Publications

by Keyword: Mutation


By year:[ 2018 | 2017 | 2016 | 2015 | 2014 | 2013 | 2012 | 2011 | 2010 | 2009 | 2008 | 2007 | 2006 | 2005 ]

A. R. Dalton, J., Lans, I., Rovira, X., Malhaire, F., Gómez-Santacana, X., Pittolo, S., Gorostiza, P., Llebaria, A., Goudet, C., Pin, J-P., Giraldo, J., (2016). Shining light on an mGlu5 photoswitchable NAM: A theoretical perspective Current Neuropharmacology 14, (5), 441-454

Metabotropic glutamate receptors (mGluRs) are important drug targets because of their involvement in several neurological diseases. Among mGluRs, mGlu5 is a particularly high-profile target because its positive or negative allosteric modulation can potentially treat schizophrenia or anxiety and chronic pain, respectively. Here, we computationally and experimentally probe the functional binding of a novel photoswitchable mGlu5 NAM, termed alloswitch-1, which loses its NAM functionality under violet light. We show alloswitch-1 binds deep in the allosteric pocket in a similar fashion to mavoglurant, the co-crystallized NAM in the mGlu5 transmembrane domain crystal structure. Alloswitch-1, like NAM 2-Methyl-6-(phenylethynyl)pyridine (MPEP), is significantly affected by P655M mutation deep in the allosteric pocket, eradicating its functionality. In MD simulations, we show alloswitch-1 and MPEP stabilize the co-crystallized water molecule located at the bottom of the allosteric site that is seemingly characteristic of the inactive receptor state. Furthermore, both NAMs form H-bonds with S809 on helix 7, which may constitute an important stabilizing interaction for NAM-induced mGlu5 inactivation. Alloswitch-1, through isomerization of its amide group from trans to cis is able to form an additional interaction with N747 on helix 5. This may be an important interaction for amide-containing mGlu5 NAMs, helping to stabilize their binding in a potentially unusual cis-amide state. Simulated conformational switching of alloswitch-1 in silico suggests photoisomerization of its azo group from trans to cis may be possible within the allosteric pocket. However, photoexcited alloswitch-1 binds in an unstable fashion, breaking H-bonds with the protein and destabilizing the co-crystallized water molecule. This suggests photoswitching may have destabilizing effects on mGlu5 binding and functionality.

Keywords: Allosteric modulation, Docking, Metabotropic glutamate receptor, Molecular dynamics, Mutation, Protein structure, Transmembrane domain


Martorell, L., Corrales, I., Ramirez, L., Parra, R., Raya, A., Barquinero, J., Vidal, F., (2015). Molecular characterization of ten F8 splicing mutations in RNA isolated from patient's leucocytes: Assessment of in silico prediction tools accuracy Haemophilia 21, (2), 249-257

Summary: Although 8% of reported FVIII gene (F8) mutations responsible for haemophilia A (HA) affect mRNA processing, very few have been fully characterized at the mRNA level and/or systematically predicted their biological consequences by in silico analysis. This study is aimed to elucidate the effect of potential splice site mutations (PSSM) on the F8 mRNA processing, investigate its correlation with disease severity, and assess their concordance with in silico predictions. We studied the F8 mRNA from 10 HA patient's leucocytes with PSSM by RT-PCR and compared the experimental results with those predicted in silico. The mRNA analysis could explain all the phenotypes observed and demonstrated exon skipping in six cases (c.222G>A, c.601+1delG, c.602-11T>G, c.671-3C>G, c.6115+9C>G and c.6116-1G>A) and activation of cryptic splicing sites, both donor (c.1009+1G>A and c.1009+3A>C) and acceptor sites (c.266-3delC and c.5587-1G>A). In contrast, the in silico analysis was able to predict the score variation of most of the affected splice site, but the precise mechanism could only be correctly determined in two of the 10 mutations analysed. In addition, we have detected aberrant F8 transcripts, even in healthy controls, so this must be taken into account as they could mask the actual contribution of some PSSM. We conclude that F8 mRNA analysis using leucocytes still constitutes an excellent approach to investigate the transcriptional effects of the PSSM in HA, whereas prediction in silico is not always reliable for diagnostic decision-making.

Keywords: Haemophilia A, Leucocytes, RNA splicing, Splice site mutation, Synonymous mutation


Zaffino, R. L., Mir, M., Samitier, J., (2014). Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor Nanotechnology 25, (10), 105501 (8)

We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications.

Keywords: Biosensor, DNA hybridization, Labelfree, Nanogap, Single nucleotide mutation


Sánchez-Danés, A., Richaud-Patin, Y., Carballo-Carbajal, I., Jiménez-Delgado, S., Caig, C., Mora, S., Di Guglielmo, C., Ezquerra, M., Patel, B., Giralt, A., Canals, J. M., Memo, M., Alberch, J., López-Barneo, J., Vila, M., Cuervo, A. M., Tolosa, E., Consiglio, A., Raya, A., (2012). Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson's disease EMBO Molecular Medicine 4, (5), 380-395

Induced pluripotent stem cells (iPSC) offer an unprecedented opportunity to model human disease in relevant cell types, but it is unclear whether they could successfully model age-related diseases such as Parkinson's disease (PD). Here, we generated iPSC lines from seven patients with idiopathic PD (ID-PD), four patients with familial PD associated to the G2019S mutation in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene (LRRK2-PD) and four age- and sex-matched healthy individuals (Ctrl). Over long-time culture, dopaminergic neurons (DAn) differentiated from either ID-PD- or LRRK2-PD-iPSC showed morphological alterations, including reduced numbers of neurites and neurite arborization, as well as accumulation of autophagic vacuoles, which were not evident in DAn differentiated from Ctrl-iPSC. Further induction of autophagy and/or inhibition of lysosomal proteolysis greatly exacerbated the DAn morphological alterations, indicating autophagic compromise in DAn from ID-PD- and LRRK2-PD-iPSC, which we demonstrate occurs at the level of autophagosome clearance. Our study provides an iPSC-based in vitro model that captures the patients' genetic complexity and allows investigation of the pathogenesis of both sporadic and familial PD cases in a disease-relevant cell type.

Keywords: Autophagy, Disease modeling, LRRK2 mutation, Neurodegeneration, Pluripotent stem cells