CENTRO DE BIOLOGÍA MOLECULAR SEVERO OCHOA

Publicaciones representativas

 
Cristina Cacho-Navas, Natalia Reglero-Real, Natalia Colás-Algora, Susana Barroso, Gema de Rivas, Kostantinos Stamatakis, Jorge Feito, Germán Andrés, Manuel Fresno, Leonor Kremer, Isabel Correas, Miguel A. Alonso & Jaime Millán

Apical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory response.

 
Andrea Fernández-Gómez, Beatriz Ramos Velasco, José M Izquierdo

T-cell intracellular antigen 1 (TIA1) is an RNA-binding protein that is primarily involved in the post-transcriptional regulation of cellular RNAs. Furthermore, it is a key component of stress granules (SGs), RNA, and protein aggregates that are formed in response to stressful stimuli to reduce cellular activity as a survival mechanism. TIA1 p.E384K mutation is the genetic cause of Welander distal myopathy (WDM), a late-onset muscular dystrophy whose pathogenesis has been related to modifying SG dynamics. In this study, we present the results obtained by analyzing two specific aspects: (i) SGs properties and dynamics depending on the amino acid at position 384 of TIA1; and (ii) the formation/disassembly time-course of TIA1WT/WDM-dependent SGs under oxidative stress. The generation of TIA1 variants-in which the amino acid mutated in WDM and the adjacent ones were replaced by lysines, glutamic acids, or alanines-allowed us to verify that the inclusion of a single lysine is necessary and sufficient to alter SGs dynamics. Moreover, time-lapse microscopy analysis allowed us to establish in vivo the dynamics of TIA1WT/WDM-dependent SG formation and disassembly, after the elimination of the oxidizing agent, for 1 and 3 h, respectively. Our observations show distinct dynamics between the formation and disassembly of TIA1WT/WDM-dependent SGs. Taken together, this study has allowed us to expand the existing knowledge on the role of TIA1 and the WDM mutation in SG formation.

 
Natalia Azpiazu, Ginés Morata

Regeneration is a response mechanism aiming to reconstruct lost or damaged structures. To achieve this, the cells repopulating the lost tissue often have to change their original identity, a process that involves chromatin remodelling.We have analysed the issue of chromatin remodelling during regeneration in the wing disc of Drosophila . In this disc the ablation of the central region (the pouch) induces the regenerative response of the cells from the lateral region (the hinge), which reconstitute the wing pouch. We have examined euchromatin and heterochromatin histone marks during the process and find that heterochromatin marks disappear but are recovered when regeneration is complete. Euchromatin marks are not modified. We also describe the transcription of two retrotransposons, Roo and F-element in the regenerating cells. We have established a temporal correlation between the alterations of heterochromatin marks and the levels of transcription of two retrotransposons, Roo and F-element, both during embryonic development and in the regeneration process.

 
Beatriz Pardo, Eduardo Herrada-Soler, Jorgina Satrústegui, Laura Contreras, Araceli Del Arco

AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named "early infantile epileptic encephalopathy 39" (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.

 
Gonzalo Garcia-Martin, Miriam Sanz-Rodriguez, Berta Alcover-Sanchez, Marta P Pereira, Francisco Wandosell, Beatriz Cubelos

Since the optic nerve is one of the most myelinated tracts in the central nervous system (CNS), many myelin diseases affect the visual system. In this sense, our laboratory has recently reported that the GTPases R-Ras1 and R-Ras2 are essential for oligodendrocyte survival and maturation. Hypomyelination produced by the absence of one or both proteins triggers axonal degeneration and loss of visual and motor function. However, little is known about R-Ras specificity and other possible roles that they could play in the CNS. In this work, we describe how a lack of R-Ras1 and/or R-Ras2 could not be compensated by increased expression of the closely related R-Ras3 or classical Ras. We further studied R-Ras1 and R-Ras2 expression within different CNS anatomical regions, finding that both were more abundant in less-myelinated regions, suggesting their expression in non-oligodendroglial cells. Finally, using confocal immunostaining colocalization, we report for the first time that R-Ras2 is specifically expressed in neurons. Neither microglia nor astrocytes expressed R-Ras1 or R-Ras2. These results open a new avenue for the study of neuronal R-Ras2's contribution to the process of myelination.

 
Laura Vallés-Saiz, Rocio Peinado-Cahuchola, Jesús Ávila, Félix Hernández

Tau is a cytoskeletal protein that is expressed mainly in neurons and is involved in several cellular processes, such as microtubule stabilization, axonal maintenance, and transport. Altered tau metabolism is related to different tauopathies being the most important Alzheimer's disease where aberrant hyperphosphorylated and aggregated tau is found in the central nervous system. Here, we have analyzed that function in kidney by using tau knockout mice generated by integrating GFP-encoding cDNA into exon 1 of MAPT (here referred to as TauGFP/GFP). IVIS Lumina from PerkinElmer demonstrated GFP expression in the kidney. We then demonstrated by qPCR that the main tau isoform in the kidney is Tau4R. The GFP reporter allowed us to demonstrate that tau is found in the glomeruli of the renal cortex, and specifically in podocytes. This was further confirmed by immunohistochemistry. TauGFP/GFP mice present a podocyte cytoskeleton more dynamic as they contain higher levels of detyrosinated tubulin than wild-type mice. In addition, transmission electron microscopy studies demonstrated glomerular damage with a decrease in urinary creatinine. Our results prove that tau has an important role in kidney metabolism under normal physiological conditions.

 
Alejandro M. Hortal, Clara L. Oeste, Claudia Cifuentes, Miguel Alcoceba, Isabel Fernández-Pisonero, Laura Clavaín, Rut Tercero, Pilar Mendoza, Verónica Domínguez, Marta García-Flores, Belén Pintado, David Abia, Carmen García-Macías, Almudena Navarro-Bailón, Xosé R. Bustelo, Marcos González & Balbino Alarcón
Background

Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRASHRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL.

Methods

Conditional knock-in mice were generated to overexpress wild type RRAS2 and prove its driver role. RT-qPCR analysis of a human CLL sample cohort was carried out to measure RRAS2 transcriptional expression. Sanger DNA sequencing was used to identify a SNP in the 3’UTR region of RRAS2 in human CLL samples. RNAseq of murine CLL was carried out to identify activated pathways, molecular mechanisms and to pinpoint somatic mutations accompanying RRAS2 overexpression. Flow cytometry was used for phenotypic characterization and shRNA techniques to knockdown RRAS2 expression in human CLL.

Results

RRAS2 mRNA is found overexpressed in its wild type form in 82% of the human CLL samples analyzed (n = 178, mean and median = 5-fold) as well as in the explored metadata. A single nucleotide polymorphism (rs8570) in the 3’UTR of the RRAS2 mRNA has been identified in CLL patients, linking higher expression of RRAS2 with more aggressive disease. Deliberate overexpression of wild type RRAS2 in mice, but not an oncogenic Q72L mutation in the coding sequence, provokes the development of CLL. Overexpression of wild type RRAS2 in mice is accompanied by a strong convergent selection of somatic mutations in genes that have been identified in human CLL. R-RAS2 protein is physically bound to the BCR and mediates BCR signals in CLL.

Conclusions

The results indicate that overexpression of wild type RRAS2 is behind the development of CLL.

 
Santos Domínguez-Zotes, Miguel Angel Fuertes, Alicia Rodríguez-Huete, Alejandro Valbuena, Mauricio G Mateu

Protein-based nanostructured materials are being developed for many biomedical and nanotechnological applications. Despite their many desirable features, protein materials are highly susceptible to disruption by mechanical stress and fatigue. This study is aimed to increase fatigue resistance and enhance self-healing of a natural protein-based supramolecular nanomaterial through permanent genetic modification. The authors envisage the conversion of a model nanosheet, formed by a regular array of noncovalently bound human immunodeficiency virus capsid protein molecules, into a supramolecular “chain mail.” Rationally engineered mutations allow the formation of a regular network of disulfide bridges in the protein lattice. This network links each molecule in the lattice to each adjacent molecule through one covalent bond, analogous to the rivetting of interlinked iron rings in the chain mail of a medieval knight. The engineered protein nanosheet shows greatly increased thermostability and resistance to mechanical stress and fatigue in particular, as well as enhanced self-healing, without undesirable stiffening compared to the original material. The results provide proof of concept for a genetic design to permanently increase fatigue resistance and enhance self-healing of protein-based nanostructured materials. They also provide insights into the molecular basis for fatigue of protein materials.

 
Sara Francisco, Jean-Marc Billod, Javier Merino, Carmen Punzón, Alicia Gallego, Alicia Arranz, Sonsoles Martin-Santamaria and Manuel Fresno

The Toll-like receptor 4 (TLR4)/myeloid differentiation protein-2 (MD-2) complex is considered the major receptor of the innate immune system to recognize lipopolysaccharides (LPSs). However, some atypical LPSs with different lipid A and core saccharide moiety structures and compositions than the well-studied enterobacterial LPSs can induce a TLR2-dependent response in innate immune cells. Ochrobactrum intermedium, an opportunistic pathogen, presents an atypical LPS. In this study, we found that O. intermedium LPS exhibits a weak inflammatory activity compared to Escherichia coli LPS and, more importantly, is a specific TLR4/TLR2 agonist, able to signal through both receptors. Molecular docking analysis of O. intermedium LPS predicts a favorable formation of a TLR2/TLR4/MD-2 heterodimer complex, which was experimentally confirmed by fluorescence resonance energy transfer (FRET) in cells. Interestingly, the core saccharide plays an important role in this interaction. This study reveals for the first time TLR4/TLR2 heterodimerization that is induced by atypical LPS and may help to escape from recognition by the innate immune system.

 
Ugo Bastolla, Patrick Chambers, David Abia, Maria-Laura Garcia-Bermejo and Manuel Fresno

Covid-19 is particularly mild with children, and its severity escalates with age. Several theories have been proposed to explain these facts. In particular, it was proposed that the lower expression of the viral receptor ACE2 in children protects them from severe Covid-19. However, other works suggested an inverse relationship between ACE2 expression and disease severity. Here we review the seemingly contradicting observations on ACE2 expression at the levels of mRNA, membrane protein and serum protein in humans and rodents and try to reconcile them at the light of the Renin-Angiotensin system (RAS) and bradykinin system, which constitute an integrated inflammatory system connected by common peptidases and interacting receptors. We find that ACE2 level is not monotonically related with age but it reaches a maximum at a young age that depends on the cell type and then decreases, consistently with almost all existing data. The increase with age of the protease Tumor necrosis factor alpha (TNF-α) converting enzyme (TACE), also known as ADAM17 (a disintegrin and metalloproteinase 17) that sheds ACE2 from the cell membrane to the serum predicts that the decrease occurs before and is steeper for ACE2 cell protein than for its mRNA. This negative relation between ACE2 level and Covid-19 severity at old age is not paradoxical but it is consistent with a mathematical model that predicts that higher viral receptor does not necessarily favour virus propagation and it can even slow it down. More importantly, the angiotensin-bradykinin system is characterized by a powerful positive feedback loop that enhances inflammation through the Angiotensin and Bradykinin receptors that upregulate ADAM17, which in turn downregulates ACE2 and upregulates TNF-α and the pro-inflammatory receptor of the cytokine interleukin 6 (IL6). Here we propose that ACE2 contributes essentially to reverse this inflammatory state by downregulating the pro-inflammatory peptides of the angiotensin-bradykinin system, and that failure to do this, possibly induced by the degradation of ACE2 by SARS-COV-2, may underlie both severe CoViD-19 infection and its many post-infection manifestations, including the multi-inflammatory syndrome of children (MIS-C). Within this view, lower severity in children despite lower ACE2 expression may be consistent with their higher expression of the alternative angiotensin II receptor ATR2 and in general of the anti-inflammatory arm of the RAS at young age.

 
Deepa S. Rajan, Sukhleen Kour, Tyler R. Fortuna, Margot A. Cousin, Sarah S. Barnett, Zhiyv Niu, Dusica Babovic-Vuksanovic, Eric W. Klee,Brian Kirmse, Micheil Innes, Siri Lynne Rydning, Kaja K. Selmer, Magnus Dehli Vigeland, Anne Kjersti Erichsen, Andrea H. Nemeth, Francisca Millan, Catherine DeVile, Katherine Fawcett, Adrien Legendre, David Sims, Ricardo Parolin Schnekenberg, Lydie Burglen, Sandra Mercier, Somayeh Bakhtiari, Rosario Francisco-Velilla, Azman Embarc-Buh, Encarnacion Martinez-Salas, Kristen Wigby, Jerica Lenberg, Jennifer R. Friedman, Michael C. Kruer and Udai Bhan Pandey

The hereditary ataxias are a heterogenous group of disorders with an increasing number of causative genes being described. Due to the clinical and genetic heterogeneity seen in these conditions, the majority of such individuals endure a diagnostic odyssey or remain undiagnosed. Defining the molecular etiology can bring insights into the responsible molecular pathways and eventually the identification of therapeutic targets. Here, we describe the identification of biallelic variants in the GEMIN5 gene among seven unrelated families with nine affected individuals presenting with spastic ataxia and cerebellar atrophy. GEMIN5, an RNA-binding protein, has been shown to regulate transcription and translation machinery. GEMIN5 is a component of small nuclear ribonucleoprotein (snRNP) complexes and helps in the assembly of the spliceosome complexes. We found that biallelic GEMIN5 variants cause structural abnormalities in the encoded protein and reduce expression of snRNP complex proteins in patient cells compared with unaffected controls. Finally, knocking out endogenous Gemin5 in mice caused early embryonic lethality, suggesting that Gemin5 expression is crucial for normal development. Our work further expands on the phenotypic spectrum associated with GEMIN5-related disease and implicates the role of GEMIN5 among patients with spastic ataxia, cerebellar atrophy, and motor predominant developmental delay.

 
Pilar López-Nieva, Iria González-Vasconcellos, Laura González-Sánchez, María A. Cobos-Fernández, Sara Ruiz-García, Raúl Sánchez Pérez, Ángel Aroca, José Fernández-Piqueras & Javier Santos

In the quest for more effective radiation treatment options that can improve both cell killing and healthy tissue recovery, combined radiation therapies are lately in the spotlight. The molecular response to a combined radiation regime where exposure to an initial low dose (priming dose) of ionizing radiation is administered prior to a subsequent higher radiation dose (challenging dose) after a given latency period have not been thoroughly explored. In this study we report on the differential response to either a combined radiation regime or a single challenging dose both in mouse in vivo and in human ex vivo thymocytes. A differential cell cycle response including an increase in the subG1 fraction on cells exposed to the combined regime was found. Together with this, a differential protein expression profiling in several pathways including cell cycle control (ATM, TP53, p21CDKN1A), damage response (γH2AX) and cell death pathways such as apoptosis (Cleaved Caspase-3, PARP1, PKCδ and H3T45ph) and ferroptosis (xCT/GPX4) was demonstrated. This study also shows the epigenetic regulation following a combined regime that alters the expression of chromatin modifiers such as DNMTs (DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L) and glycosylases (MBD4 and TDG). Furthermore, a study of the underlying cellular status six hours after the priming dose alone showed evidence of retained modifications on the molecular and epigenetic pathways suggesting that the priming dose infers a “radiation awareness phenotype” to the thymocytes, a sensitization key to the differential response seen after the second hit with the challenging dose. These data suggest that combined-dose radiation regimes could be more efficient at making cells respond to radiation and it would be interesting to further investigate how can these schemes be of use to potential new radiation therapies.

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