Desarrollo de una estrategia de captación de estudiantes de excelencia para Máster de Investigación

Memoria ID-147. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2018-2019

PCNA Deubiquitylases Control DNA Damage Bypass at Replication Forks

[EN]DNA damage tolerance plays a key role in protecting cell viability through translesion synthesis and template switching-mediated bypass of genotoxic polymerase-blocking base lesions. Both tolerance pathways critically rely on ubiquitylation of the proliferating-cell nuclear antigen (PCNA) on lysine 164 and have been proposed to operate uncoupled from replication. We report that Ubp10 and Ubp12 ubiquitin proteases differentially cooperate in PCNA deubiquitylation, owing to distinct activities on PCNAlinked ubiquitin chains. Ubp10 and Ubp12 associate with replication forks in a fashion determined by Ubp10 dependency on lagging-strand PCNA residence, and they downregulate translesion polymerase recruitment and template switch events engaging nascent strands. These findings reveal PCNAK164 deubiquitylation as a key mechanism for the modulation of lesion bypass during replication, which might set a framework for establishing strand-differential pathway choices. We propose that damage tolerance is tempered at replication forks to limit the extension of bypass events and sustain chromosome replication rates

Aprendizaje de estrategias de investigación científica en temas de biología fundamental y biomedicina

Memoria ID-0178. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2017-2018

Impacto de la implementación del aprendizaje basado en problemas en combinación con las prácticas de laboratorio clínico y virtual de Fisiopatología para el desarrollo de competencias profesionales

Sección Deptal. de Fisiología (Farmacia)Fac. de FarmaciaFALSEsubmitte

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Molecular cloning and characterization of the 5′ region of the mouse trkA proto-oncogene

[EN] The trkA proto-oncogene encodes a high-affinity NGF receptor that is essential for the survival, differentiation and maintenance of many neural and non-neural cell types. Altered expression of the trkA gene or trkA receptor malfunction have been implicated in neurode-generation, tumor progression and oncogenesis. We have cloned and characterized the 5' region of the mouse trkA gene and have identified its promoter. trkA promoter sequences are GC-rich, lack genuine TATA or CAAT boxes, and are contained within a CpG island which extends over the entire first coding exon. The mouse trkA transcription start site is located 70/71 bp upstream to the AUG translation initiation codon. Sequence analysis showed that the gene encoding the insulin receptor-related receptor, IRR, is located just 1.6 kbp upstream to the trkA gene and is transcribed in the opposite direction. We have used trkA-CAT transcriptional fusions to study trkA promoter function in transient transfection experiments. RNase protection assays and CAT protein ELISA analyses showed that a 150 bp long DNA segment, immediately upstream to the start site, is sufficient to direct accurate transcription in trkA-expressing cells. Dissection of this fragment allowed us to identify a 13 bp cis-regulatory element essential for both promoter activity and cell-type specific expression. Deletion of this 13 bp segment as well as modification of its sequence by site-directed mutagenesis led to a dramatic decline in promoter activity. Gel mobility shift assays carried out with double-stranded oligonucleotides containing the 13 bp element revealed several specific DNA-protein complexes when nuclear extracts from trkA-expressing cells were used. Supershift experiments showed that the Sp1 transcription factor was a component of one of these complexes. Our results identify a minimal trkA gene promoter, located very close to the transcription start site, and define a 13 bp enhancer within this promoter sequence

Ubiquitin protease Ubp1 cooperates with Ubp10 and Ubp12 to revert Lysine-164 PCNA ubiquitylation at replication forks

[EN]Proliferating cell nuclear antigen (PCNA) is essential for the faithful duplication of eukaryotic genomes. PCNA also orchestrates events necessary to deal with threats to genomic integrity, such as the DNA damage tolerance (DDT) response, a mechanism by which eukaryotic cells bypass replication-blocking lesions to maintain replisome stability. DDT is regulated by the ubiquitylation of PCNA and the consequent recruitment of specialized polymerases that guarantee the continuity of replication. We have recently described that the deubiquitylases Ubp10 and Ubp12 modulate DDT events by reverting the ubiquitylation of PCNA in Saccharomyces cerevisiae. This study unveils Ubp1 as a new PCNA deubiquitylase, which cooperates with Ubp10 and Ubp12 in the regulation of DDT during DNA replication. Ubp1, so far known as a cytoplasmic protein, also localizes to the nucleus where associates with DNA replication forks. In addition, Ubp1 interacts with and deubiquitylates PCNA. Importantly, we provide the first evidence that S. cerevisiae PCNA is ubiquitylated during an unperturbed S phase and that Ubp1, Ubp10, and Ubp12 work together facilitating DNA replication by efficiently reverting PCNA ubiquitylation at replication forks. Thus, the ablation of Ubp1, Ubp10, and Ubp12 causes a permanent ubiquitylation of PCNA and a marked delay in the S phase progression

Cdc6 cooperates with Sic1 and Hct1 to inactivate mitotic cyclin-dependent kinases

[EN] Exit from mitosis requires the inactivation of mitotic cyclindependent kinases (CDKs). In the budding yeast, Saccharomyces cerevisiae, inactivation of CDKs during late mitosis involves degradation of B-type cyclins as well as direct inhibition of cyclin-CDK complexes by the CDK-inhibitor protein Sic1. Several striking similarities exist between Sic1 and Cdc6, a DNA replication factor essential for the formation of pre-replicative complexes at origins ofDNA replication. Transcription of both genes is activated during late mitosis by a process dependent on Swi5. Like Sic1, Cdc6 binds CDK complexes in vivo and downregulates them in vitro. Here we show that Cdc6, like Sic1, also contributes to inactivation of CDKs during late mitosis in S. cerevisiae. Deletion of the CDK-interacting domain of Cdc6 does not inhibit the function of origins of DNA replication during S phase, but instead causes a delay in mitotic exit; this delay is accentuated in the absence of Sic1 or of cyclin degradation. By contributing to mitotic exit and inactivation of CDKs, Cdc6 helps to create the conditions that are required for its subsequent role in the formation of pre-replicative complexes at origins of DNA replication

T‐Cadherin 2: Molecular characterization, function in cell adhesion, and coexpression with T‐cadherin and N‐cadherin

[EN] Cadherins are integral membrane glycoproteins that mediate calcium-dependent, hornophilic cell-cell adhesion and are implicated in controlling tissue morphogenesis. T-cadherin is anchored to the membrane through a glycosyl phosphatidylinositol and expressed in a restricted pattern in developing embryos. We report here the molecular and functional characterization of the T-cadherin isoform, T-cadherin 2 (Tcad-2) and the expression of the corresponding mRNA. Tcad-2 cDNA differs in its 3’ nucleotide sequence from T-cadherin cDNA and encodes a protein in which the carboxy terminal Leu of T-cadherin is substituted by Lys and extended by the amino acids SerPheProTyrVal. By RNase protection, mRNAs encoding the T-cadherin isoforms are coexpressed in heart, muscle, liver, skin, somites, and in neural tissue. Many tissues contain both T-cadherin and Tcad-2 mRNAs in conjunction with N-cadherin transcripts, and T-cadherins and N-cadherin proteins are coexpressed on the surface of individual neurons in vitro. Expression in Chinese hamster ovary cells (CHO) revealed that Tcad-2 is a glycosyl phosphatidylinositol-anchored membrane protein that functions in calcium-dependent, homophilic cell adhesion. The identification of a functional T-cadherin isoform and the coexpression of T-cadherins and N-cadherin by individual cells suggest that specific adhesive interactions of embryonic cells may involve a complex interplay between multiple cadherins

Human Cdc14A Reverses CDK1 Phosphorylation of Cdc25A on Serines 115 and 320

[EN] Human Cdc14A is an evolutionary conserved dual‑specificity protein phosphatase that reverses the modifications effected by cyclin‑dependent kinases and plays an important role in centrosome duplication and mitotic regulation. Few substrates of Cdc14A have been identified, some of them with homologues in yeast that, in turn, are substrates of the Saccharomyces cerevisiae Cdc14 homologue, a protein phosphatase essential for yeast cell viability owing its role in mitotic exit regulation. Identification of the physiological substrates of human Cdc14A is an immediate goal in order to elucidate which cellular processes it regulates. Here, we show that human Cdc14A can dephosphorylate Cdc25A in vitro. Specifically, the Cdk1/Cyclin‑B1‑dependent phosphate groups on Ser115 and Ser320 of Cdc25A were found to be removed by Cdc14A. Cdc25A is an important cell cycle‑regulatory protein involved in several cell cycle transitions and checkpoint responses and whose function and own regulation depend on complex phosphorylation/dephosphorylation‑mediated processes. Importantly, we also show that the upregulation of Cdc14A phosphatase affects Cdc25A protein levels in human cells. Our results suggest that Cdc14A may be involved in the cell cycle regulation of Cdc25A stability