Publicaciones representativas
n1. APC_ali: a tool for improved multiple alignments and evolutionary inference based on a hybrid protein sequence and structure similarity score
Ugo Bastolla, David Abia, Oscar Piette
Motivation: Evolutionary inference depends crucially on the quality of multiple sequence alignments (MSA), which is problematic for distantly re-lated proteins. Since protein structure is more conserved than sequence, it seems natural to use structure alignments for distant homologs. However, structure alignments may not be suitable for inferring evolutionary relationships.
Results: Here we examined four protein similarity measures that depend on sequence and structure (fraction of aligned residues, sequence identity, fraction of superimposed residues, and contact overlap), finding that they are intimately correlated but none of them provides a com-plete and unbiased picture of conservation in proteins. Therefore, we propose the new hybrid protein sequence and structure similarity score PC_sim based on their main principal component. The corresponding divergence measure PC_div shows the strongest correlation with divergen-ces obtained from individual similarities, suggesting that it infers accurate evolutionary divergences. We developed the program PC_ali that con-structs protein MSAs either de novo or modifying an input MSA, using a similarity matrix based on PC_sim. The program constructs a starting MSA based on the maximal cliques of the graph of these PAs and it refines it through progressive alignments along the tree reconstructed with PC_div. Compared with eight state-of-the-art multiple structure or sequence alignment tools, PC_ali achieves higher or equal aligned fraction and structural scores, sequence identity higher than structure aligners although lower than sequence aligners, highest score PC_sim, and highest similarity with the MSAs produced by other tools and with the reference MSA Balibase.
Availability and implementation: https://github.com/ugobas/PC_ali
Publication Date: 17 October 2023 https://doi.org/10.1093/bioinformatics/btad630
n2. The BRCA2 R2645G variant increases DNA binding and induces hyper-recombination
Lucia Alvaro-Aranda, Ambre Petitalot, Yasmina Djeghmoum, Davide Panig ada, Jenny Kaur Singh, Åsa Ehlén, Domagoj Vugic,Charlotte Martin, Simona Miron, Aida Contreras-Perez, Naima Nhiri, Virginie Boucherit, Philippe Lafitte, Isaac Dumoulin, Etienne Rouleau, Eric Jacquet, Lidia Feliubadaló, Jesús del Valle, Dominique Stoppa-Lyonnet, Sophie Zinn-Justin, Conxi Lázaro, Sandrine M. Caputo and Aura Carreira
BRCA2 tumor suppressor protein ensures genome integrity by mediating DNA repair via homologous recombination ( HR) . This function is e x ecuted in part by its canonical DNA binding domain located at the C-terminus ( BRCA2 CTD) , the only folded domain of the protein. Most germline pathogenic missense variants are located in this highly conserved region which binds to single-stranded DNA ( ssDNA) and to the acidic protein DSS1. These interactions are essential for the HR function of BRCA2. Here, we report that the variant R2645G, identified in breast cancer and located at the DSS1 interf ace, une xpectedly increases the ssDNA binding activity of BRCA2 CTD in vitro. Human cells expressing this variant displa y a h yper-recombination phenot ype, chromosomal inst abilit y in the form of chromatid gaps when exposed to DNA damage, and increased P ARP inhibitor sensitivity. In mouse embryonic stem cells ( mES) , this variant alters viability and confers sensitivity to cisplatin and Mitom y cin C. These results suggest that BRCA2 interaction with ssDNA needs to be tightly regulated to limit HR and prevent chromosomal inst abilit y and we propose that this control mechanism in v olv es DSS1. Giv en that se v eral missense v ariants located within this region ha v e been identified in breast cancer patients, these findings might ha v e clinical implications for carriers.
Accepted: December 12, 2023 https://doi.org/10.1093/nar/gkad1222
n3. Spatial regulation of DNA damage tolerance protein Rad5 interconnects genome stability maintenance and proteostasis networks
Carl P. Lehmann, Paula González-Fernández and José Antonio Tercero
The Rad5 / HLTF protein has a central role in the tolerance to DNA damage by mediating an error-free mode of bypassing unrepaired DNA lesions, and is therefore critical for the maintenance of genome st abilit y. We show in this w ork that, f ollo wing cellular stress, Rad5 is regulated by relocalization into t wo t ypes of nuclear foci that coexist within the same cell, which we termed ‘S’ and ‘I’. Rad5 S-f oci f orm in response to genotoxic stress and are associated with Rad5’s function in maintaining genome stability, whereas I-foci form in the presence of proteotoxic stress and are related to Rad5’s own proteostasis. Rad5 accumulates into S-foci at DNA damage tolerance sites by liquid-liquid phase separation, while I-foci constitute sites of chaperone-mediated sequestration of Rad5 at the intranuclear quality control compartment ( INQ) . Relocalization of Rad5 into each type of foci involves different pathways and recruitment mechanisms, but in both cases is driven by the evolutionarily conserved E2 ubiquitin-conjugating enzyme Rad6. This coordinated differential relocalization of Rad5 interconnects DNA damage response and proteostasis net works, highlighting the import ance of studying these homeost asis mechanisms in t andem. Spatial regulation of Rad5 under cellular stress conditions thus provides a useful biological model to study cellular homeostasis as a whole.
Published: 06 December 2023 https://doi.org/10.1093/nar/gkad1176
n4. Simultaneous Targeting of IL-1–Signaling and IL-6–Trans-Signaling Preserves Human Pulmonary Endothelial Barrier Function During a Cytokine Storm—Brief Report
Natalia Colás-Algora, Pablo Muñoz-Pinillos, Cristina Cacho-Navas , José Avendaño-Ortiz , Gema de Rivas, Susana Barroso, Eduardo López-Collazo, Jaime Millán
Systemic inflammatory diseases, such as sepsis and severe COVID-19, provoke acute respiratory distress syndrome in which the pathological hyperpermeability of the microvasculature, induced by uncontrolled inflammatory stimulation, causes pulmonary edema. Identifying the inflammatory mediators that induce human lung microvascular endothelial cell barrier dysfunction is essential to find the best anti-inflammatory treatments for critically ill acute respiratory distress syndrome patients.
Key Words: endothelial cell interleukin interferon permeability respiratory distress syndrome sepsis
Published: 21 Sep 2023 https://doi.org/10.1161/ATVBAHA.123.319695
n5. Lost in mTORC1-related translation limits healing, repair and regeneration in mammals
José M. Izquierdo
Wound healing is a coordinated process that can be divided into three general phases: inflammatory processes, tissue formation and tissue remodeling. The molecular and cellular events involved in healing, repair and regeneration are still poorly understood, and current therapies are limited. As a result, defective wound healing affects millions of people worldwide every year. Beyond the current wound healing dogma, new mediators and regulatory nodes are continuously being discovered, opening new therapeutic avenues.
Key Words: mTORC1, de novo translation, wound healing, repair, regeneration, regenerative medicine
Accepted: 2023 https://doi-org/10.3389/fcell.2023.1294934
n6. Multimerization of HIF enhances transcription of target genes containing the hypoxia ancillary sequence
Tamara Rosell-Garcia, Sergio Rivas-Muñoz, Koryu Kin, Verónica Romero-Albillo, Silvia Alcaraz, Carlos Fernandez-Tornero, Fernando Rodriguez-Pascual
Transcriptional activity of the hypoxia inducible factor (HIF) relies on the formation of a heterodimer composed of an oxygen-regulated α-subunit and a stably expressed β-subunit. Heterodimeric HIF activates expression by binding to RCGTG motifs within promoters of hypoxia-activated genes. Some hypoxia targets also possess an adjacent HIF ancillary sequence (HAS) reported to increase transcription but whose function remains obscure. Here, we investigate the contribution of the HAS element to the hypoxia response and its mechanism of action, using the HAS-containing prolyl 4-hydroxylase subunit α1 (P4HA1) as a gene model in NIH/3T3 mouse em-bryonic fibroblasts and HEK293 human embryonic kidney cells. Our HIF overexpression experiments demon-strate that the HAS motif is essential for full induction by hypoxia and that the presence of the tandem HAS/HIF, as opposed to HIF-only sequences, provides HIF proteins with the capacity to form complexes of stoichiometry beyond the classical heterodimer, likely tetramers, to cooperatively potentiate hypoxia-induced transcription. We also provide evidence of the crucial role played by the Fα helix of the PAS-B domain of the HIF1β subunit to support the interaction between heterodimers. Functional analysis showed that human genes containing the HAS/HIF motifs are better responders to hypoxia, and their promoters are enriched for specific transcription factor binding sites. Gene ontology enrichment revealed a predominance of HAS/HIF in genes primarily related to tissue formation and development. Our findings add an extra level of regulation of the hypoxia/HIF signaling through multimerization of HIF proteins on regulatory elements containing the HAS/HIF motifs.
Key Words: Hypoxia inducible factor, Hypoxia response, Cooperation, HIF ancillary sequence, HIF binding site
Available online 25 July 2023 https://doi.org/10.1016/j.bbagrm.2023.194963
n7. Perinatal exposure to pesticides alters synaptic plasticity signaling and induces behavioral deficits associated with neurodevelopmental disorders
Esperanza López‑Merino, María I. Cuartero, José A. Esteban, Víctor Briz
Increasing evidence from animal and epi-demiological studies indicates that perinatal exposure to pesticides cause developmental neurotoxicity and may increase the risk for psychiatric disorders such as autism and intellectual disability. However, the under-lying pathogenic mechanisms remain largely elusive. This work was aimed at testing the hypothesis that developmental exposure to different classes of pesti-cides hijacks intracellular neuronal signaling contrib-uting to synaptic and behavioral alterations associ-ated with neurodevelopmental disorders (NDD). Low concentrations of organochlorine (dieldrin, endosul-fan, and chlordane) and organophosphate (chlorpyri-fos and its oxon metabolite) pesticides were chroni-cally dosed ex vivo (organotypic rat hippocampal slices) or in vivo (perinatal exposure in rats), and then biochemical, electrophysiological, behavioral, and protzeomic studies were performed. All the pes-ticides tested caused prolonged activation of MAPK/ERK pathway in a concentration-dependent manner. Additionally, some of them impaired metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). In the case of the pesticide chlor-dane, the effect was attributed to chronic modulation of MAPK/ERK signaling. These synaptic alterations were reproduced following developmental in vivo exposure to chlordane and chlorpyrifos-oxon, and were also associated with prototypical behavioral phenotypes of NDD, including impaired motor devel-opment, increased anxiety, and social and memory deficits. Lastly, proteomic analysis revealed that these pesticides differentially regulate the expres-sion of proteins in the hippocampus with pivotal roles in brain development and synaptic signaling, some of which are associated with NDD. Based on these results, we propose a novel mechanism of syn-aptic dysfunction, involving chronic overactivation of MAPK and impaired mGluR-LTD, shared by differ-ent pesticides which may have important implications for NDD.
Key Words: Contaminants · Kinase signaling · Psychiatric disorders · Neurodevelopment · MAPK · mGluR LTD
Published: 08 February 2022 https://doi.org/10.1007/s10565-022-09697-2
n8. Enhanced fatty acid oxidation through metformin and baicalin as therapy for COVID-19 and associated inflammatory states in lung and kidney
Verónica Miguel, Carlos Rey-Serra, Jessica Tituaña, Belén Sirera, Elena Alcalde-Estévez, Ignacio Herrero, Irene Ranz, Laura Fernández, Carolina Castillo, Lucía Sevilla, James Nagai, Katharina C. Reimer, Jitske Jansen, Rafael Kramann, Ivan G. Costa, Ana Castro, David Sancho, José Miguel Rodríguez González-Moro, Santiago Lamas
Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome of the acute respiratory distress syndrome (ARDS), characterized by an initial exacerbated inflammatory response, metabolic derangement and ultimate tissue scarring. A positive balance of cellular energy may result crucial for the recovery of clinical COVID-19. Hence, we asked if two key pathways involved in cellular energy generation, AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation (FAO) could be beneficial. We tested the drugs metformin (AMPK activator) and baicalin (CPT1A activator) in different experimental models mimicking COVID-19 associated inflammation in lung and kidney. We also studied two different cohorts of COVID-19 patients that had been previously treated with metformin. These drugs ameliorated lung damage in an ARDS animal model, while activation of AMPK/ ACC signaling increased mitochondrial function and decreased TGF-β-induced fibrosis, apoptosis and inflam- mation markers in lung epithelial cells. Similar results were observed with two indole derivatives, IND6 and IND8 with AMPK activating capacity. Consistently, a reduced time of hospitalization and need of intensive care was observed in COVID-19 patients previously exposed to metformin. Baicalin also mitigated the activation of pro-inflammatory bone marrow-derived macrophages (BMDMs) and reduced kidney fibrosis in two animal models of kidney injury, another key target of COVID-19. In human epithelial lung and kidney cells, both drugs improved mitochondrial function and prevented TGF-β-induced renal epithelial cell dedifferentiation. Our results support that favoring cellular energy production through enhanced FAO may prove useful in the prevention of COVID-19-induced lung and renal damage.
Accepted 1 November 2023 https://doi.org/10.1016/j.redox.2023.102957
n9. Clearance of β-amyloid mediated by autophagy is enhanced by MTORC1 inhibition but not AMPK activation in APP/PSEN1 astrocytes
Marta García-Juan, Lara Ordóñez-Gutiérrez, Francisco Wandosell
Proteostasis mechanisms mediated by macroautophagy/autophagy are altered in neurodegenerative diseases such as Alzheimer disease (AD) and their recovery/enhancement has been proposed as a therapeutic approach. From the two central nodes in the anabolism–catabolism balance, it is generally accepted that mechanistic target of rapamycin kinase complex 1 (MTORC1)_ activation leads to the inhibition of autophagy, whereas adenosine 50-monophosphate (AMP)-activated protein kinase (AMPK) has the opposite role. In AD, amyloid beta (Aβ) production disturbs the optimal neuronal/glial proteostasis. As astrocytes are essential for brain homeostasis, the purpose of this work was to analyze if the upregulation of autophagy in this cell type, either by MTORC1 inhibition or AMPK activation, could modulate the generation/degradation of β-amyloid. By using primary astrocytes from amyloid beta precursor protein (APP)/Presenilin 1 (PSEN1) mouse model of AD, we confirmed that MTORC1 inhibition reduced Aβ secretion through moderate autophagy induction. Surprisingly, pharmacologically increased activity of AMPK did not enhance autophagy but had different effects on Aβ secretion. Conversely, AMPK inhibition did not affect autop-hagy but reduced Aβ secretion. These puzzling data were confirmed through the overexpression of different mutant AMPK isoforms: while only the constitutively active AMPK increased autophagy, all versions augmented Aβ secretion. We con-clude that AMPK has a significantly different role in primary astrocytes than in other reported cells, similar to our previous findings in neurons. Our data support that per-haps only a basal AMPK activity is needed to maintain autophagy whereas the increased activity, either physiologically or pharmacologically, has no direct effect on autophagy-dependent amyloidosis. These results shed light on the controversy about the therapeutic effect of AMPK activation on autophagy induction.
Key Words: AICAR, Alzheimer, amyloid accumulation, autophagy, cultured astrocytes, metformin, rapamycin
Published: 27 November 2023 https://doi.org/10.1002/glia.24492
n10. Characterization of Three Somatic Mutations in the 3´UTR of RRAS2 and Their Inverse Correlation with Lymphocytosis in Chronic Lymphocytic Leukemia
Marta Lacuna, Alejandro M. Hortal, Claudia Cifuentes, Tania Gonzalo, Miguel Alcoceba, Miguel Bastos, Xosé R. Bustelo, Marcos González and Balbino Alarcón
Chronic lymphocytic leukemia (CLL) is a hematologic malignancy characterized by progres-sive accumulation of a rare population of CD5+ B-lymphocytes in peripheral blood, bone marrow, and lymphoid tissues. CLL exhibits remarkable clinical heterogeneity, with some patients presenting with indolent disease and others progressing rapidly to aggressive CLL. The significant heterogeneity of CLL underscores the importance of identifying novel prognostic markers. Recently, the RAS-related gene RRAS2 has emerged as both a driver oncogene and a potential marker for CLL progression, with higher RRAS2 expression associated with poorer disease prognosis. Although missense somatic mutations in the coding sequence of RRAS2 have not been described in CLL, this study reports the frequent detection of three somatic mutations in the 3´ untranslated region (3´UTR) affecting positions+26, +53, and +180 downstream of the stop codon in the mRNA. An inverse relationship was observed between these three somatic mutations and RRAS2 mRNA expression, which correlated with lower blood lymphocytosis. These findings highlight the importance of RRAS2 overexpression in CLL development and prognosis and point to somatic mutations in its 3´UTR as novel mechanistic clues. Our results may contribute to the development of targeted therapeutic strategies and improved risk stratification for CLL patients.
Key Words: RRAS2; somatic mutations; 3´ untranslated region; Chronic Lymphocytic Leukemia (CLL); clinical implications
Published: 22 November 2023 https://doi.org/10.3390/cells12232687
n11. Biodegradation of Choline NTF2 by Pantoea agglomerans in Different Osmolarity. Characterization and Environmental Implications of the Produced Exopolysaccharide
Abrusci Concepción, Amils Ricardo and Sánchez-León Enrique
A specific microorganism, Pantoea agglomerans uam8, was isolated from the ionic liquid (IL) Choline NTF2 and identified by molecular biology. A biodegradation study was performed at osmolarity conditions (0.2, 0.6, 1.0 M). These had an important influence on the growth of the strain, exopolysaccharide (EPS) production, and biodegradation (1303 mg/L max production and 80% biodegradation at 0.6 M). These conditions also had an important influence on the morphology of the strain and its EPSs, but not in the chemical composition. The EPS (glucose, mannose and galactose (6:0.5:2)) produced at 0.6 M was further characterized using different techniques. The obtained EPSs presented important differences in the behavior of the emulsifying activity for vegetable oils (olive (86%), sunflower (56%) and coconut (90%)) and hydrocarbons (diesel (62%), hexane (60%)), and were compared with commercial emulsifiers. The EPS produced at 0.6 M had the highest emulsifying activity overall. This EPS did not show cytotoxicity against the tested cell line (<20%) and presented great advantages as an antioxidant (1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) (85%), hydroxyl radical (OH) (99%), superoxide anion (O2−) (94%), chelator (54%), and antimicrobial product (15 mm). The osmolarity conditions directly affected the capacity of the strain to biodegrade IL and the subsequently produced EPS. Furthermore, the EPS produced at 0.6 M has potential for environmental applications, such as the removal of hazardous materials by emulsification, whilst resulting in positive health effects such as antioxidant activity and non-toxicity.
Keywords: bacteria; ionic liquid; toxicity; emulsifying; antioxidant
Published: 3 October 2023 https://doi.org/10.3390/polym15193974
n12. Single-Molecule Analysis of Genome Uncoating from Individual Human Rhinovirus Particles, and Modulation by Antiviral Drugs
Alejandro Valbuena, Klara Strobl, Juan Carlos Gil-Redondo, Luis Valiente, Pedro J. de Pablo, Mauricio G. Mateu
Infection of humans by many viruses is typically initiated by the internalization of a single virion in each of a few susceptible cells. Thus, the outcome of the infection process may depend on stochastic single-molecule events. A crucial process for viral infection, and thus a target for developing antiviral drugs, is the uncoating of the viral genome. Here a force spectroscopy procedure using an atomic force microscope is implemented to study uncoating for individual human rhinovirus particles. Application of an increasing mechanical force on a virion led to a high force-induced structural transition that facilitated extrusion of the viral RNA molecule without loss of capsid integrity. Application of force to virions that h ad previously extruded the RNA, or to RNA-free capsids, led to a lower force-induced event associated with capsid disruption. The kinetic parameters are determined for each reaction. The high-force event is a stochastic process governed by a moderate free energy barrier (≈20 kcal mol−1), which results in a heterogeneous population of structurally weakened virions in which different fractions of the RNA molecule are externalized. The effects of antiviral compounds or capsid mutation on the kinetics of this reaction reveal a correlation between the reaction rate and virus infectivity.
Published: 08 October 2023 https://doi.org/10.1002/smll.202304722