Representative publications
1. BRCA1/2 Haploinsufficiency: Exploring the Impact of Losing one Allele
Anna Minello and Aura Carreira
Since their discovery in the late 20th century, significant progress has been made in elucidating the func-tions of the tumor suppressor proteins BRCA1 and BRCA2. These proteins play vital roles in maintaining genome integrity, including DNA repair, replication fork protection, and chromosome maintenance. It is well-established that germline mutations in BRCA1 and BRCA2 increase the risk of breast and ovarian cancer; however, the precise mechanism underlying tumor formation in this context is not fully under-stood. Contrary to the long-standing belief that the loss of the second wild-type allele is necessary for tumor development, a growing body of evidence suggests that tumorigenesis can occur despite the pres-ence of a single functional allele. This entails that heterozygosity in BRCA1/2 confers haploinsufficiency, where a single copy of the gene is not sufficient to fully suppress tumor formation. Here we provide an overview of the findings and the ongoing debate regarding BRCA haploinsufficiency. We further put out the challenges in studying this topic and discuss its potential relevance in the prevention and treatment of BRCA-related cancers.
Available online 14 September 2023 https://doi.org/10.1016/j.jmb.2023.168277
2. Telomeres Fuse During Cardiomyocyte Maturation
Esther Aix, PhD*; Alex Gallinat , PhD*; Carla Yago-Díez , PhD; Javier Lucas, MS; Manuel José Gómez , PhD; Alberto Benguría , PhD; Patricia Freitag; Elizabeth Cortez-Toledo; Laura Fernández de Manuel, PhD; Lucía García-Cuasimodo, BS; Héctor Sánchez-Iranzo , PhD; María C. Montoya , PhD; Ana Dopazo, PhD; Fátima Sánchez-Cabo, PhD; Nadia Mercader, PhD; Javier E. López, MD; Bernd K. Fleischmann , MD; Michael Hesse, PhD; Ignacio Flores , PhD
Although polyploidization is a hallmark of adult mammalian cardiomyocytes and may constrain their proliferation, the mechanisms leading to ploidy increase in cardiomyocytes remain elusive.1 Our laboratory and others have reported the formation of DNA bridges between daughter nuclei as a potential route to cardiomyocyte polyploidization.2,3 These earlier in vivo studies found DNA bridges on thin tissue sections, but this approach does not cover all events because not all cardiomyocytes were oriented in the same 2-dimen-sional plane.
Accepted: May 23, 2023 Circulation. 2023;147:1634–1636. DOI: 10.1161/CIRCULATIONAHA.122.062229
3. Refinement of Leishmania donovani Genome Annotations in the Light of Ribosome-Protected mRNAs Fragments (Ribo-Seq Data)
Alejandro Sánchez-Salvador, Sandra González-de la Fuente, Begoña Aguado, Phillip A. Yates and Jose M. Requena
Advances in next-generation sequencing methodologies have facilitated the assembly of an ever-increasing number of genomes. Gene annotations are typically conducted via specialized software, but the most accurate results require additional manual curation that incorporates insights derived from functional and bioinformatic analyses (e.g., transcriptomics, proteomics, and phyloge-netics). In this study, we improved the annotation of the Leishmania donovani (strain HU3) genome using publicly available data from the deep sequencing of ribosome-protected mRNA fragments (Ribo-Seq). As a result of this analysis, we uncovered 70 previously non-annotated protein-coding genes and improved the annotation of around 600 genes. Additionally, we present evidence for small upstream open reading frames (uORFs) in a significant number of transcripts, indicating their poten-tial role in the translational regulation of gene expression. The bioinformatics pipelines developed for these analyses can be used to improve the genome annotations of other organisms for which Ribo-Seq data are available. The improvements provided by these studies will bring us closer to the ultimate goal of a complete and accurately annotated L. donovani genome and will enhance future transcriptomics, proteomics, and genetics studies.
Keywords: Leishmania; ribosome profiling; Ribo-seq; uORFs; genome; transcriptome
Accepted: 15 August 2023 https://doi.org/10.3390/genes14081637
4. An acytokinetic cell division creates PIP2-enriched membrane asymmetries leading to slit diaphragm assembly in Drosophila nephrocytes
Marta Carrasco-Rando, Joaquim Culi, Sonsoles Campuzano and Mar Ruiz-Gómez*
Vertebrate podocytes and Drosophila nephrocytes display slit diaphragms, specialised cell junctions that are essential for the execution of the basic excretory function of ultrafiltration. To elucidate the mechanisms of slit diaphragm assembly we have studied their formation in Drosophila embryonic garland nephrocytes. These cells of mesenchymal origin lack overt apical-basal polarity. We find that their initial membrane symmetry is broken by an acytokinetic cell division that generates PIP2-enriched domains at their equator. The PIP2-enriched equatorial cortex becomes a favourable domain for hosting slit diaphragm proteins and the assembly of the first slit diaphragms. Indeed, when this division is either prevented or forced to complete cytokinesis, the formation of diaphragms is delayed to larval stages. Furthermore, although apical polarity determinants also accumulate at the equatorial cortex, they do not appear to participate in the recruitment of slit diaphragm proteins. The mechanisms we describe allow the acquisition of functional nephrocytes in embryos, which may confer on them a biological advantage similar to the formation of the first vertebrate kidney, the pronephros.
Key Words: Acytokinetic cell division, Membrane symmetry breaking, Slit diaphragm, Nephrocyte, Drosophila
Accepted: 15 August 2023 Development (2023) 150, dev201708. doi:10.1242/dev.201708
5. Targeting carcinoma-associated mesothelial cells with antibody–drug conjugates in ovarian carcinomatosis
Lucía Pascual-Antón1† , Pilar Sandoval1*† , Guadalupe T González-Mateo1, Valeria Kopytina1, Henar Tomero-Sanz1, Eva María Arriero-País1, José Antonio Jiménez-Heffernan2, Myriam Fabre3, Isabel Egaña3, Cristina Ferrer3, Laureano Simón3, Lucía González-Cortijo4, Ricardo Sainz de la Cuesta4 and Manuel López-Cabrera
Ovarian carcinomatosis is characterized by the accumulation of carcinoma-associated mesothelial cells (CAMs) in the peritoneal stroma and mainly originates through a mesothelial-to-mesenchymal transition (MMT) process. MMT has been proposed as a therapeutic target for peritoneal metastasis. Most ovarian cancer (OC) patients present at diagnosis with peritoneal seeding, which makes tumor progression control difficult by MMT modulation. An alternative approach is to use antibody–drug conjugates (ADCs) targeted directly to attack CAMs. This strategy could represent the cornerstone of precision-based medicine for peritoneal carcinomatosis. Here, we performed complete transcriptome analyses of ascitic fluid-isolated CAMs in advanced OC patients with primary-, high-, and low-grade, serous subtypes and following neoadjuvant chemotherapy. Our findings suggest that both cancer biological aggressiveness and chemotherapy-induced tumor mass reduction reflect the MMT-associated changes that take place in the tumor surrounding microenvironment. Accordingly, MMT-related genes, including fibroblast activation protein (FAP), mannose receptor C type 2 (MRC2), interleukin-11 receptor alpha (IL11RA), myristoylated alanine-rich C-kinase substrate (MARCKS), and sulfatase-1 (SULF1), were identified as specific actionable targets in CAMs of OC patients, which is a crucial step in the de novo design of ADCs. These cell surface target receptors were also validated in peritoneal CAMs of colorectal cancer peritoneal implants, indicating that ADC-based treatment could extend to other abdominal tumors that show peritoneal colonization. As proof of concept, a FAP-targeted ADC reduced tumor growth in an OC xenograft mouse model with peritoneal metastasis-associated fibroblasts. In summary, we propose MMT as a potential source of ADC-based therapeutic targets for peritoneal carcinomatosis.
Key Words: mesothelial-to-mesenchymal transition; peritoneal carcinomatosis; ovarian cancer; carcinoma-associated mesothelial cells; antibody–drug conjugates
Accepted: 22 June 2023 https://pathsocjournals.onlinelibrary.wiley.com/doi/full/10.1002/path.6170242/dev.201708
6. Lack of apoptosis leads to cellular senescence and tumorigenesis in Drosophila epithelial cells
Juan Manuel Garcia-Arias 1, Noelia Pinal1, Sara Cristobal-Vargas 2,3, Carlos Estella 2 and Ginés Morata
Programmed cell death (apoptosis) is a homeostasis program of animal tissues designed to remove cells that are unwanted or are damaged by physiological insults. To assess the functional role of apoptosis, we have studied the consequences of subjecting Drosophila epithelial cells defective in apoptosis to stress or genetic perturbations that normally cause massive cell death. We find that many of those cells acquire persistent activity of the JNK pathway, which drives them into senescent status, characterized by arrest of cell division, cell hypertrophy, Senescent Associated ß-gal activity (SA-ß-gal), reactive oxygen species (ROS) production, Senescent Associated Secretory Phenotype (SASP) and migratory behaviour. We have identified two classes of senescent cells in the wing disc: 1) those that localize to the appendage part of the disc, express the upd, wg and dpp signalling genes and generate tumour overgrowths, and 2) those located in the thoracic region do not express wg and dpp nor they induce tumour overgrowths. Whether to become tumorigenic or non-tumorigenic depends on the original identity of the cell prior to the transformation. We also find that the p53 gene contributes to senescence by enhancing the activity of JNK.
Accepted: 26 July 2023 https://www.nature.com/articles/s41420-023-01583-y
7. Kidins220 sets the threshold for survival of neural stem cells and progenitors to sustain adult neurogenesis
Ana del Puerto1,2,12,14, Coral Lopez-Fonseca 3,4,5,14, Ana Simón-García 1,2,14, Beatriz Martí-Prado2,6, Ana L. Barrios-Muñoz3,4,5, Julia Pose-Utrilla1,2,13, Celia López-Menéndez 1,2, Berta Alcover-Sanchez 3,4,5, Fabrizia Cesca 7, Giampietro Schiavo 8,9, Miguel R. Campanero4,10,11, Isabel Fariñas 2,6, Teresa Iglesias 1,2,15 and Eva Porlan 3,4,5,11,15
In the adult mammalian brain, neural stem cells (NSCs) located in highly restricted niches sustain the generation of new neurons that integrate into existing circuits. A reduction in adult neurogenesis is linked to ageing and neurodegeneration, whereas dysregulation of proliferation and survival of NSCs have been hypothesized to be at the origin of glioma. Thus, unravelling the molecular underpinnings of the regulated activation that NSCs must undergo to proliferate and generate new progeny is of considerable relevance. Current research has identified cues promoting or restraining NSCs activation. Yet, whether NSCs depend on external signals to survive or if intrinsic factors establish a threshold for sustaining their viability remains elusive, even if this knowledge could involve potential for devising novel therapeutic strategies. Kidins220 (Kinase D-interacting substrate of 220 kDa) is an essential effector of crucial pathways for neuronal survival and differentiation. It is dramatically altered in cancer and in neurological and neurodegenerative disorders, emerging as a regulatory molecule with important functions in human disease. Herein, we discover severe neurogenic deficits and hippocampal-based spatial memory defects accompanied by increased neuroblast death and high loss of newly formed neurons in Kidins220 deficient mice. Mechanistically, we demonstrate that Kidins220-dependent activation of AKT in response to EGF restraints GSK3 activity preventing NSCs apoptosis. We also show that NSCs with Kidins220 can survive with lower concentrations of EGF than the ones lacking this molecule. Hence, Kidins220 levels set a molecular threshold for survival in response to mitogens, allowing adult NSCs growth and expansion. Our study identifies Kidins220 as a key player for sensing the availability of growth factors to sustain adult neurogenesis, uncovering a molecular link that may help paving the way towards neurorepair.
Accepted: 13 July 2023 https://doi.org/10.1038/s41419-023-05995-7
8. GSK‑3β orchestrates the inhibitory innervation of adult‑born dentate granule cells in vivo
E. P. Moreno‑Jiménez1,2,3 · M. Flor‑García1,2,3 · A. Hernández‑Vivanco4 · J. Terreros‑Roncal1,2,3 · C. B. Rodríguez‑Moreno1,2,3 · N. Toni5 · P. Méndez4 · María Llorens‑Martín1,2,3
Adult hippocampal neurogenesis enhances brain plasticity and contributes to the cognitive reserve during aging. Adult hippocampal neurogenesis is impaired in neurological disorders, yet the molecular mechanisms regulating the maturation and synaptic integration of new neurons have not been fully elucidated. GABA is a master regulator of adult and develop-mental neurogenesis. Here we engineered a novel retrovirus encoding the fusion protein Gephyrin:GFP to longitudinally study the formation and maturation of inhibitory synapses during adult hippocampal neurogenesis in vivo. Our data reveal the early assembly of inhibitory postsynaptic densities at 1 week of cell age. Glycogen synthase kinase 3 Beta (GSK-3β) emerges as a key regulator of inhibitory synapse formation and maturation during adult hippocampal neurogenesis. GSK-3β-overexpressing newborn neurons show an increased number and altered size of Gephyrin+ postsynaptic clusters, enhanced miniature inhibitory postsynaptic currents, shorter and distanced axon initial segments, reduced synaptic output at the CA3 and CA2 hippocampal regions, and impaired pattern separation. Moreover, GSK-3β overexpression triggers a depletion of Parvalbumin+ interneuron perineuronal nets. These alterations might be relevant in the context of neurological diseases in which the activity of GSK-3β is dysregulated.
Keywords: Adult hippocampal neurogenesis · Gephyrin · Alzheimer´s disease · GSK-3β · Retrovirus · Electrophysiology · Behavior
Accepted: 13 July 2023 https://doi.org/10.1007/s00018-023-04874-w
9. Reciprocal regulation between the molecular clock and kidney injury
Carlos Rey-Serra1 , Jessica Tituaña1 , Terry Lin2 , J Ignacio Herrero1 , Verónica Miguel1, Coral Barbas3 , Anna Meseguer4 , Ricardo Ramos5, Amandine Chaix2 , Satchidananda Panda2, Santiago Lamas1
Tubulointerstitial fibrosis is the common pathological substrate for many etiologies leading to chronic kidney disease. Although perturbations in the circadian rhythm have been associated with renal disease, the role of the molecular clock in the pathogenesis of fibrosis remains incompletely understood. We investigated the relationship between the molecular clock and renal damage in experimental models of injury and fibrosis (unilateral ureteral obstruction, folic acid, and adenine nephrotoxicity), using ge-netically modified mice with selective deficiencies of the clock components Bmal1, Clock, and Cry. We found that the molecular clock pathway was enriched in damaged tubular epithelial cells with marked metabolic alterations. In human tubular epithelial cells, TGFβ significantly altered the expression of clock compo-nents. Although Clock played a role in the macrophage-mediated inflammatory response, the combined absence of Cry1 and Cry2 was critical for the recruitment of neutrophils, correlating with a worsening of fibrosis and with a major shift in the expression of metabolism-related genes. These results support that renal damage disrupts the kidney peripheral molecular clock, which in turn promotes metabolic derangement linked to inflammatory and fibrotic responses.
Accepted: 3 July 2023 https://doi.org/10.26508/lsa.202201886
10. Signal peptide and N-glycosylation of N-terminal-CD2v determine the hemadsorption of African swine fever virus
Daniel Pérez-Núñez,1 Raquel García-Belmonte,1 Elena Riera,1 Marta H. Fernández-Sesma,1 Gonzalo Vigara-Astillero,1 Yolanda Revilla1
African swine fever virus (ASFV) is the cause of African swine fever (ASF), a devastating disease that affects domestic pigs and wild boar and is currently responsible for the largest animal epidemic. One of the characteristics of ASFV infection, but absent in naturally attenuated strains, is hemadsorption (HAD), a phenomenon that has been linked to virulence. In this study, we have shown that ASFV HAD depends exclusively on the Nt domain of the ASFV CD2v protein during infection. CD2v is a highly glycosylated protein, and we found that glycosylation is essential for HAD. However, despite the higher degree of CD2v glycosylation, only simultaneous N-glycosylation of two Asp residues on the Nt region is the determinant for HAD. On the contrary, we have demonstrated that the presence of a specific signal peptide sequence on CD2v not only influences the degree of CD2v glycosylation but is also critical for HAD, although not for CD2v localization. Finally, we have shown that the CD2v expression during infection of the non-HAD NH/P68 strain is not sufficient for HAD, despite its glycosylation and cell surface localization. Complementation studies of CD2v from NH/P68 with CD2v signal peptides from HAD+ strains indicate (i) that different signal peptides from differen t genotypes are able to restore HAD and (ii) the existence of a HAD-inhibitory sequence in NH/P68-CD2v. This study lays the molecular basis for ASFV HAD, which could be key for the study of virulent and pathogenic aspects of the virus, as well as the rational development of new vaccines against ASFV.
Keywords: ASFV, hemoadsorption, CD2v, N-glycosylation, signal peptide, NH/P68, Arm/07/CBM/c2
Accepted: 26 July 2023 https://pubmed.ncbi.nlm.nih.gov/37768082/
11. Charge Engineering of the Nucleic Acid Binding Cleft of a Thermostable HIV-1 Reverse Transcriptase Reveals Key Interactions and a Novel Mechanism of RNase H Inactivation
Javier Martínez del Río 1, Nerea López-Carrobles 1, Jesús I. Mendieta-Moreno 2, Oscar Herrera-Chacón 1, Adrián Sánchez-Ibáñezez 1, Jesús Mendieta 3 and Luis Menéndez-Arias
Coupled with PCR, reverse transcriptases (RTs) have been widely used for RNA detection and gene expression analysis. Increased thermostability and nucleic acid binding affinity are desirable RT proper-ties to improve yields and sensitivity of these applications. The effects of amino acid substitutions in the RT RNase H domain were tested in an engineered HIV-1 group O RT, containing mutations K358R/A359G/S360A and devoid of RNase H activity due to the presence of E478Q (O3MQ RT). Twenty mutant RTs with Lys or Arg at positions interacting with the template-primer (i.e., at positions 473–477, 499–502 and 505) were obtained and characterized. Most of them produced significant amounts of cDNA at 37, 50 and 65 C, as determined in RT-PCR reactions. However, a big loss of activity was observed with mutants A477K/R, S499K/R, V502K/R and Y505K/R, particularly at 65 C. Binding affinity experi-ments confirmed that residues 477, 502 and 505 were less tolerant to mutations. Amino acid substitutions Q500K and Q500R produced a slight increase of cDNA synthesis efficiency at 50 and 65 C, without alter-ing the KD for model DNA/DNA and RNA/DNA heteroduplexes. Interestingly, molecular dynamics simu-lations predicted that those mutations inactivate the RNase H activity by altering the geometry of the catalytic site. Proof of this unexpected effect was obtained after introducing Q500K or Q500R in the wild-type HIV-1BH10 RT and mutant K358R/A359G/S360A RT. Our results reveal a novel mechanism of RNase H inactivation that preserves RT DNA binding and polymerization efficiency without substituting RNase H active site residues.
Accepted: xx mm 2023 https://doi.org/10.1016/j.jmb.2023.168219
12. Dark Fermentation in the Dark Biosphere: The Case of Citrobacter sp. T1.2D-1
Violeta Gallego-Rodríguez 1,†, Adrián Martínez-Bonilla 1,†, Nuria Rodríguez 1,2 and Ricardo Amils 1,2,*
Microbial diversity that thrives in the deep subsurface remains largely unknown. In this work, we present the characterization of Citrobacter sp. T1.2D-1, isolated from a 63.6 m-deep core sample extracted from the deep subsurface of the Iberian Pyrite Belt (IPB). A genomic analysis was performed to identify genes that could be ecologically significant in the IPB. We identified all the genes that encoded the formate–hydrogen lyase and hydrogenase-2 complexes, related to hydrogen production, as well as those involved in glycerol fermentation. This is particularly relevant as some of the substrates and byproducts of this process are of industrial interest. Additionally, we conducted a phylogenomic study, which led us to conclude that our isolate was classified within the Citrobacter telavivensis species. Experimentally, we verified the strain’s ability to produce hydrogen from glucose and glycerol and, thus, of performing dark fermentation. Moreover, we assessed the activity of the nitrate and tetrathionate reductase complexes and the isolate’s ability to tolerate high concentrations of heavy metals, especially Zn. These results suggest that C. telavivensis T1.2D-1 can play a role in the carbon, hydrogen, iron, nitrogen, and sulfur cycles that occur in the deep subsurface of the IPB, making it a candidate worthy of further study for possible biotechnological applications.
Keywords: Citrobacter; deep subsurface; Iberian pyrite belt; biohydrogen; dark fermentation
Accepted: 27 September 2023 https://doi.org/10.3390/fermentation9100887
1. SOCS3 deregulation contributes to aberrant activation of the JAK/STAT pathway in precursor T- cell neoplasmsSOCS3 deregulation contributes to aberrant activation of the JAK/STAT pathway in precursor T- cell neoplasms
Antonio Lahera, Pilar López- Nieva, Hernán Alarcón, José L. Marín-Rubio, María Á. Cobos-Fernández, Pablo Fernández-Navarro, Agustín F. Fernández, Laura Vela-Martín, Isabel Sastre, Sara Ruiz- García, Pilar Llamas, José L. López-Lorenzo, Javier Cornago, Javier Santos, José Fernández-Piqueras, María Villa-Morales
Despite the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway being frequently altered in T- ALL/LBL, no specific therapy has been approved for T- ALL/LBL patients with constitutive signalling by JAK/STAT, so there is an urgent need to identify pathway members that may be potential therapeutic tar-gets. In the present study, we searched for JAK/STAT pathway members potentially modulated through aberrant methylation and identified SOCS3 hypermethylation as a recurrent event in T- ALL/LBL. Additionally, we explored the implications of SOCS3 deregulation in T- ALL/LBL and demonstrated that SOCS3 counteracts the constitutive activation of the JAK/STAT pathway through different molecular mech-anisms. Therefore, SOCS3 emerges as a potential therapeutic target in T- ALL/LBL.
Accepted: 31 January 2023 https://doi.org/10.1111/bjh.18694
2. Telomeres Fuse During Cardiomyocyte Maturation
Esther Aix, PhD*; Alex Gallinat , PhD*; Carla Yago-Díez , PhD; Javier Lucas, MS; Manuel José Gómez , PhD; Alberto Benguría , PhD; Patricia Freitag; Elizabeth Cortez-Toledo; Laura Fernández de Manuel, PhD; Lucía García-Cuasimodo, BS; Héctor Sánchez-Iranzo , PhD; María C. Montoya , PhD; Ana Dopazo, PhD; Fátima Sánchez-Cabo, PhD; Nadia Mercader, PhD; Javier E. López, MD; Bernd K. Fleischmann , MD; Michael Hesse, PhD; Ignacio Flores , PhD
Although polyploidization is a hallmark of adult mammalian cardiomyocytes and may constrain their proliferation, the mechanisms leading to ploidy increase in cardiomyocytes remain elusive. Our laboratory and others have reported the formation of DNA bridges between daughter nuclei as a potential route to cardiomyocyte polyploidization. These earlier in vivo studies found DNA bridges on thin tissue sections, but this approach does not cover all events because not all cardiomyocytes were oriented in the same 2-dimensional plane.
Accepted: 23 May 2023 Published on line: 07 March 2023 https://doi.org/10.1161/CIRCULATIONAHA.122.062229
3. Comprehensive characterization of a novel, oncogenic and targetable SEPTIN6::ABL2 fusion in T- ALL
Antonio Lahera, Laura Vela- Martín, Pilar López- Nieva, Rocío N. Salgado, Sandra Rodríguez-Perales, Raúl Torres-Ruiz, José L. López-Lorenzo, Javier Cornago, Pilar Llamas, Pablo Fernández-Navarro, Rebeca Sánchez-Domínguez, José C. Segovia, Isabel Sastre, María Á. Cobos-Fernández, Pablo Menéndez, Javier Santos, José Fernández-Piqueras, María Villa- Morales
T- cell acute lymphoblastic leukaemia (T- ALL) arises from the malignant transformation of T- cell progenitors.1,2 Current standard- of- care treatment for T- ALL consists of high- dose multi- agent chemotherapy followed by haematopoietic stem cell transplantation (HSCT) in standard- high risk patients.3 Despite reasonable rates of initial complete responses (CR), patients who relapse or do not respond to first-l ine treat-ments show a dismal outcome with cure rates below 10% and limited therapeutic options.2 Actually, since the approval of nelarabine in 2005 no pharmacological inhibitors have been approved for relapsed/refractory T- ALL patients.4 Therefore, there is an urgent need to identify the molecular alterations that are present in such patients and that promote leukemo-genesis in order to implement personalized-t herapies with higher efficacy and fewer adverse effects.
Accepted: 23 May 2023 https://doi.org/10.1111/bjh.18901
