Artículos (Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER))

URI permanente para esta colecciónhttps://hdl.handle.net/11441/11090

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Mostrando 1 - 20 de 216

Salivary estrone and estradiol are associated with oral microbiome profiles in aging women
(Taylor & francis LTD, 2026-06-21) J. Rus, María; R. Nieto, María; Oh, Hyo Jung; Yoo, Hosuon; Areal Quecuty, Victoria; Duarte Faria, Flavio; Lendines-Cordero, Debora; Simon-Soro, Aurea; Estomatología; Centro Andaluz de Investigaciones en Biología Molecular y Medicina Regenerativa (CABIMER); Ministerio de Ciencia e Innovación de España; CTS941: Patología Dentaria, Operatoria Dental y Endodoncia
Objectives: To explore whether salivary estrogens (estrone and estradiol) are associated with oral microbiome composition in aging women, and to assess the oral cavity as a potential sentinel of systemic hormonal changes during midlife. Materials and methods: Cross-sectional study including 30 women aged 40-65 years. Saliva and microbial specimens were collected from four oral ecological niches (buccal mucosa, tongue dorsum, supragingival plaque, subgingival plaque). Microbiome composition and diversity were assessed by 16S rRNA gene sequencing, ecological indices, and co-occurrence network analysis. Salivary estrone and estradiol were quantified, and associations with oral health and microbial profiles were evaluated. Results: Estrone levels declined significantly with age and were associated with hyposalivation and lower oral health scores. Estrone was linked to increased microbial diversity on the tongue dorsum and enrichment of taxa such as Porphyromonas. In contrast, estradiol was positively associated with commensal genera (Streptococcus, Lactobacillus) and negatively with periodontal-associated taxa (Fusobacterium, Prevotella). Co-occurrence networks revealed niche-specific microbial shifts associated with estrogen levels. Conclusions: Salivary estrogens, particularly estrone, shape oral microbial communities in aging women. The oral cavity may act as a window into systemic hormonal changes, supporting its role as a non-invasive sentinel of women's health during midlife.
Deciphering the role of ammonia in MASLD: From a neurotoxin to a metabolic by-product
(Elsevier, 2026-04) Zurita Palomo, Carola; Martín Bermudo, Franz; Maya Miles, Douglas; Ampuero Herrojo, Javier; Romero Gómez, Manuel; Gallego Durán, Rocío; Fisiología; Medicina; Instituto de Salud Carlos III; Agencia Estatal de Investigación. España; European Union (UE)
Dbf4-dependent kinase finetunes Ino80 function at chromosome replication origins
(Springer Science and Business Media LLC, 2026) Bansal, Priyanka; Lahiri, Shibojyoti; Kumar, Chadni Natalia; Furtmeier, Jessica; Spechtenhauser, Lorenz; Galanti, Lorenzo; Ortíz-Bazán, María Ángeles; Aguilera López, Andrés; Gómez González, Belén; Kurat, Christoph F.; Genética; Deutsche Forschungsgemeinschaft / German Research Foundation (DFG); European Union (UE); Junta de Andalucía
The highly conserved Dbf4-Dependent Kinase (DDK) plays a pivotal role during S phase. It phosphorylates the replicative helicase (minichromosome maintenance, MCM complex), which leads to the initiation of replication. However, few other targets, besides the MCM complex, are known, leaving DDK an understudied kinase. Here, we determine the nuclear DDK-dependent phosphoproteome by a two-pronged mass spectrometry approach. Among ~ 400 DDK-dependent phosphorylation targets, we find the Arp8 subunit of the INO80 chromatin remodeling complex. Arp8 phosphorylation stabilises INO80’s complex integrity, finetunes its nucleosome spacing at replication origins, stimulates replication and improves the replication stress response. Taken together, we report the regulation of a chromatin remodeler with nucleosome-spacing activity by the cell-cycle machinery. DDK not only regulates the core replication machinery but also regulates a factor that generates replication-conducive chromatin architecture at replication origins.
Characterization of a novel interaction of the Nup159 nucleoporin with asymmetrically localized spindle pole body proteins and its link with autophagy
(Public Library of Science, 2023-08-03) García de Oya, Inés; Manzano López, Javier; Álvarez Llamas, Alejandra; Vázquez Aroca, María de la Paz; Cepeda García, Cristina; Monje Casas, Fernando; Fisiología
Both the spindle microtubule-organizing centers and the nuclear pore complexes (NPCs) are convoluted structures where many signaling pathways converge to coordinate key events during cell division. Interestingly, despite their distinct molecular conformation and overall functions, these structures share common components and collaborate in the regulation of essential processes. We have established a new link between microtubule-organizing centers and nuclear pores in budding yeast by unveiling an interaction between the Bfa1/Bub2 complex, a mitotic exit inhibitor that localizes on the spindle pole bodies, and the Nup159 nucleoporin. Bfa1/Bub2 association with Nup159 is reduced in metaphase to not interfere with proper spindle positioning. However, their interaction is stimulated in anaphase and assists the Nup159-dependent autophagy pathway. The asymmetric localization of Bfa1/Bub2 during mitosis raises the possibility that its interaction with Nup159 could differentially promote Nup159-mediated autophagic processes, which might be relevant for the maintenance of the replicative lifespan.
The Multiple Roles of the Cdc14 Phosphatase in Cell Cycle Control
(MDPI, 2020-01-20) Manzano López, Javier; Monje Casas, Fernando; Fisiología; European Union (UE); Ministerio de Economia, Industria y Competitividad (MINECO). España
The Cdc14 phosphatase is a key regulator of mitosis in the budding yeast Saccharomyces cerevisiae. Cdc14 was initially described as playing an essential role in the control of cell cycle progression by promoting mitotic exit on the basis of its capacity to counteract the activity of the cyclin-dependent kinase Cdc28/Cdk1. Acompiling body of evidence, however, has later demonstrated that this phosphatase plays other multiple roles in the regulation of mitosis at di erent cell cycle stages. Here, we summarize our current knowledge about the pivotal role of Cdc14 in cell cycle control, with a special focus in the most recently uncovered functions of the phosphatase.
Polo-like kinase acts as a molecular timer that safeguards the asymmetric fate of spindle microtubule-organizing centers
(eLife Sciences Publications, 2020-11-02) Matellán Fernández, Laura; Manzano López, Javier; Monje Casas, Fernando; Fisiología; European Union (UE); Ministerio de Economía y Competitividad (MINECO). España
The microtubules that form the mitotic spindle originate from microtubule-organizing centers (MTOCs) located at either pole. After duplication, spindle MTOCs can be differentially inherited during asymmetric cell division in organisms ranging from yeast to humans. Problems with establishing predetermined spindle MTOC inheritance patterns during stem cell division have been associated with accelerated cellular aging and the development of both cancer and neurodegenerative disorders. Here, we expand the repertoire of functions Polo-like kinase family members fulfill in regulating pivotal cell cycle processes. We demonstrate that the Plk1 homolog Cdc5 acts as a molecular timer that facilitates the timely and sequential recruitment of two key determinants of spindle MTOCs distribution, that is the g-tubulin complex receptor Spc72 and the protein Kar9, and establishes the fate of these structures, safeguarding their asymmetric inheritance during Saccharomyces cerevisiae mitosis.
Asymmetric inheritance of spindle microtubule-organizing centers preserves replicative lifespan
(Nature Research, 2019-07-29) Manzano López, Javier; Matellán, Laura; Álvarez Llamas, Alejandra; Blanco Mira, José Carlos; Monje Casas, Fernando; Fisiología; European Union (UE); Ministerio de Ciencia, Innovación y Universidades (MICIU). España
The differential distribution of the microtubule-organizing centers (MTOCs) that orchestrate spindle formation during cell division is a fascinating phenomenon originally described in Saccharomyces cerevisiae and later found to be conserved during stem cell divisions in organisms ranging from Drosophila to humans. Whether pre-determined MTOC inheritance patterns fulfill any biological function is however so far unknown. Using a genetically-designed S. cerevisiae strain that displays a constitutively inverted MTOC fate, we demonstrate that the asymmetric segregation of these structures is critical to ensure normal levels of the Sir2 sirtuin and a correct localization of the mitochondrial inheritance regulator Mfb1, and therefore to properly distribute functional mitochondria and protein aggregates between the mother and daughter cells. Consequently, interfering with this process severely accelerates cellular aging.
FANCD2 restrains fork progression and prevents fragility at early origins upon re-replication
(Springer Nature, 2026-02-07) Badra Fajardo, Nibal; Karydi, Elena; Bayona Feliu, Aleix; Gómez González, Belén; Preza, Ourania; Arbi, Marina; Kalogeropoulou, Argyro; Rantala, Juha K.; Taraviras, Stavros; Aguilera López, Andrés; Lygerou, Zoi; Genética; European Union (UE); Hellenic Foundation for Research and Innovation; Agencia Estatal de Investigación. España; Caixa Research Foundation
DNA replication is tightly regulated to ensure a single round of chromosome duplication per cell division. DNA licensing restricts origin firing to once-per-cell-cycle while aberrant licensing promotes re-replication and genome instability. Here, we investigate the mechanisms that protect genome integrity following re-replication induced by depletion of the licensing inhibitor Geminin. We find that re-replicating cells require FANCD2 to prevent genome instability. FANCD2 is rapidly recruited to chromatin upon Geminin loss, where it limits unrestrained fork progression and prevents single strand DNA gap accumulation and fork breakage. Genome-wide analyses reveal that upon re-replication, FANCD2 localizes to early origins within highly transcribed regions prone to accumulate R-loops and enriched in early replicating fragile sites. Importantly, reducing transcription and R-loops alleviates re-replication-induced genome fragility, whereas PARP inhibition exacerbates it. Our study uncovers a role for FANCD2 in safeguarding genome integrity during re-replication, offering avenues for selective targeting of cancer cells.
PSMA-targeted delivery of docetaxel in prostate cancer using small-sized PDA-based micellar nanovectors
(Elsevier, 2025-01-30) Rosales Barrios, Cristian; González Sánchez, Zaira; Zuliani, Alessio; Jiménez Vacas, Juan M.; Luque, Raul M.; Pozo Pérez, David; Khiar, Noureddine; Química Inorgánica; Bioquímica Médica y Biología Molecular e Inmunología; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España; Junta de Andalucía; European Union (UE)
In this study, we present the first comparative analysis of active and passive drug delivery systems for docetaxel (DTX) in prostate cancer using supramolecular self-assembled micellar nanovectors. Specifically, we developed two novel micelles based on polydiacetylenic amphiphiles (PDA) for passive and active targeting. The active targeting micelles were designed with a prostate-specific membrane antigen (PSMA) ligand, ACUPA, to facilitate recognition by PSMA-positive cancer cells. These PDA-based micelles feature a well-defined structure with a hydrophobic PDA core and a surface functionalized with PEG, and for active targeting, ACUPA. Our micelles demonstrated excellent encapsulation capacity, significantly improving DTX solubility in water, a crucial factor for clinical drug use. In vitro studies confirmed the safety and cytotoxic profiles of both systems, with ACUPA-functionalized micelles showing notable internalization into PSMA-positive LNCaP cells, mediated through the PSMA-ACUPA interaction. In vivo imaging revealed preferential accumulation of ACUPA-functionalized nanomicelles in LNCaP xenograft tumors, suggesting enhanced retention via specific ACUPA-PSMA interactions and active uptake by LNCaP cells. Notably, Balb/c-Foxn1nu/nu early in vivo studies showed a marked reduction in tumor volume and tumor expression levels of proliferation, cell cycle progression, cell survival and anti-apoptotic markers with DTX-loaded micelles functionalized with ACUPA compared to those without ACUPA. Overall, our studies collect initial evidence regarding the feasibility of supramolecular self-assembly of ACUPA-PDA-based nanomicelles for PSMA-targeted drug chemotherapy delivery developments.
Regulation of NTRK2 alternative splicing by PRPF40B controls neural differentiation and synaptic plasticity
(Springer Nature, 2025-12-08) Duarte-Ruiz, María; Moreno-Castillo, Adela; El Yousfi, Younes; Moreno-Castro, Cristina; Martínez-Martínez, Noelia; Jiménez-Lozano, Sandra; Kennel, Marion; Ruiz-Rodríguez, Candela; Rodríguez-Caparrós, Alonso; López-Ros, Jennifer; de la Grange, Pierre; Hernández-Munain, Cristina; Suñé, Carlos; Genética; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Junta de Andalucía
BDNF signaling through its receptor TRKB plays a critical role in brain development, neuroplasticity, and homeostasis. Alternative splicing of the TRKB gene, NTRK2, generates either the full-length receptor (TRKB-FL) or a truncated isoform (TRKB-T1) that inhibits BDNF signaling and has been implicated in neurodegenerative diseases, psychiatric disorders, and cognitive impairments. Here, we show that PRPF40B, a splicing factor associated with neuronal dysfunction, promotes the production of the TRKB-FL isoform during neuronal differentiation. Silencing PRPF40B increases TRKB-T1 expression and impairs the expression of genes important for neuronal differentiation and synaptic plasticity, both in vitro and in vivo, during early embryogenesis. Our data thus identify PRPF40B as a key regulator of the balance between TRKB receptor isoforms, crucial for fine-tuning neuronal responses and for preventing neuroplasticity or survival impairments, providing also a mechanism for the role of PRPF40B in the pathogenesis of various human neurodegenerative diseases and psychiatric disorders. (Figure presented.)
Patulin and Xestoquinol are inhibitors of DNA topoisomerase 1
(National Academy of Sciences, 2025-04-24) Tumini, Emanuela; Wellinger, Ralf Erik; Herrera Moyano, Emilia; Navarro Cansino, Patricia; García Rubio, María Luisa; Salas Lloret, Daniel; Losada, Alejandro; Gaillard, Hélène; Luna Varo, Rosa María; Aguilera López, Andrés; Genética; Ministerio de Economía y Competitividad (MINECO). España; European Research Council (ERC); Agencia Estatal de Investigación. España
DNA topoisomerase 1 (TOP1) is essential for transcription, replication, and repair. Its function relies on two catalytic steps, DNA breakage and rejoining. Inhibitors of the second step prevent DNA rejoining and lead to persistent DNA breaks, acting as topoisomerase poisons, used as anticancer drugs. However, reliable inhibitors of the first step are not available. Here, we provide genetic and molecular evidence supporting that Patulin and, to a lesser extent, Xestoquinol inhibit the first catalytic step of TOP1 in vitro, in yeast and in human cells. Particularly, Patulin prevents the accumulation of TOP1 cleavage complexes caused by the TOP1 poison camptothecin (CPT) in human cells. Moreover, Patulin pretreatment of human or yeast cells reduces DNA damage and the accumulation of DNA breaks upon CPT exposure. Consistent with the protective role of TOP1 against harmful R-loops, Patulin treatment increases R-loops and R-loop-associated cytotoxicity, mimicking the effect of TOP1 silencing. Altogether our findings indicate that Patulin and Xestoquinol are nonpoisoning inhibitors of TOP1, which should potentiate new research approaches in molecular biology and medicine.
Subretinal Transplant of Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelium on Nanostructured Fibrin-Agarose
(SAGE Publications, 2019-11-04) García Delgado, Ana Belén; De la Cerda, Berta; Alba Amador, Julia; Valdés Sánchez, Maria Lourdes; Fernández Muñoz, Beatriz; Relimpio López, Isabel; Rodríguez de la Rúa Franch, Enrique; Díaz Corrales, Francisco J.; Farmacia y Tecnología Farmacéutica; Cirugía; Junta de Andalucía; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)
Damage to the retinal pigment epithelium (RPE) in age‐related macular degeneration (AMD) and other diseases results in photoreceptor cell death and blindness. Replacement of RPE is therefore being explored as a therapy for several retinal diseases. To move towards a future personalized autologous transplant approach, we have prepared a biocompatible implant using RPE derived from induced pluripotent stem cells (iPSC) reprogrammed from a healthy donor´s monocytes. The correct positioning of the polarized RPE is essential to fulfill its role and protect photoreceptors from degeneration. Hence, we have used a biocompatible hydrogel matrix of fibrin and agarose (FAH) that allows the surgical placement of an RPE sheet in the subretinal space, keeping its functional orientation. Our aim was to demonstrate safety and viability of the transplant in preclinical models. Pigs were used to test the feasibility of a regular vitreo‐retinal surgery. Our results show that this implant is suitable for subretinal transplantation allowing human RPE cells to survive and maintain their phenotype and orientation without any local or systemic adverse events. The ability to transplant the iPSC‐derived RPE sheet in its natural orientation will surely increase the chance to obtain a therapeutic effect in future translational studies.
Transcription and FACT facilitate the restoration of replication-coupled chromatin assembly defects
(Nature Research, 2023-07-14) Barrientos Moreno, Marta; Maya Miles, Douglas; Murillo Pineda, Marina; Fontalva Ostio, Sara; Pérez Alegre, Mónica; Andújar, Eloísa; Prado, Félix; Fisiología; Ministerio de Ciencia e Innovación (MICIN). España; Agencia Estatal de Investigación. España; European Union (UE); Junta de Andalucía
Genome duplication occurs through the coordinated action of DNA replication and nucleosome assembly at replication forks. Defective nucleosome assembly causes DNA lesions by fork breakage that need to be repaired. In addition, it causes a loss of chromatin integrity. These chromatin alterations can be restored, even though the mechanisms are unknown. Here, we show that the process of chromatin restoration can deal with highly severe chromatin defects induced by the absence of the chaperones CAF1 and Rtt106 or a strong reduction in the pool of available histones, and that this process can be followed by analyzing the topoisomer distribution of the 2µ plasmid. Using this assay, we demonstrate that chromatin restoration is slow and independent of checkpoint activation, whereas it requires the action of transcription and the FACT complex. Therefore, cells are able to “repair” not only DNA lesions but also chromatin alterations associated with defective nucleosome assembly.
TOP2B modulates DNA supercoiling and chromatin contacts during transcriptional induction
(American Association for the Advancement of Science, 2025-11-26) Terrón Bautista, José; Bejarano Franco, Marina; Martínez Sánchez, María del Mar; López Hernández, Laura; Díaz Maldonado, Héctor; Santiago Gómez, Angélica; Kidane, Sara; Aguilera López, Andrés; Millán Zambrano, Gonzalo; Cortés Ledesma, Felipe; Genética; European Union (UE); Ministerio de Ciencia e Innovación (MICIN). España; European Research Council (ERC); Junta de Andalucía; National Institutes of Health. United States; Agencia Estatal de Investigación. España
Human type-II topoisomerases, TOP2A and TOP2B, resolve transcription-associated DNA supercoiling, therebyinfluencing gene expression programs, and have been recently linked to three-dimensional genome architecturethrough yet poorly understood mechanisms. Here, we investigate the regulatory roles of TOP2 paralogs usingestrogen signaling, which triggers an acute transcriptional induction that involves extensive rewiring of genomeorganization, as a model system. Unexpectedly, we find that estrogen treatment strongly inhibits TOP2B catalyticactivity—although not its binding—specifically at estrogen-responsive enhancers and promoters. This inhibitionresults in an accumulation of negative DNA supercoiling and promotes the formation of regulatory chromatincontacts. Estrogen-mediated inhibition of TOP2B activity depends on estrogen receptor α, a noncatalytic functionof TOP2A, and, most importantly, the action of zinc finger protein associated with tyrosyl-DNA phosphodiesterase2 and TOP2, an atypical small ubiquitin-like modifier ligase that directly inhibits TOP2 activity. This mechanism oftranscriptional control, involving the fine-tuning of DNA supercoiling levels, highlights the role of DNA topoisom-erases as central regulators of genome dynamics.
PLAMseq enables the proteo-genomic characterization of chromatin-associated proteins and protein interactions in a single workflow
(American Association for the Advancement of Science, 2025-11-07) González Vinceiro, Lourdes; Espejo Serrano, Carmen; Soler Oliva, María Eugenia; Soto Hidalgo, Emily; Mateos Martín, María Luisa; Rico, Daniel; Gonzalez Aguilera, Cristina; González Prieto, Román; Biología Celular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; European Union (UE); Junta de Andalucía
Chromatin immunoprecipitation and coimmunoprecipitation assays are common approaches to characterize thegenomic localization and protein interactors, respectively, for a protein of interest. However, these approachesrequire the use of specific antibodies, which often face sensitivity and specificity issues. On the basis of TurboID,we developed proximity-labeled affinity-purified mass spectrometry and sequencing (PLAMseq), which enables,in the same workflow, identification of the genomic loci and the interacting proteome of a protein of interest.Moreover, PLAMseq can also be applied to specifically map protein interactions and ubiquitin(-like)–modifiedproteins. We validated PLAMseq with two well-characterized proteins, RNA polymerase II, and CTCF, with excellentrobustness and reproducibility. Next, we applied PLAMseq to characterize histone H1 SUMOylation, in whichstudy has remained elusive due to the lack of specific reagents, and found that SETDB1 binds to SUMOylated his-tones H1.2 and H1.4 that also colocalize with H3K9me3 at repetitive regions of the genome.
Enhanced non-enzymatic H2S generation extends lifespan and healthspan in male mice
(Elsevier, 2025-12-19) Cáliz-Molina, María Ángeles; López-Fernández-Sobrino, Raúl; Pino-Pérez, Inmaculada; Panadero-Morón, Concepción; Vilches-Pérez, María del Carmen; Camacho-Cabrera, María; García-Ruiz, Almudena; Pérez-Rosendo, Leopoldo; Espadas, Isabel; Venegas Calerón, Mónica; Aroca Aguilar, Ángeles; González Prieto, Román; Bernabeu Wittel, Máximo; Martín-Montalvo, Alejandro; Fisiología Médica y Biofísica; Bioquímica Vegetal y Biología Molecular; Biología Celular; Medicina; Ministerio de Ciencia e Innovación (MICIN). España; Junta de Andalucía; Sociedad Española de Diabetes (SED); Instituto de Salud Carlos III
Hydrogen sulfide is a gasotransmitter with biological functions, including roles in antioxidant defenses, mitochondrial bioenergetics, and cellular signaling via cysteine persulfidation. Several longevity-promoting interventions enhance endogenous hydrogen sulfide generation. However, whether enhanced hydrogen sulfide generation extends healthspan and lifespan in mammals remains unknown. Here, we investigated the in vivo effects of the non-enzymatic hydrogen sulfide generation promoted by natural diallyl sulforated compounds. Diallyl sulforated compounds extended lifespan and improved the main aspects of healthspan, including glucoregulation, locomotor function, and neurocognition in wild-type male mice across their lifespan. At the histological and molecular levels, we observed reductions in hepatic lipid-droplet size, attenuation of transcriptional and proteomic signatures associated with mTOR and immune-related pathways, and increased cysteine persulfidation in proteins. In humans, greater protein persulfidation in individuals with polypathological conditions was associated with increased muscle strength and lower triglyceride levels, supporting its physiological relevance. Our findings uncover the potential of enhanced hydrogen sulfide generation to promote healthy aging.
DNA topoisomerase II promotes N6-adenosine mRNA methylation
(Elsevier, 2025-10-30) Megías Fernández, Clara; Delgado Sainz, Irene; León Halcón, Alberto; Buglioni, Valentina; Bruno, Federica; González Aguilera, Cristina; Maslon, Magdalena M.; Jimeno González, Silvia; Genética; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Agencia Estatal de Investigación. España; Junta de Andalucía; European Union (UE)
DNA topoisomerase II (TOP2) is an enzyme that regulates DNA topology, primarily by removing DNA supercoiling. This function is crucial during transcription, as the movement of RNA polymerase II (RNAPII) generates torsional stress. However, the specific role of TOP2 in the regulation of gene expression remains to be fully elucidated, as both TOP2 inhibitors and poisons have been shown to upregulate specific genes. In this study, we show that TOP2 poisoning negatively affects transcription elongation of genes repressed at the level of promoter-proximal pausing. Importantly, this effect is counteracted by defective mRNA N6 -denosine methylation (m6A), which results in altered RNA turnover and pre-mRNA splicing. We propose that TOP2 serves a dual function, supporting the maintenance of basal transcription elongation while simultaneously promoting m6A modification in pre-mRNAs to reduce the overall gene expression output.
Aurora B and INCENP co-overexpression Severely Disrupts Mitosis and Distinctly Modifies the Global Transcriptional Landscape
(Cell Press, 2025) Galindo Moreno, María; Muñoz Barrera, Marta; Marcozzi, Chiara; Bruno, Federica; Maya Álvarez, Cristina; Cortés Ledesma, Felipe; Ríos, Rosa María; González Aguilera, Cristina; Monje Casas, Fernando; Genética; Microbiología; Biología Celular; Ministerio de Ciencia, Innovación y Universidades (MICIU). España; Junta de Andalucía; European Commission (EC). Fondo Europeo de Desarrollo Regional (FEDER)
Aurora B kinase, as part of the chromosomal passenger complex (CPC), controls key processes during the cell cycle such as DNA compaction, genome partitioning, or cytokinesis. Nonetheless, increased Aurora B levels are a potential threat for the cells and have been linked to different tumor types. We have carried out an exhaustive characterization of the global consequences of the overexpression of Aurora B and INCENP, the scaffold of the CPC and an activator of Aurora B kinase activity, in non-transformed human cells. Our data demonstrate not only that an individual increase in the levels of Aurora B or INCENP have a different impact on the cells, but more importantly that their simultaneous overexpression stabilizes both CPC components, exacerbates Aurora B activity, severely impairs mitotic progression and chromosome dynamics, and has a distinctive and more dramatic effect on the transcriptional landscape of the cells.
A Rfa1-MN-based system reveals new factors involved in the rescue of broken replication forks
(Public Library of Science, 2025-04-01) Amiama Roig, Ana; Barrientos Moreno, Marta; Cruz Zambrano, Esther; López Ruiz, Luz M.; González Prieto, Román; Ríos Orelogio, Gabriel; Prado, Félix; Biología Celular; Ministerio de Ciencia e Innovación (MICIN). España
The integrity of the replication forks is essential for an accurate and timely completion of genome duplication. However, little is known about how cells deal with broken replication forks. We have generated in yeast a system based on a chimera of the largest subunit of the ssDNA binding complex RPA fused to the micrococcal nuclease (Rfa1-MN) to induce double-strand breaks (DSBs) at replication forks and searched for mutants affected in their repair. Our results show that the core homologous recombination (HR) proteins involved in the formation of the ssDNA/Rad51 filament are essential for the repair of DSBs at forks, whereas non-homologous end joining plays no role. Apart from the endonucleases Mus81 and Yen1, the repair process employs fork-associated HR factors, break-induced replication (BIR)-associated factors and replisome components involved in sister chromatid cohesion and fork stability, pointing to replication fork restart by BIR followed by fork restoration. Notably, we also found factors controlling the length of G1, suggesting that a minimal number of active origins facilitates the repair by converging forks. Our study has also revealed a requirement for checkpoint functions, including the synthesis of Dun1-mediated dNTPs. Finally, our screening revealed minimal impact from the loss of chromatin factors, suggesting that the partially disassembled nucleosome structure at the replication fork facilitates the accessibility of the repair machinery. In conclusion, this study provides an overview of the factors and mechanisms that cooperate to repair broken forks.
Radiotherapy Resistance Driven by Asparagine Endopeptidase through ATR Pathway Modulation in Breast Cancer
(BioMed Central, 2025) Morillo Huesca, Macarena; G. López Cepero, Ignacio; Conesa Bakkali, Ryan; Tomé, Mercedes; Watts, Colin; Huertas Sánchez, Pablo; Moreno Bueno, Gema; Durán, Raúl V.; Martínez Fábregas, Jonathan; Bioquímica Vegetal y Biología Molecular; Genética; European Union (UE). H2020; Ministerio de Ciencia e Innovación (MICIN). España; Universidad de Sevilla; Instituto de Salud Carlos III
Background: Tumor resistance represents a major challenge in the current oncology landscape. Asparagine endopeptidase (AEP) overexpression correlates with worse prognosis and reduced overall survival in most human solid tumors. However, the underlying mechanisms of the connection between AEP and reduced overall survival in cancer patients remain unclear. Methods: High-throughput proteomics, cellular and molecular biology approaches and clinical data from breast cancer (BC) patients were used to identify novel, biologically relevant AEP targets. Immunoblotting and qPCR analyses were used to quantify protein and mRNA levels. Flow cytometry, confocal microscopy, chemical inhibitors, siRNA- and shRNA-silencing and DNA repair assays were used as functional assays. In-silico analyses using the TCGA BC dataset and immunofluorescence assays in an independent cohort of invasive ductal (ID) BC patients were used to validate the clinical relevance of our findings. Results: Here we showed a dual role for AEP in genomic stability and radiotherapy resistance in BC patients by suppressing ATR and PPP1R10 levels. Reduced ATR and PPP1R10 levels were found in BC patients expressing high AEP levels and correlated with worst prognosis. Mechanistically, AEP suppresses ATR levels, reducing DNA damage-induced cell death, and PPP1R10 levels, promoting Chek1/P53 cell cycle checkpoint activation, allowing BC cells to efficiently repair DNA. Functional studies revealed AEP-deficiency results in genomic instability, increased DNA damage signaling, reduced Chek1/P53 activation, impaired DNA repair and cell death, with phosphatase inhibitors restoring the DNA damage response in AEP-deficient BC cells. Furthermore, AEP inhibition sensitized BC cells to the chemotherapeutic reagents cisplatin and etoposide. Immunofluorescence assays in an independent cohort of IDBC patients showed increased AEP levels in ductal cells. These analyses showed that higher AEP levels in radioresistant IDBC patients resulted in ATR nuclear eviction, revealing AEPhigh/ATRlow protein levels as an efficient predictive biomarker for the stratification of radioresistant patients. Conclusion: The newly identified AEP/ATR/PPP1R10 axis plays a dual role in genomic stability and radiotherapy resistance in BC. Our work provides new clues to the underlying mechanisms of tumor resistance and strong evidence validating the AEP/ATR axis as a novel predictive biomarker and therapeutic target for the stratification and treatment of radioresistant BC patients.

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