1. Replication gap suppression depends on the double-strand DNA binding activity of BRCA2
Domagoj Vugic, Isaac Dumoulin, Charlotte Martin, Anna Minello, Lucia Alvaro-Aranda, Jesus Gomez-Escudero, Rady Chaaban, Rana Lebdy, Catharina von Nicolai, Virginie Boucherit, Cyril Ribeyre, Angelos Constantinou & Aura Carreira.
Replication stress (RS) is a major source of genomic instability and is intrinsic to cancer cells. RS is also the consequence of chemotherapeutic drugs for treating cancer. However, adaptation to RS is also a mechanism of resistance to chemotherapy. BRCA2 deﬁciency results in replication stress in human cells. BRCA2 protein’s main functions include DNA repair by homologous recom-bination (HR) both at induced DNA double-strand breaks (DSB) and sponta-neous replicative lesions. At stalled replication forks, BRCA2 protects the DNA from aberrant nucleolytic degradation and is thought to limit the appearance of ssDNA gaps by arresting replication and via post-replicative HR. However, whether and how BRCA2 acts to limit the formation of ssDNA gaps or mediate their repair, remains ill-deﬁned. Here, we use breast cancer variants affecting different domains of BRCA2 to shed light on this function. We demonstrate that the N-terminal DNA binding domain (NTD), and speciﬁcally, its dsDNA binding activity, is required to prevent and repair/ﬁll-in ssDNA gaps upon nucleotide depletion but not to limit PARPi-induced ssDNA gaps. Thus, these ﬁndings suggest that nucleotide depletion and PARPi trigger gaps via distinct mechanisms and that the NTD of BRCA2 prevents nucleotide depletion-induced ssDNA gaps.
Accepted: 17 January 2023 Published on line: 27 January 2023 https://doi.org/10.1038/s41467-023-36149-0
2. Distinct roles of Arabidopsis ORC1 proteins in DNA replication and heterochromatic H3K27me1 deposition
Zaida Vergara, María S.Gomez, Bénédicte Desvoyes, Joana Sequeira-Mendes, Kinda Masoud, Celina Costas, Sandra Noir, Elena Caro, Victoria Mora-Gil, Pascal Genschik & Crisanto Gutierrez
Most cellular proteins involved in genome replication are conserved in all eukaryotic lineages including yeast, plants and animals. However, the mechanisms controlling their availability during the cell cycle are less well deﬁned. Here we show that the Arabidopsis genome encodes for two ORC1 proteins highly similar in amino acid sequence and that have partially over-lapping expression domains but with distinct functions. The ancestral ORC1b gene, present before the partial duplication of the Arabidopsis genome, has retained the canonical function in DNA replication. ORC1b is expressed in both proliferating and endoreplicating cells, accumulates during G1 and is rapidly degraded upon S-phase entry through the ubiquitin-proteasome pathway. In contrast, the duplicated ORC1a gene has acquired a specialized function in heterochromatin biology. ORC1a is required for efﬁcientdepositionofthe heterochromatic H3K27me1 mark by the ATXR5/6 histone methyltransferases. Thedistinctroles of thetwoORC1 proteinsmaybeafeaturecommontoother organisms with duplicated ORC1 genes and a major difference with
Accepted: 24 February 2023 Published on line: 07 March 2023 https://doi.org/10.1038/s41467-023-37024-8
3. The enterohemorrhagic Escherichia coli insertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity
Patricia A. Calvo, Víctor Mateo-Cáceres, Silvia Díaz-Arco, Modesto Redrejo-Rodríguez and Miguel de Vega
Bacterial genomes contain an abundance of trans-posable insertion sequence (IS) elements that are essential for genome evolution and ﬁtness. Among them, IS629 is present in most strains of enterohe-morrhagic Escherichia coli O157 and accounts for many polymorphisms associated with gene inacti-vation and/or genomic deletions. The excision of IS629 from the genome is promoted by IS-excision enhancer (IEE) protein. Despite IEE has been identi-ﬁed in the most pathogenic serotypes of E. coli,its biochemical features that could explain its role in IS excision are not yet understood. We show that IEE is present in >30% of all available E. coli genome as-semblies, and is highly conserved and very abundant within enterohemorrhagic, enteropathogenic and en-terotoxigenic genomes. In vitro analysis of the re-combinant protein from E. coli O157:H7 revealed the presence of a Mn2+-dependent error-prone DNA poly-merase activity in its N-terminal archaeo-eukaryotic primase (AEP) domain able to promote dislocations of the primer and template strands. Importantly, IEE could efﬁciently perform in vitro an end-joining reac-tion of 3’-single-strand DNA overhangs with ≥4bp of homology requiring both the N-terminal AEP and C-terminal helicase domains. The proposed role for IEE in the novel IS excision mechanism is discussed.
Accepted: 05 January 2023 Published on line: 30 January 2023 https://academic.oup.com/nar/article/51/3/1189/7009115
4. Glial regenerative response in the imaginal discs of Drosophila melanogaster
Sergio B. Velarde, Antonio Baonza
Glial cells play a key role during nervous system development and actively participate in all the cellular processes involved in maintaining its structural robustness and functional plasticity. In response to neuronal damage, glial cells proliferate, migrate to the injured region and change their morphology, function, and behavior (Gallo and Deneen, 2014; Kato et al., 2018). This glial regenerative response is associated with the repairing function of these cells and is found across species, suggesting that it may reflect a common underlying genetic mechanism (Kato et al., 2018). In mammals, while the central nervous system has very limited capacity to regenerate after traumatic injury or disease, the peripheral nervous system (PNS) exhibits a far greater capacity for regeneration and damaged peripheral nerves can be totally restored (Brosius Lutz and Barres, 2014; Gallo and Deneen, 2014). The PNS largely owes its regenerative potential to the ability of the main glial cells present in the PNS, myelin, and non-myelin (Remak) Schwann cells, to convert to cells devoted to repairing after injury (Nocera and Jacob, 2020). During the regeneration of peripheral nerves in vertebrates, Schwann cells function as a central hub, collecting signals from neurons and other cell types and undergoing a complex process of reprogramming which converts them into a specialized cell for repair. Even though many aspects of regeneration in peripheral nerves have been studied, there is still a lack of understanding regarding the genetic network that controls the flexible differentiation state of PNS neurons and Schwann. The identification of those signals is essential for getting new insight to develop innovative regenerative therapies. In this scenario, the use of relatively simple model organisms, amenable to genetic, cellular, and molecular analysis is fundamental to study the behavior of glial cells in response to damage in their natural context.
NEURAL REGENERATION RESEARCH｜Vol 18｜No. 1｜January 2023
5. RNAi screen in the Drosophila wing of genes encoding proteins related to cytoskeleton organization and cell division
Cristina M. Ostalé, Patricia Vega-Cuesta, Tamara González, Ana López-Varea, Jose F. de Celis
Cell division and cytoskeleton organization are fundamental processes participating in the development of Drosophila imaginal discs. In this manuscript we describe the phenotypes in the adult ﬂy wing generated by knockdowns of 85% of Drosophila genes encoding proteins likely related to the regulation of cell division and cytoskeleton organization. We also compile a molecular classiﬁcation of these proteins into classes that describe their expected or known main biochemical characteristics, as well as mRNA expression in the wing disc and likely protein subcellular localization for a subset of these genes. Finally, we analyze in more detail one protein family of cytoskeleton genes (Arp2/3 complex), and deﬁne the consequences of interfering with cell division for wing growth and patterning.
Developmental Biology 498 (2023) 61–76
6. Extremely Differentiated T Cell Subsets Contribute to Tissue Deterioration During Aging
Gonzalo Soto-Heredero, Manuel M. Gómez de las Heras, J. Ignacio Escrig-Larena and María Mittelbrunn
There is a dramatic remodeling of the T cell compartment during aging. The most notorious changes are the reduction of the naive T cell pool and the accumulation of memory-like T cells. Memory-like T cells in older people acquire a phenotype of terminally differentiated cells, lose the expression of costimulatory molecules, and acquire properties of senescent cells. In this review, we focus on the different subsets of age-associated T cells that accu-mulate during aging. These subsets include extremely cytotoxic T cells with natural killer properties, exhausted T cells with altered cytokine production, and regulatory T cells that gain proinflammatory features. Importantly, all of these subsets lose their lymph node homing capacity and migrate preferen-tially to nonlymphoid tissues, where they contribute to tissue deterioration and inflammaging.
Keywords: immunosenescence, thymus involution, senescence, inflammation, lymphocyte
Annu. Rev. Immunol. 2023. 41:181–205
7. Pathogenic Mis-splicing of CPEB4 in Schizophrenia
Ivana Ollà, Antonio F. Pardiñas, Alberto Parras, Ivó H. Hernández, María Santos-Galindo, Sara Picó, Luis F. Callado, Ainara Elorza, Claudia Rodríguez-López, Gonzalo Fernández-Miranda, Eulàlia Belloc, James T. R. Walters, Michael C. O'Donovan, Raúl Méndez, Claudio Toma, J. Javier Meana, Michael J. Owen and José J. Lucas
Background: Schizophrenia (SCZ) is caused by an interplay of polygenic risk and environmental factors, which might alter regulators of gene expression leading to pathogenic mis-expression of SCZ-risk genes. The CPEB family of RNA-binding proteins (CPEB1-4) regulates translation of target RNAs (approximately 40% of overall genes). We previously identified CPEB4 as a key dysregulated translational regulator in autism spectrum disorder (ASD), as its neuronal-specific microexon (exon4) is mis-spliced in ASD brains, causing underexpression of numerous ASD-risk genes. The genetic and pathogenic mechanisms shared between SCZ and ASD led us to hypothesize CPEB4 mis-splicing in SCZ, leading to underexpression of multiple SCZ-related genes.
Methods: We performed MAGMA-enrichment analysis in Psychiatric Genomics Consortium GWAS data and analyzed RNA-seq data from the PsychENCODE Consortium. RT-PCR and Western blot were performed on post-mortem brain tissue in which presence/absence of antipsychotics was assessed through toxicological analysis. Finally, mice with mild overexpression of exon4-lacking CPEB4 (CPEB4Δ4) were generated and biochemically and behaviorally analyzed.
Results: We first found enrichment of SCZ-associated genes for CPEB4-binder transcripts. We also found decreased usage of CPEB4 microexon in SCZ probands, correlating with decreased protein levels of CPEB4-target SCZ-associated genes, selectively in antipsychotics-free individuals. Interestingly, differentially expressed genes fit those reported for SCZ, specifically in the SCZ probands with decreased CPEB4-microexon inclusion. Finally, we demonstrate that mice with mild overexpression of CPEB4Δ4 show decreased protein levels of CPEB4-target SCZ genes and SCZ-linked behaviors.
Conclusion: We identify aberrant CPEB4 splicing and downstream mis-expression of SCZ-risk genes as a novel etiological mechanism in SCZ.
Keywords: CPEB; GWAS; RNA-binding protein (RBP); microexon; mouse model; splicing.
Biol Psychiatry 2023 Mar 21;S0006-3223(23)01161-7. doi: 10.1016/j.biopsych.2023.03.010.
8. Dysregulated Cell Homeostasis and miRNAs in Human iPSC-Derived Cardiomyocytes from a Propionic Acidemia Patient with Cardiomyopathy
Mar Álvarez, Pedro Ruiz-Sala, Belén Pérez, Lourdes Ruiz Desviat and Eva Richard
Propionic acidemia (PA) disorder shows major involvement of the heart, among other alterations. A significant number of PA patients develop cardiac complications, and available evidence suggests that this cardiac dysfunction is driven mainly by the accumulation of toxic metabolites. To contribute to the elucidation of the mechanistic basis underlying this dysfunction, we have successfully generated cardiomyocytes through the differentiation of induced pluripotent stem cells (iPSCs) from a PCCB patient and its isogenic control. In this human cellular model, we aimed to examine microRNAs (miRNAs) profiles and analyze several cellular pathways to determine miRNAs activity patterns associated with PA cardiac phenotypes. We have identified a series of upregulated cardiac-enriched miRNAs and alterations in some of their regulated signaling pathways, including an increase in the expression of cardiac damage markers and cardiac channels, an increase in oxidative stress, a decrease in mitochondrial respiration and autophagy; and lipid accumulation. Our findings indicate that miRNA activity patterns from PA iPSC-derived cardiomyocytes are biologically informative and advance the understanding of the molecular mechanisms of this rare disease, providing a basis for identifying new therapeutic targets for intervention strategies.
Keywords: propionic acidemia; PCCB; iPSC; iPSC-derived cardiomyocytes; microRNAs
Accepted: 18 January 2023 Published: 22 January 2023 Cardiomyopathy. Int. J. Mol. Sci. 2023, 24, 2182. https://doi.org/10.3390/ijms24032182
9. Experimental and Bioinformatic Insights into the Effects of Epileptogenic Variants on the Function and Trafficking of the GABA Transporter GAT-1
Dolores Piniella, Ania Canseco, Silvia Vidal, Clara Xiol, Aránzazu Díaz de Bustamante, Itxaso Martí-Carrera, Judith Armstrong, Ugo Bastolla and Francisco Zafra
In this article, we identified a novel epileptogenic variant (G307R) of the gene SLC6A1, which encodes the GABA transporter GAT-1. Our main goal was to investigate the pathogenic mechanisms of this variant, located near the neurotransmitter permeation pathway, and compare it with other variants located either in the permeation pathway or close to the lipid bilayer. The mutants G307R and A334P, close to the gates of the transporter, could be glycosylated with variable efficiency and reached the membrane, albeit inactive. Mutants located in the center of the permeation pathway (G297R) or close to the lipid bilayer (A128V, G550R) were retained in the endoplasmic reticulum. Applying an Elastic Network Model, to these and to other previously characterized variants, we found that G307R and A334P significantly perturb the structure and dynamics of the intracellular gate, which can explain their reduced activity, while for A228V and G362R, the reduced translocation to the membrane quantitatively accounts for the reduced activity. The addition of a chemical chaperone (4-phenylbutyric acid, PBA), which improves protein folding, increased the activity of GAT-1WT, as well as most of the assayed variants, including G307R, suggesting that PBA might also assist the conformational changes occurring during the alternative access transport cycle.
Keywords: GABA transporter; epilepsy; intracellular trafficking; 4-phenylbutyrate
Accepted: 31 December 2022 Published: 4 January 2023 nt. J. Mol. Sci. 2023, 24, 955. https://doi.org/10.3390/ijms24020955
10. Monitoring monkeypox virus in saliva and air samples in Spain: a cross-sectional study
Bruno Hernaez, Ana Muñoz Gómez, Africa Sanchiz, Eva Orviz, Adrian Valls Carbo, Íñigo Sagastagoitia, Oskar Ayerdi, Rocío Martín, Teresa Puerta, Mar Vera, Noemí Cabello, Jorge Vergas, Cristina Prieto, María Pardo-Figuerez, Anabel Negredo, José María Lagarón, Jorge Del Romero, Vicente Estrada, Antonio Alcamí.
Background: The transmission of monkeypox virus occurs through direct contact, but transmission through saliva or exhaled droplets and aerosols has not yet been investigated. We aimed to assess the presence of monkeypox virus DNA and infectious virus in saliva samples and droplets and aerosols exhaled from patients infected with monkeypox virus.
Methods: We did a cross-sectional study in patients with monkeypox confirmed by PCR who attended two health centres in Madrid, Spain. For each patient, we collected samples of saliva, exhaled droplets within a mask, and aerosols captured by air filtration through newly developed nanofiber filters. We evaluated the presence of monkeypox virus in the samples by viral DNA detection by quantitative PCR (qPCR) and isolation of infectious viruses in cell cultures.
Findings: Between May 18 and July 15, 2022, 44 patients with symptomatic monkeypox attended two health centres in Madrid and were included in the study. All were cisgender men, with a median age of 35·0 years (IQR 11·3). We identified high loads of monkeypox virus DNA by qPCR in 35 (85%) of 41 saliva samples. Infectious monkeypox virus was recovered from 22 (67%) of 33 saliva samples positive for monkeypox virus DNA. We also found a significant association between the number of affected cutaneous areas or general symptoms and the viral load present in saliva samples. Droplets exhaled from patients with monkeypox, detected inside a mask, contained monkeypox virus DNA in 32 (71%) of 45 samples, with two of the 32 positive samples showing the presence of the infectious virus. Monkeypox virus DNA in aerosols, collected from the medical consultation room, were detected in 27 (64%) of 42 samples, despite patients wearing an FFP2 mask during the visit. Infectious virus was not recovered from aerosol samples. High levels of monkeypox virus DNA were identified in aerosols collected from a hospital isolation room housing a patient with monkeypox.
Interpretation: The identification of high viable monkeypox virus loads in saliva in most patients with monkeypox and the finding of monkeypox virus DNA in droplets and aerosols warrants further epidemiological studies to evaluate the potential relevance of the respiratory route of infection in the 2022 monkeypox virus outbreak.
Published Online November 24, 2022 https://doi.org/10.1016/S2666-5247(22)00291-9
11. Non-coding RNAs derived from the foot-and-mouth disease virus genome trigger broad antiviral activity against coronaviruses
Miguel Rodríguez-Pulido, Eva Calvo-Pinilla, Miryam Polo, Juan-Carlos Saiz, Raúl Fernández-González, Eva Pericuesta, Alfonso Gutiérrez-Adán, Francisco Sobrino, Miguel A. Martín-Acebes and Margarita Sáiz
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking speciﬁc domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efﬁcacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection signiﬁcantly decreased the viral load and the levels of pro-inﬂammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efﬁcacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to ﬁght future emerging variants.
Keywords: non-coding RNA, foot-and-mouth disease virus (FMDV), SARS-CoV-2, COVID-19, type-I IFN, antiviral immunity, RNA-based therapy, coronaviruses
Aepted 20 March 2023 Published 29 March 2023 Front. Immunol. 14:1166725. doi: 10.3389/fimmu.2023.1166725
12. Nitro-oleic acid regulates T cell activation through post-translational modiﬁcation of calcineurin
Ángel Bago, M. Laura Cayuela, Alba Gil, Enrique Calvo, Jesús Vázquez, Antonio Queiro, Francisco J. Schopfere, Rafael Radi, Juan M. Serrador and Miguel A. Íñiguez
Nitro-fatty acids (NO2-FAs) are unsaturated fatty acid nitration products that exhibit anti-inﬂammatory actions in experimental mouse models of autoimmune and allergic diseases. These electrophilic molecules interfere with intracellular signaling pathways by reversible post-translational modiﬁcation of nucleophilic amino-acid residues.
Several regulatory proteins have been identiﬁed as targets of NO2-FAs, modifying their activity and promoting gene expression changes that result in anti-inﬂammatory eﬀects. Herein, we report the eﬀects of nitro-oleic acid (NO2-OA) on pro-inﬂammatory T cell functions, showing that 9- and 10-NOA, but not their oleic acid precursor, decrease T cell proliferation, expression of activation markers CD25 and CD71 on the plasma membrane, and IL-2, IL-4, and IFN-γ cytokine gene expres-sions. Moreover, we have found that NO2-OA inhibits the transcriptional activity of nuclear factor of activated T cells (NFAT) and that this inhibition takes place through the regulation of the phosphatase activity of calcineurin (CaN), hindering NFAT dephosphorylation, and nuclear translocation in activated T cells. Finally, using mass spectrometry-based approaches, we have found that NO2-OA nitroalkylates CaNA on four Cys (Cys129, 228, 266, and 372), of which only nitroalkylation on Cys372 was of importance for the regulation of CaN phosphatase activity in cells, disturbing functional CaNA/CaNB heterodimer formation. These results provide evidence for an additional mechanism by which NO2-FAs exert their anti-inﬂammatory actions, pointing to their potential as therapeutic bioactive lipids for the modulation of harmful T cell-mediated immune responses.
Keywords: nitro-fatty acids | T cells | NFAT | calcineurin | inflammation
Accepted December 10, 2022 Published January 18, 2023 https://doi.org/10.1073/pnas.2208924120