in the presence of high concentrations of diverse toxic organic compounds. This whole-cell biosensor is capable to detect a wide range of structurally diverse antibiotics, as well as compounds such as toluene or flavonoids.

2026-02-16T17:03:17Z Cañadas-Garre, Marisa Fernández-Escamilla, Ana Mª Fernández-Ballester, Gregorio Becerra-Massare, Patricia García-Calvente, Carlos Ramos, Juan Luis Llamas-Elvira, José Manuel https://hdl.handle.net/11000/39332 2026-02-17T02:08:17Z 2026-02-16T16:53:51Z

Título : Novel BRAFI599Ins Mutation Identified in a Follicular Variant of Papillary Thyroid Carcinoma: A Molecular Modeling Approach Autor : Cañadas-Garre, Marisa; Fernández-Escamilla, Ana Mª; Fernández-Ballester, Gregorio; Becerra-Massare, Patricia; García-Calvente, Carlos; Ramos, Juan Luis; Llamas-Elvira, José Manuel Resumen : Objective: BRAF mutations are the most common genetic alteration found in papillary thyroid carcinoma (PTC). Approximately, 90% correspond to BRAFV600E, although other less common BRAF mutations have been described. The aim of this study was to describe a new mutation on BRAF gene discovered on the previous thyroid cytology of a patient diagnosed with a follicular variant of PTC (FV-PTC). Methods: The mutation was identified by independent cloning of the 2 alleles and direct sequencing in the previous cytology and tumor tissue samples from a patient diagnosed with FV-PTC. To elucidate the effect of the mutation on the structure and hence on the activating mechanism of the protein, the structures of BRAFI599Ins, BRAFT599Ins, BRAFV599Ins and BRAFV600E were modeled by using the reconstructed wild-type BRAF (BRAFWT) crystal structure. Results: The novel mutation in BRAF consisted in the in-frame insertion of 3 nucleotides (TAA) after nucleotide 1795, resulting in the incorporation of an extra isoleucine residue at position 599 (BRAFI599Ins) of the protein. The structural comparison of BRAFI599Ins, BRAFT599Ins, BRAFV599Ins with BRAFWT, and BRAFV600E models revealed that the overall shape of the kinase was conserved in the protein produced by this novel mutation, except for the displacement of the activation loop (A-loop), as a direct consequence of the increase in loop size, and the exposition of 1 of the 2 residues involved in BRAF activation (T599), probably facilitating its phosphorylation. Conclusion: BRAFI599Ins mutation constitutes a new BRAF mutation affecting the length of the A-loop, which most likely facilitates BRAF activation by altering the A-loop conformation.

2026-02-16T16:53:51Z Ramos, Juan Luis Daniels, Craig Krell, Tino Duque, Estrella Godoy, Patricia de la Torre, Jesús Fernández-Escamilla, Ana Mª Daddaoua, Abdelali Navarro-Avilés, Gloria Fillet, Sandy Pini, Cecilia Molina-Henares, María Antonia Lacal, Jesús Busch, Andreas Silva-Jiménez, Hortencia Rodríguez, Sara Molina, Lázaro Bursakov, Sergey A. Roca, Amalia Segura, Ana https://hdl.handle.net/11000/39315 2026-02-14T02:07:05Z 2026-02-13T19:14:44Z

Título : 8.3 Molecular Responses to Solvent Stress: Strategies for Living in Unpalatable Substrates Autor : Ramos, Juan Luis; Daniels, Craig; Krell, Tino; Duque, Estrella; Godoy, Patricia; de la Torre, Jesús; Fernández-Escamilla, Ana Mª; Daddaoua, Abdelali; Navarro-Avilés, Gloria; Fillet, Sandy; Pini, Cecilia; Molina-Henares, María Antonia; Lacal, Jesús; Busch, Andreas; Silva-Jiménez, Hortencia; Rodríguez, Sara; Molina, Lázaro; Bursakov, Sergey A.; Roca, Amalia; Segura, Ana Resumen : This review article covers some general conclusions reached in research studies with respect to a number of solvent-tolerant Pseudomonas strains. The seminal observation was performed by Inoue and Korikoshi 1989 when they described in Nature a bacterium, belonging to the genus Pseudomonas, which was able to thrive in the presence of high concentrations of toluene. This property of extreme tolerance to solvents makes this Pseudomonas strain the first extremophile identified as able to survive in the presence of highly toxic solvents. Following this seminal observation, other solvent-tolerant strains, such as Pseudomonas putida DOT-T1E, S12, GM1, and MTB6, were described as able to grow in the presence of highly toxic solvents such as p-xylene (log Pow 3.15), styrene (log Pow 3.0), octanol (log Pow 2.92), and toluene (log Pow 2.69) (Aono et al. 1992; Cruden et al. 1992; Huertas et al. 2000; Isken and de Bont 1996; Kim et al. 1998; Ramos et al. 1995; Weber et al. 1994). These microbes can reach high cell densities in culture medium in the presence of 0.3% (v/v) of the mentioned solvents, a concentration that kills most of the microbes we work with in our laboratories. This solvent-tolerant trait allows us to consider this set of Pseudomonas strains as extremophiles. Normally solvent toxicity is due to these chemicals dissolving in the cell membranes, disorganizing them and altering the electron flow. They also prevent ATP synthesis and irrevocably provoke cell death. Currently, there is an inherent interest in deciphering the basis for survival and growth of extremophile microbes in such harsh conditions, particularly for the exploitation of these microbes in the bioremediation of heavily-polluted sites and in the biotransformation of water-insoluble compounds into added-value products. The growing interest in biodegradation derives from the fact that many pollutants are toxic above a certain threshold, killing many living organisms. The use of solvent-tolerant microorganisms, provided with an arsenal of enzymes to deal with pollutants, represents a powerful tool for in situ pollutant removal. In the case of biotransformation, many substrates of interest are poorly soluble in water and the use of a double-phase system, made of water and an organic solvent for the production of high added-value chemicals is of great interest in green chemistry. Among some examples we can cite the use of solvent-tolerant microorganisms to produce catechols or to carry out biotransformation of aromatic hydrocarbons including nitro substituted ones (Ju and Parales 2006; Neumann et al. 2005, 2006; Ramos-Gonza´lez et al. 2003; Ruhl et al. 2009; Rojas et al. 2004; Verhoef et al. 2009; Wierckx et al. 2005). Notas: Editor: Koki Horikoshi

2026-02-13T19:14:44Z Fernández-Escamilla, Ana Mª Villegas, Virtudes Martínez, José C. van Nuland, Nico A. J. Conejero-Lara, Francisco Avilés, Francisco X. Serrano, Luis Filimonov, Vladimir V. Mateo, Pedro L. https://hdl.handle.net/11000/39314 2026-02-14T02:07:07Z 2026-02-13T18:58:21Z

Título : Thermodynamic analysis of helix-engineered forms of the activation domain of human procarboxypeptidase A2 Autor : Fernández-Escamilla, Ana Mª; Villegas, Virtudes; Martínez, José C.; van Nuland, Nico A. J.; Conejero-Lara, Francisco; Avilés, Francisco X.; Serrano, Luis; Filimonov, Vladimir V.; Mateo, Pedro L. Resumen : Thermodynamic characterization of the activation domain of human procarboxypeptidase A2, ADA2h, and its helix-engineered mutants was carried out by differential scanning calorimetry. The mutants were engineered by changing residues in the exposed face of the two a helices in order to increase their stability. At neutral and alkaline pH the three mutants, a-helix 1 (M1), a-helix 2 (M2) and a-helix 1 and a-helix 2 (DM), were more stable than the wild-type domain, in the order DM, M2, M1 and wild-type. Under these conditions the CD and NMR spectra of all the variants are very similar, indicating that this increase in stability is not the result of gross structural changes. Calorimetric analysis shows that the stabilizing effect of mutating the water-exposed surfaces of the helices seems to be mainly entropic, because the mutations do not change the enthalpy or the increase in heat capacity of denaturation. The unfolding behavior of all variants changes under acidic conditions: whereas wild-type and M1 have a strong tendency to aggregate, giving rise to a b conformation upon unfolding, M2 and DM unfold reversibly, M2 being more stable than DM. CD and NMR experiments at pH 3.0 suggest that a region involving residues of the second and third b strands as well as part of a-helix 1 changes its conformation. It seems that the enhanced stability of the altered conformation of M2 and DM reduces the aggregation tendency of ADA2h at acidic pH.

2026-02-13T18:58:21Z Prieto-Vega, Samuel Berzal-Herranz, Alfredo Garrido, Juan José Arias, Armando Grande-Pérez, Ana Fernández-Escamilla, Ana Mª Montoya, María https://hdl.handle.net/11000/39313 2026-02-14T02:07:07Z 2026-02-13T18:43:43Z

Título : West Nile virus unmasked: from gene variability to future challenges Autor : Prieto-Vega, Samuel; Berzal-Herranz, Alfredo; Garrido, Juan José; Arias, Armando; Grande-Pérez, Ana; Fernández-Escamilla, Ana Mª; Montoya, María Resumen : West Nile virus (WNV) is a mosquito-borne orthoflavivirus with a complex transmission cycle involving avian reservoirs and mosquito vectors. Although no precise global infection figure exists, conservative estimates based on seroprevalence data suggest between 4 and 16 million infections annually. With an approximate mortality rate of 6–7% among reported cases, WNV poses a significant public health concern across continents. This review provides a comprehensive overview of WNV molecular biology, including genome organization, protein maturation, replication mechanisms, the functional roles of untranslated regions (UTRs) and post-translational modifications in viral adaptation. Particular attention is given to intrahost genetic variability and the quasispecies nature of WNV as key drivers of immune evasion and viral evolution. The ecological and epidemiological dynamics of WNV are also discussed in the context of climate change and its impact on vector distribution and global viral spread. Additionally, the review details clinical manifestations, pathogenesis, diagnostic tools, and current therapeutic strategies. Emerging approaches for prevention and control are explored, including entomological surveillance, vaccine development, and novel antiviral candidates such as targeted peptides, antibodies and lethal mutagenesis. Given the pressing challenges associated with WNV, this review underscores the importance of integrated One Health surveillance systems and accelerated vaccine development to mitigate future outbreaks, highlighting the intersection of virology, immunology, ecology, and global health.

2026-02-13T18:43:43Z Molina-Henares, María Antonia Ramos-González, María Isabel Daddaoua, Abdelali Fernández-Escamilla, Ana Mª Espinosa-Urgel, Manuel https://hdl.handle.net/11000/39312 2026-02-14T02:07:05Z 2026-02-13T18:35:09Z

Título : FleQ of Pseudomonas putida KT2440 is a multimeric cyclic diguanylate binding protein that differentially regulates expression of biofilm matrix components Autor : Molina-Henares, María Antonia; Ramos-González, María Isabel; Daddaoua, Abdelali; Fernández-Escamilla, Ana Mª; Espinosa-Urgel, Manuel Resumen : The intracellular signal molecule cyclic di-GMP (c-di-GMP) is an important element in regulation of biofilm formation by bacteria. In Pseudomonas aeruginosa, FleQ functions as a c-di-GMP-dependent transcriptional regulator of expression of flagellar genes and the exopolysaccharide (EPS) Pel, a component of the biofilm extracellular matrix. In the plant-beneficial bacterium Pseudomonas putida KT2440, a mutation in fleQ reduces biofilm formation and colonization of plant surfaces. Using isothermal titration calorimetry and electrophoretic mobility shift assays, we show in this work that FleQ of P. putida interacts with c-di-GMP and directly binds the promoter regions of flagellar and EPS genes. Data obtained by analytical gel filtration and ultracentrifugation indicate that FleQ is in multiple oligomeric states in solution (dimers, tetramers and hexamers), which do not show altered equilibrium in the presence of c-di-GMP. DNA binding is independent of c-diGMP, although it is favored by the second messenger in the case of the promoter of the operon responsible for synthesis of the species-specific EPS Pea. Analysis of expression using transcriptional fusions showed an influence of FleQ upon two of the four EPS operons under regular growth conditions. Finally, a consensus sequence for promoter recognition by FleQ in P. putida is also proposed.

2026-02-13T18:35:09Z Sánchez-Hidalgo, Marina Martínez-Bueno, Manuel Fernández-Escamilla, Ana Mª Valdivia, Eva Serrano, Luis Maqueda, Mercedes https://hdl.handle.net/11000/39311 2026-02-14T02:07:04Z 2026-02-13T18:34:46Z

Título : Effect of replacing glutamic residues upon the biological activity and stability of the circular enterocin AS-48 Autor : Sánchez-Hidalgo, Marina; Martínez-Bueno, Manuel; Fernández-Escamilla, Ana Mª; Valdivia, Eva; Serrano, Luis; Maqueda, Mercedes Resumen : Background: Bacteriocins are antimicrobial peptides produced by bacteria and have a relatively narrow range of activity against closely related strains. AS-48 is a circular bacteriocin produced by Enterococcus faecalis that acts against many Gram-positive and some Gram-negative bacteria, and could well serve as a natural food preservative and antimicrobial agent. The structure of AS-48 is a fivehelix bundle in which a hypothetical plane containing the Ca atoms of E4, E20, E49 and E58 segregates a patch of positively charged residues from the rest of the hydrophobic or uncharged surface residues. Objectives: The aim of this study is to investigate the significance of the four glutamic residues with regard to the potency, stability and functionality of enterocin AS-48. Methods: Four genetically engineered variants of AS-48 were obtained by replacing each glutamic residue with alanine by site-directed mutagenesis. Each mutant peptide was purified from E. faecalis cultures. The activity of highly concentrated samples and the MIC were determined against nine bacterial strains by the spot-assay method. Structural studies were made with circular dichroism (CD) spectroscopy. Results: Occasional alterations to the net charge of AS-48 did not significantly affect its activity when high concentrations of bacteriocin were used. Nevertheless, according to the MIC values, three of the four mutated peptides showed weaker activity against the majority of the Gram-positive bacteria tested. CD spectroscopy showed that the derivatives were well structured, in a similar way to those of the native molecule, with no modifications in their helix content. Conclusions: The spatial location of the Glu residues rather than their negative charge played a critical role in AS-48 target-cell specificity and bactericidal activity, because the replacement of Glu with Ala modify the interactions between neighbouring residues through their side chains and the interaction to the solvent affecting the protein stability and causing variations in the activity levels against identical organisms.

2026-02-13T18:34:46Z