Publications by authors named "Pablo Fuentes-Prior"

56 Publications

Beyond copy number: A new, rapid, and versatile method for sequencing the entire SMN2 gene in SMA patients.

Hum Mutat 2021 Mar 19. Epub 2021 Mar 19.

Medicine Genetics Group, Vall d'Hebron Research Institute (VHIR), Barcelona, Spain.

Spinal muscular atrophy (SMA) is caused by bi-allelic loss or pathogenic variants in the SMN1 gene. SMN2, the highly homologous copy of SMN1, is considered the major phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish robust genotype-phenotype correlations and predict disease evolution, to stratify patients for clinical trials, as well as to define those eligible for treatment. Discordant genotype-phenotype correlations are not uncommon in SMA, some of which are due to intragenic SMN2 variants that may influence the amount of complete SMN transcripts and, therefore, of full-length SMN protein. Detection of these variants is crucial to predict SMA phenotypes in the present scenario of therapeutic advances and with the perspective of SMA neonatal screening and early diagnosis to start treatments. Here, we present a novel, affordable, and versatile method for complete sequencing of the SMN2 gene based on long-range polymerase chain reaction and next-generation sequencing. The method was validated by analyzing samples from 53 SMA patients who lack SMN1, allowing to characterize paralogous, rare variants, and single-nucleotide polymorphisms of SMN2 as well as SMN2-SMN1 hybrid genes. The method identifies partial deletions and can be adapted to determine rare pathogenic variants in patients with at least one SMN1 copy.
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http://dx.doi.org/10.1002/humu.24200DOI Listing
March 2021

Practical guidelines to manage discordant situations of copy number in patients with spinal muscular atrophy.

Neurol Genet 2020 Dec 18;6(6):e530. Epub 2020 Nov 18.

Medicine Genetics Group (I.C., L.B.-P., M.C., E.F.T.), Vall dHebron Research Institute (VHIR), Barcelona; Department of Clinical and Molecular Genetics (I.C., L.B.-P., M.C., E.F.T.), Hospital Vall dHebron, Barcelona; Department of Genetics (S.B., L.A.), Hospital de la Santa Creu i Sant Pau, Barcelona; Biomedical Research Institute Sant Pau (IIB Sant Pau) (S.B., L.A.), Hospital de la Santa Creu i Sant Pau, Barcelona; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER-ISCIII, U-705 Barcelona) (S.B., L.A.), Madrid; Molecular Bases of Disease (P.F.-P.), Biomedical Research Institute Sant Pau (IIB Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

Objective: Assessment of copy number in patients with spinal muscular atrophy (SMA) is essential to establish careful genotype-phenotype correlations and predict disease evolution. This issue is becoming crucial in the present scenario of therapeutic advances with the perspective of SMA neonatal screening and early diagnosis to initiate treatment, as this value is critical to stratify patients for clinical trials and to define those eligible to receive medication. Several technical pitfalls and interindividual variations may account for reported discrepancies in the estimation of copy number and establishment of phenotype-genotype correlations.

Methods: We propose a management guide based on a sequence of specified actions once copy number is determined for a given patient. Regardless of the method used to estimate the number of copies, our approach focuses on the manifestations of the patient to recommend how to proceed in each case.

Results: We defined situations according to copy number in a presymptomatic scenario of screening, in which we predict the possible evolution, and when a symptomatic patient is genetically confirmed. Unexpected discordant cases include patients having a single copy but noncongenital disease forms, 2 copies compatible with type II or III SMA, and 3 or 4 copies of the gene showing more severe disease than expected.

Conclusions: Our proposed guideline would help to systematically identify discordant SMA cases that warrant further genetic investigation. The gene, as the main modifier of SMA phenotype, deserves a more in-depth study to provide more accurate genotype-phenotype correlations.
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http://dx.doi.org/10.1212/NXG.0000000000000530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7713720PMC
December 2020

A new de novo SYT2 mutation presenting as distal weakness. Neuropathy or neuromuscular junction dysfunction?

J Peripher Nerv Syst 2021 03 22;26(1):113-117. Epub 2020 Dec 22.

Neuromuscular Diseases Unit, Department of Neurology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Barcelona, Spain.

We report the case of a patient with a clinical phenotype characterized by distal lower limb weakness and pes cavus. The electrophysiological study showed slightly reduced or normal amplitude of motor potentials, a decremental response to repetitive nerve stimulation and post-exercise facilitation. Muscle biopsy showed only mild neurogenic features. Genetic analysis included a clinical exome sequencing, followed by Sanger analysis. Three-dimensional (3D) models were generated with a SwissModel (https://swissmodel.expasy.org/) to explain the clinical observations and reinforce the pathogenic nature of the genetic variant identified. Genetic analysis demonstrated a new de novo heterozygous in frame deletion of the SYT2 gene (NM_177402.4: c.1082_1096del), confirmed by Sanger sequencing, which removes five aminoacids in the C2B domain of synaptotagmin-2 protein, that cause a profound effect on the structure and function of this synaptic vesicle protein. We identified a de novo genetic variant in the SYT2 gene, further supporting its association with a highly stereotyped clinical and electrophysiological phenotype. Our case showed electrophysiological features consistent with a presynaptic dysfunction in the neuromuscular junction with normal post-exercise amplitudes, not supporting the presence of predominant axonal damage. Although the analysis of SYT2 gene should be included in genetic analysis of patients presenting with this clinical phenotype that mimics motor neuropathy, clinicians have to consider the study of neuromuscular transmission to early identify this potentially treatable condition.
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http://dx.doi.org/10.1111/jns.12425DOI Listing
March 2021

Novel PLEKHG5 mutations in a patient with childhood-onset lower motor neuron disease.

Ann Clin Transl Neurol 2021 01 4;8(1):294-299. Epub 2020 Dec 4.

Neurology Department, Complejo Universitario de Navarra, IdisNa, Navarra, 31008, Spain.

The PLEKHG5 gene encodes a protein that activates the nuclear factor kappa B (NFκB) signaling pathway. Mutations in this gene have been associated with distal spinal muscular atrophy IV and intermediate axonal neuropathy C, both with an autosomal recessive mode of inheritance. Two families with low motor neuron disease (LMND) caused by mutations in PLEKHG5 have been reported to date. We present a third LMND family, the first nonconsanguineous, due to two not previously reported PLEKHG5 mutations. Our results confirm and extend previous findings linking PLEKHG5 mutations to lower motor neuron diseases.
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http://dx.doi.org/10.1002/acn3.51265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7818229PMC
January 2021

Priming of SARS-CoV-2 S protein by several membrane-bound serine proteinases could explain enhanced viral infectivity and systemic COVID-19 infection.

J Biol Chem 2020 12 2. Epub 2020 Dec 2.

Molecular Bases of Disease, Biomedical Research Institute Sant Pau (IIB Sant Pau), Hospital de la Santa, Barcelona, Spain.

The ongoing COVID-19 pandemic has already caused over a million deaths worldwide, and this death toll will be much higher before effective treatments and vaccines are available. The causative agent of the disease, the coronavirus SARS-CoV-2, shows important similarities with the previously emerged SARS-CoV-1, but also striking differences. First, SARS-CoV-2 possesses a significantly higher transmission rate and infectivity than SARS-CoV-1 and has infected in a few months over 60 million people. Moreover, COVID-19 has a systemic character, as in addition to the lungs it also affects heart, liver, and kidneys among other organs of the patients, and causes frequent thrombotic and neurological complications. In fact, the term "viral sepsis" has been recently coined to describe the clinical observations. Here I review current structure-function information on the viral spike proteins and the membrane fusion process to provide plausible explanations for these observations. I hypothesize that several membrane-associated serine proteinases (MASPs), in synergy with or in place of TMPRSS2, contribute to activate the SARS-CoV-2 spike protein. Relative concentrations of the attachment receptor, ACE2, MASPs, their endogenous inhibitors (the Kunitz-type transmembrane inhibitors, HAI-1/SPINT1 and HAI-2/SPINT2, as well as major circulating serpins) would determine the infection rate of host cells. The exclusive or predominant expression of major MASPs in specific human organs suggests a direct role of these proteinases in e.g. heart infection and myocardial injury, liver dysfunction, kidney damage, as well as neurological complications. Thorough consideration of these factors could have a positive impact on the control of the current COVID-19 pandemic.
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http://dx.doi.org/10.1074/jbc.REV120.015980DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7834812PMC
December 2020

Non-canonical dimerization of the androgen receptor and other nuclear receptors: implications for human disease.

Endocr Relat Cancer 2019 08;26(8):R479-R497

Department of Biochemistry and Molecular Biomedicine, Institute of Biomedicine (IBUB) of the University of Barcelona (UB), Barcelona, Spain.

Nuclear receptors are transcription factors that play critical roles in development, homeostasis and metabolism in all multicellular organisms. An important family of nuclear receptors comprises those members that respond to steroid hormones, and which is subdivided in turn into estrogen receptor (ER) isoforms α and β (NR3A1 and A2, respectively), and a second subfamily of so-called oxosteroid receptors. The latter includes the androgen receptor (AR/NR3C4), the glucocorticoid receptor (GR/NR3C1), the mineralocorticoid receptor (MR/NR3C2) and the progesterone receptor (PR/NR3C3). Here we review recent advances in our understanding of the structure-and-function relationship of steroid nuclear receptors and discuss their implications for the etiology of human diseases. We focus in particular on the role played by AR dysregulation in both prostate cancer (PCa) and androgen insensitivity syndromes (AIS), but also discuss conditions linked to mutations of the GR gene as well as those in a non-steroidal receptor, the thyroid hormone receptor (TR). Finally, we explore how these recent results might be exploited for the development of novel and selective therapeutic strategies.
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http://dx.doi.org/10.1530/ERC-19-0132DOI Listing
August 2019

TET2 missense variants in human neoplasia. A proposal of structural and functional classification.

Mol Genet Genomic Med 2019 07 11;7(7):e00772. Epub 2019 Jun 11.

Molecular Bases of Disease, The Biomedical Research Institute Sant Pau (IIB Sant Pau), Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

Background: The human TET2 gene plays a pivotal role in the epigenetic regulation of normal and malignant hematopoiesis. Somatic TET2 mutations have been repeatedly identified in age-related clonal hematopoiesis and in myeloid neoplasms ranging from acute myeloid leukemia (AML) to myeloproliferative neoplasms. However, there have been no attempts to systematically explore the structural and functional consequences of the hundreds of TET2 missense variants reported to date.

Methods: We have sequenced the TET2 gene in 189 Spanish AML patients using Sanger sequencing and NGS protocols. Next, we performed a thorough bioinformatics analysis of TET2 protein and of the expected impact of all reported TET2 missense variants on protein structure and function, exploiting available structure-and-function information as well as 3D structure prediction tools.

Results: We have identified 38 TET2 allelic variants in the studied patients, including two frequent SNPs: p.G355D (10 cases) and p.I1762V (28 cases). Four of the detected mutations are reported here for the first time: c.122C>T (p.P41L), c.4535C>G (p.A1512G), c.4760A>G (p.D1587G), and c.5087A>T (p.Y1696F). We predict a complex multidomain architecture for the noncatalytic regions of TET2, and in particular the presence of well-conserved α+β globular domains immediately preceding and following the actual catalytic unit. Further, we provide a rigorous interpretation of over 430 missense SNVs that affect the TET2 catalytic domain, and we hypothesize explanations for ~700 additional variants found within the regulatory regions of the protein. Finally, we propose a systematic classification of all missense mutants and SNPs reported to date into three major categories (severe, moderate, and mild), based on their predicted structural and functional impact.

Conclusions: The proposed classification of missense TET2 variants would help to assess their clinical impact on human neoplasia and may guide future structure-and-function investigations of TET family members.
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http://dx.doi.org/10.1002/mgg3.772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6625141PMC
July 2019

Metazoan evolution of glutamate receptors reveals unreported phylogenetic groups and divergent lineage-specific events.

Elife 2018 11 22;7. Epub 2018 Nov 22.

Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau, Barcelona, Spain.

Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings.
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http://dx.doi.org/10.7554/eLife.35774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307864PMC
November 2018

A rare STAP1 mutation incompletely associated with familial hypercholesterolemia.

Clin Chim Acta 2018 Dec 9;487:270-274. Epub 2018 Oct 9.

Institut de Recerca de l'HSCSP - IIB Sant Pau, Spain. Electronic address:

Autosomal dominant hypercholesterolemia, being referred to as familial hypercholesterolemia (FH), is mainly due to defective LDL receptor (LDLR) function, but is also associated with variants in genes encoding APOB (LDLR ligand) and PCSK9, the catabolic regulator of LDLR. The signal-transducing adaptor family member 1 (STAP1) gene has been recently linked to FH. We describe the case of a 56-year-old male patient found to have hypercholesterolemia at age 34, but who did not continue follow-up nor received treatment with lipid-lowering drugs. At age 55 he suffered a myocardial infarction. A systematic NGS analysis did not show point mutations in the LDLR, APOB, LDLRAP1, or PCSK9 genes, nor large rearrangements of the LDLR gene, but revealed the heterozygous missense variant rs199787258 of STAP1 (c.526C > T; p.Pro176Ser). This variant was also found in heterozygosis in the two siblings of the index case, who also had hypercholesterolemia, but did not cosegregate in his progeny. A bioinformatics analysis and available structural information predicts p.Pro176Ser as the most damaging of all STAP1 missense variants associated with familial hypercholesterolemia. Our findings confirm and extend the linkage between STAP1 variants and FH, and point to an important role of this adaptor protein within a signaling pathway that affects cholesterol homeostasis.
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http://dx.doi.org/10.1016/j.cca.2018.10.014DOI Listing
December 2018

Diversity of Quaternary Structures Regulates Nuclear Receptor Activities.

Trends Biochem Sci 2019 01 4;44(1):2-6. Epub 2018 Oct 4.

Department of Biochemistry and Molecular Biomedicine, Institute of Biomedicine (IBUB), University of Barcelona (UB), 08028 Barcelona, Spain; Equally contributing authors. Electronic address:

Nuclear receptors (NRs) form homo- and/or heterodimers as central scaffolds of multiprotein complexes, which activate or repress gene transcription to regulate development, homeostasis, and metabolism. Recent studies on NR quaternary structure reveal novel mechanisms of receptor dimerization, the existence of tetrameric chromatin-bound NRs, and previously unanticipated protein-protein/protein-DNA interactions.
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http://dx.doi.org/10.1016/j.tibs.2018.09.005DOI Listing
January 2019

Utility of two SMN1 variants to improve spinal muscular atrophy carrier diagnosis and genetic counselling.

Eur J Hum Genet 2018 10 14;26(10):1554-1557. Epub 2018 Jun 14.

CIBERER (CB06/07/0011 group), Barcelona, Spain.

Spinal muscular atrophy (SMA) is caused by deletions/mutations in SMN1. Most heterozygous SMA carriers have only one SMN1 copy in one of the alleles (1/0 carriers). However, a few carriers lack SMN1 in one of their chromosomes, but present two gene copies in the other. These "2/0 carriers" are undistinguishable from non-carrier individuals (1/1) with currently available methods. Previous association of SMN1 variants c.*3 + 80 T > G and c.*211_*212del with two SMN1 copies in cis in Ashkenazi population prompted us to analyze them in 270 Spanish individuals (SMA carriers, patients and general population). Both variants were much more frequently detected in chromosomes with 2 SMN1 copies in cis in comparison with chromosomes carrying one copy (17.9 vs. 0.7%; p < 0.001). In particular, one-fifth of 2/0 SMA carriers harboured one or both variants compared to none of 99 non-carriers with two SMN1 copies (p < 0.001). The c.*211_*212del variant was also much more frequent in exon 8 of SMN2-SMN1 hybrids than in that of intact SMN1 genes (20 vs. 0.83%, p < 0.001), suggesting its association with chromosomal rearrangements. Although absence of these variants does not exclude that a particular individual is a 2/0 SMA carrier, their presence is valuable to substantially increase residual risk in putative carriers, thus improving genetic counselling.
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http://dx.doi.org/10.1038/s41431-018-0193-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138687PMC
October 2018

C-Reactive Protein as a Therapeutic Target in Age-Related Macular Degeneration.

Front Immunol 2018 19;9:808. Epub 2018 Apr 19.

Academic Unit of Ophthalmology, School of Clinical Sciences, University of Bristol, Bristol, United Kingdom.

Age-related macular degeneration (AMD), a retinal degenerative disease, is the leading cause of central vision loss among the elderly population in developed countries and an increasing global burden. The major risk is aging, compounded by other environmental factors and association with genetic variants for risk of progression. Although the etiology of AMD is not yet clearly understood, several pathogenic pathways have been proposed, including dysfunction of the retinal pigment epithelium, inflammation, and oxidative stress. The identification of AMD susceptibility genes encoding complement factors and the presence of complement and other inflammatory mediators in drusen, the hallmark deposits of AMD, support the concept that local inflammation and immune-mediated processes play a key role in AMD pathogenesis that may be accelerated through systemic immune activation. In this regard, increased levels of circulating C-reactive protein (CRP) have been associated with higher risk of AMD. Besides being a risk marker for AMD, CRP may also play a role in the progression of the disease as it has been identified in drusen, and we have recently found that its monomeric form (mCRP) induces blood retinal barrier disruption . In this review, we will address recent evidence that links CRP and AMD pathogenesis, which may open new therapeutic opportunities to prevent the progression of AMD.
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http://dx.doi.org/10.3389/fimmu.2018.00808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5916960PMC
June 2019

Correlation between SMA type and SMN2 copy number revisited: An analysis of 625 unrelated Spanish patients and a compilation of 2834 reported cases.

Neuromuscul Disord 2018 03 11;28(3):208-215. Epub 2018 Jan 11.

Medicine Genetics Group, Vall d'Hebron Research Institute (VHIR), 08035 Barcelona, Spain; CB06/07/0011 group, CIBERER, Barcelona, Spain; Department of Clinical and Molecular Genetics and Rare Diseases Unit, Hospital Vall d'Hebron, 08035 Barcelona, Spain. Electronic address:

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by loss or mutations in SMN1. According to age of onset, achieved motor abilities, and life span, SMA patients are classified into type I (never sit), II (never walk unaided) or III (achieve independent walking abilities). SMN2, the highly homologous copy of SMN1, is considered the most important phenotypic modifier of the disease. Determination of SMN2 copy number is essential to establish careful genotype-phenotype correlations, predict disease evolution, and to stratify patients for clinical trials. We have determined SMN2 copy numbers in 625 unrelated Spanish SMA patients with loss or mutation of both copies of SMN1 and a clear assignation of the SMA type by clinical criteria. Furthermore, we compiled data from relevant worldwide reports that link SMN2 copy number with SMA severity published from 1999 to date (2834 patients with different ethnic and geographic backgrounds). Altogether, we have assembled a database with a total of 3459 patients to delineate more universal prognostic rules regarding the influence of SMN2 copy number on SMA phenotype. This issue is crucial in the present scenario of therapeutic advances with the perspective of SMA neonatal screening and early diagnosis to initiate treatments.
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http://dx.doi.org/10.1016/j.nmd.2018.01.003DOI Listing
March 2018

Structure of the homodimeric androgen receptor ligand-binding domain.

Nat Commun 2017 02 6;8:14388. Epub 2017 Feb 6.

Department of Biochemistry and Molecular Biomedicine, Institute of Biomedicine (IBUB) of the University of Barcelona (UB), Barcelona 08028, Spain.

The androgen receptor (AR) plays a crucial role in normal physiology, development and metabolism as well as in the aetiology and treatment of diverse pathologies such as androgen insensitivity syndromes (AIS), male infertility and prostate cancer (PCa). Here we show that dimerization of AR ligand-binding domain (LBD) is induced by receptor agonists but not by antagonists. The 2.15-Å crystal structure of homodimeric, agonist- and coactivator peptide-bound AR-LBD unveils a 1,000-Å large dimerization surface, which harbours over 40 previously unexplained AIS- and PCa-associated point mutations. An AIS mutation in the self-association interface (P767A) disrupts dimer formation in vivo, and has a detrimental effect on the transactivating properties of full-length AR, despite retained hormone-binding capacity. The conservation of essential residues suggests that the unveiled dimerization mechanism might be shared by other nuclear receptors. Our work defines AR-LBD homodimerization as an essential step in the proper functioning of this important transcription factor.
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http://dx.doi.org/10.1038/ncomms14388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303882PMC
February 2017

Complement factor H binding of monomeric C-reactive protein downregulates proinflammatory activity and is impaired with at risk polymorphic CFH variants.

Sci Rep 2016 Mar 10;6:22889. Epub 2016 Mar 10.

Academic Unit of Ophthalmology, School of Clinical Sciences and School of Cellular and Molecular Medicine, University of Bristol, Bristol, BS8 1TH, UK.

Inflammation and immune-mediated processes are pivotal to the pathogenic progression of age-related macular degeneration (AMD). Although plasma levels of C-reactive protein (CRP) have been shown to be associated with an increased risk for AMD, the pathophysiological importance of the prototypical acute-phase reactant in the etiology of the disease is unknown, and data regarding the exact role of CRP in ocular inflammation are limited. In this study, we provide mechanistic insight into how CRP contributes to the development of AMD. In particular, we show that monomeric CRP (mCRP) but not the pentameric form (pCRP) upregulates IL-8 and CCL2 levels in retinal pigment epithelial cells. Further, we show that complement factor H (FH) binds mCRP to dampen its proinflammatory activity. FH from AMD patients carrying the "risk" His402 polymorphism displays impaired binding to mCRP, and therefore proinflammatory effects of mCRP remain unrestrained.
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http://dx.doi.org/10.1038/srep22889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4785391PMC
March 2016

A novel gain-of-function STAT1 mutation resulting in basal phosphorylation of STAT1 and increased distal IFN-γ-mediated responses in chronic mucocutaneous candidiasis.

Mol Immunol 2015 Dec;68(2 Pt C):597-605

Department of Immunology, Hospital de la Santa Creu i Sant Pau, Universitat Autònoma de Barcelona, Spain. Electronic address:

Gain-of-function STAT1 mutations have recently been associated with autosomal dominant chronic mucocutaneous candidiasis (CMC). The purpose of this study was to characterize the three members of a non-consanguineous family, the father and his two sons, who presented with recurrent oral thrush and ocular candidiasis since early childhood. The three patients had reduced levels of IL-17-producing T cells. This reduction affected specifically IL-17(+)IFN-γ(-) T cells, because the levels of IL-17(+)IFN-γ(+) T cells were similar to controls. We found that PBMC (peripheral blood mononuclear cells) from the patients did not respond to Candida albicans ex vivo. Moreover, after polyclonal activation, patients' PBMC produced lower levels of IL-17 and IL-6 and higher levels of IL-4 than healthy controls. Genetic analyses showed that the three patients were heterozygous for a new mutation in STAT1 (c.894A>C, p.K298N) that affects a highly conserved residue of the coiled-coil domain of STAT1. STAT1 phosphorylation levels were significantly higher in patients' cells than in healthy controls, both in basal conditions and after IFN-γ stimulation, suggesting a permanent activation of STAT1. Cells from the patients also presented increased IFN-γ-mediated responses measured as MIG and IP-10 production. In conclusion, we report a novel gain-of-function mutation in the coiled-coil domain of STAT1, which increases STAT1 phosphorylation and impairs IL-17-mediated immunity. The mutation is responsible for CMC in this family with autosomal dominant inheritance of the disease.
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http://dx.doi.org/10.1016/j.molimm.2015.09.014DOI Listing
December 2015

K Domain CR9 of Low Density Lipoprotein (LDL) Receptor-related Protein 1 (LRP1) Is Critical for Aggregated LDL-induced Foam Cell Formation from Human Vascular Smooth Muscle Cells.

J Biol Chem 2015 Jun 27;290(24):14852-65. Epub 2015 Apr 27.

From the Cardiovascular Research Center, CSIC-ICCC, Biomedical Research Institute Sant Pau (IIB Sant Pau), 08025 Barcelona, Spain and

Low density lipoprotein receptor-related protein (LRP1) mediates the internalization of aggregated LDL (AgLDL), which in turn increases the expression of LRP1 in human vascular smooth muscle cells (hVSMCs). This positive feedback mechanism is thus highly efficient to promote the formation of hVSMC foam cells, a crucial vascular component determining the susceptibility of atherosclerotic plaque to rupture. Here we have determined the LRP1 domains involved in AgLDL recognition with the aim of specifically blocking AgLDL internalization in hVSMCs. The capacity of fluorescently labeled AgLDL to bind to functional LRP1 clusters was tested in a receptor-ligand fluorometric assay made by immobilizing soluble LRP1 "minireceptors" (sLRP1-II, sLRP1-III, and sLRP1-IV) recombinantly expressed in CHO cells. This assay showed that AgLDL binds to cluster II. We predicted three well exposed and potentially immunogenic peptides in the CR7-CR9 domains of this cluster (termed P1 (Cys(1051)-Glu(1066)), P2 (Asp(1090)-Cys(1104)), and P3 (Gly(1127)-Cys(1140))). AgLDL, but not native LDL, bound specifically and tightly to P3-coated wells. Rabbit polyclonal antibodies raised against P3 prevented AgLDL uptake by hVSMCs and were almost twice as effective as anti-P1 and anti-P2 Abs in reducing intracellular cholesteryl ester accumulation. Moreover, anti-P3 Abs efficiently prevented AgLDL-induced LRP1 up-regulation and counteracted the down-regulatory effect of AgLDL on hVSMC migration. In conclusion, domain CR9 appears to be critical for LRP1-mediated AgLDL binding and internalization in hVSMCs. Our results open new avenues for an innovative anti-VSMC foam cell-based strategy for the treatment of vascular lipid deposition in atherosclerosis.
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http://dx.doi.org/10.1074/jbc.M115.638361DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4463433PMC
June 2015

Rare variants in calcium homeostasis modulator 1 (CALHM1) found in early onset Alzheimer's disease patients alter calcium homeostasis.

PLoS One 2013 17;8(9):e74203. Epub 2013 Sep 17.

Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, University Pompeu Fabra, Barcelona, Spain.

Calcium signaling in the brain is fundamental to the learning and memory process and there is evidence to suggest that its dysfunction is involved in the pathological pathways underlying Alzheimer's disease (AD). Recently, the calcium hypothesis of AD has received support with the identification of the non-selective Ca(2+)-permeable channel CALHM1. A genetic polymorphism (p. P86L) in CALHM1 reduces plasma membrane Ca(2+) permeability and is associated with an earlier age-at-onset of AD. To investigate the role of CALHM1 variants in early-onset AD (EOAD), we sequenced all CALHM1 coding regions in three independent series comprising 284 EOAD patients and 326 controls. Two missense mutations in patients (p.G330D and p.R154H) and one (p.A213T) in a control individual were identified. Calcium imaging analyses revealed that while the mutation found in a control (p.A213T) behaved as wild-type CALHM1 (CALHM1-WT), a complete abolishment of the Ca(2+) influx was associated with the mutations found in EOAD patients (p.G330D and p.R154H). Notably, the previously reported p. P86L mutation was associated with an intermediate Ca(2+) influx between the CALHM1-WT and the p.G330D and p.R154H mutations. Since neither expression of wild-type nor mutant CALHM1 affected amyloid ß-peptide (Aß) production or Aß-mediated cellular toxicity, we conclude that rare genetic variants in CALHM1 lead to Ca(2+) dysregulation and may contribute to the risk of EOAD through a mechanism independent from the classical Aß cascade.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0074203PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775809PMC
May 2014

Structural and functional analysis of APOA5 mutations identified in patients with severe hypertriglyceridemia.

J Lipid Res 2013 Mar 10;54(3):649-61. Epub 2013 Jan 10.

Institute for Biomedical Research-IIB Sant Pau, 08025 Barcelona, Spain.

During the diagnosis of three unrelated patients with severe hypertriglyceridemia, three APOA5 mutations [p.(Ser232_Leu235)del, p.Leu253Pro, and p.Asp332ValfsX4] were found without evidence of concomitant LPL, APOC2, or GPIHBP1 mutations. The molecular mechanisms by which APOA5 mutations result in severe hypertriglyceridemia remain poorly understood, and the functional impairment/s induced by these specific mutations was not obvious. Therefore, we performed a thorough structural and functional analysis that included follow-up of patients and their closest relatives, measurement of apoA-V serum concentrations, and sequencing of the APOA5 gene in 200 nonhyperlipidemic controls. Further, we cloned, overexpressed, and purified both wild-type and mutant apoA-V variants and characterized their capacity to activate LPL. The interactions of recombinant wild-type and mutated apoA-V variants with liposomes of different composition, heparin, LRP1, sortilin, and SorLA/LR11 were also analyzed. Finally, to explore the possible structural consequences of these mutations, we developed a three-dimensional model of full-length, lipid-free human apoA-V. A complex, wide array of impairments was found in each of the three mutants, suggesting that the specific residues affected are critical structural determinants for apoA-V function in lipoprotein metabolism and, therefore, that these APOA5 mutations are a direct cause of hypertriglyceridemia.
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http://dx.doi.org/10.1194/jlr.M031195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3617940PMC
March 2013

Unique thrombin inhibition mechanism by anophelin, an anticoagulant from the malaria vector.

Proc Natl Acad Sci U S A 2012 Dec 5;109(52):E3649-58. Epub 2012 Dec 5.

Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, 4150-180 Porto, Portugal.

Anopheles mosquitoes are vectors of malaria, a potentially fatal blood disease affecting half a billion humans worldwide. These blood-feeding insects include in their antihemostatic arsenal a potent thrombin inhibitor, the flexible and cysteine-less anophelin. Here, we present a thorough structure-and-function analysis of thrombin inhibition by anophelin, including the 2.3-Å crystal structure of the human thrombin·anophelin complex. Anophelin residues 32-61 are well-defined by electron density, completely occupying the long cleft between the active site and exosite I. However, in striking contrast to substrates, the D50-R53 anophelin tetrapeptide occupies the active site cleft of the enzyme, whereas the upstream residues A35-P45 shield the regulatory exosite I, defining a unique reverse-binding mode of an inhibitor to the target proteinase. The extensive interactions established, the disruption of thrombin's active site charge-relay system, and the insertion of residue R53 into the proteinase S(1) pocket in an orientation opposed to productive substrates explain anophelin's remarkable specificity and resistance to proteolysis by thrombin. Complementary biophysical and functional characterization of point mutants and truncated versions of anophelin unambiguously establish the molecular mechanism of action of this family of serine proteinase inhibitors (I77). These findings have implications for the design of novel antithrombotics.
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http://dx.doi.org/10.1073/pnas.1211614109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3535606PMC
December 2012

In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation.

Nat Med 2011 Aug 21;17(9):1142-6. Epub 2011 Aug 21.

Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Coagulase-positive Staphylococcus aureus (S. aureus) is the major causal pathogen of acute endocarditis, a rapidly progressing, destructive infection of the heart valves. Bacterial colonization occurs at sites of endothelial damage, where, together with fibrin and platelets, the bacteria initiate the formation of abnormal growths known as vegetations. Here we report that an engineered analog of prothrombin could be used to detect S. aureus in endocarditic vegetations via noninvasive fluorescence or positron emission tomography (PET) imaging. These prothrombin derivatives bound staphylocoagulase and intercalated into growing bacterial vegetations. We also present evidence for bacterial quorum sensing in the regulation of staphylocoagulase expression by S. aureus. Staphylocoagulase expression was limited to the growing edge of mature vegetations, where it was exposed to the host and co-localized with the imaging probe. When endocarditis was induced with an S. aureus strain with genetic deletion of coagulases, survival of mice improved, highlighting the role of staphylocoagulase as a virulence factor.
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http://dx.doi.org/10.1038/nm.2423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169740PMC
August 2011

Structural basis of thrombin-mediated factor V activation: the Glu666-Glu672 sequence is critical for processing at the heavy chain-B domain junction.

Blood 2011 Jun 9;117(26):7164-73. Epub 2011 May 9.

Institute for Biomedical Research, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

Thrombin-catalyzed activation of coagulation factor V (FV) is an essential positive feedback reaction within the blood clotting system. Efficient processing at the N- (Arg(709)-Ser(710)) and C-terminal activation cleavage sites (Arg(1545)-Ser(1546)) requires initial substrate interactions with 2 clusters of positively charged residues on the proteinase surface, exosites I and II. We addressed the mechanism of activation of human factor V (FV) using peptides that cover the entire acidic regions preceding these cleavage sites, FV (657-709)/ (FVa2) and FV(1481-1545)/(FVa3). FVa2 appears to interact mostly with exosite I, while both exosites are involved in interactions with the C-terminal linker. The 1.7-Å crystal structure of irreversibly inhibited thrombin bound to FVa2 unambiguously reveals docking of FV residues Glu(666)-Glu(672) to exosite I. These findings were confirmed in a second, medium-resolution structure of FVa2 bound to the benzamidine-inhibited proteinase. Our results suggest that the acidic A2-B domain linker is involved in major interactions with thrombin during cofactor activation, with its more N-terminal hirudin-like sequence playing a critical role. Modeling experiments indicate that FVa2, and likely also FVa3, wrap around thrombin in productive thrombin·FV complexes that cover a large surface of the activator to engage the active site.
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http://dx.doi.org/10.1182/blood-2010-10-315309DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3143557PMC
June 2011

Clinical and genetic findings in five female patients with haemophilia A: Identification of a novel missense mutation, p.Phe2127Ser.

Thromb Haemost 2010 Oct 20;104(4):718-23. Epub 2010 Jul 20.

Haematology Department, Hospital Universitario La Paz, Madrid, Spain.

Severe manifestations of X-linked recessive disorders such as haemophilia A (HA) are rare in females. Here we describe the clinical and genetic findings in five female HA patients from two different Spanish families. Three sisters born to consanguineous parents presented moderate bleeding due to a known mutation (p.Ser1791Pro) detected in a homozygous state. In the second family, two sisters with Morris syndrome (46,XY) and mild/moderate illness were hemizygous for a novel missense mutation, p.Phe2127Ser. The mutation is predicted to impair binding to the factor VIII (FVIII) carrier protein, von Willebrand factor, and thus increased clearance of FVIII from plasma. Clinical and molecular characterisation of these patients is essential to optimise follow-up, genetic counselling and treatment of the disease.
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http://dx.doi.org/10.1160/TH10-02-0085DOI Listing
October 2010

Leech-derived thrombin inhibitors: from structures to mechanisms to clinical applications.

J Med Chem 2010 May;53(10):3847-61

Institut de Recerca, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

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http://dx.doi.org/10.1021/jm901743xDOI Listing
May 2010

Tick-derived Kunitz-type inhibitors as antihemostatic factors.

Insect Biochem Mol Biol 2009 Sep 24;39(9):579-95. Epub 2009 Jul 24.

Institut de Recerca, Hospital de la Santa Creu i Sant Pau, Sant Antoni Maria Claret 167, 08025 Barcelona, Spain.

Endogenous Kunitz-type inhibitors target a large number of serine proteinases, including coagulation factors VIIa and Xa, but not thrombin. By contrast, several two-domain Kunitz inhibitors of this major procoagulant proteinase have been isolated from both soft ticks (e.g., ornithodorin from Ornithodoros moubata) and hard ticks (e.g., boophilin from Rhipicephalus (Boophilus) microplus). Surprisingly, these anticoagulants do not follow the canonical mechanism of proteinase inhibition. Instead, their N-terminal residues bind across the thrombin active-site cleft, while C-terminal modules interact with the basic exosite I. The reactive-site loop of boophilin remains fully accessible in its complex with thrombin, and might interact with FXa according to the standard mechanism. A conceptually similar inhibition mechanism is employed by a related inhibitor of the TF-FVIIa complex isolated from Ixodes scapularis, ixolaris. Significant variations to the Kunitz fold are encountered in several of these factors, and are particularly evident in the single-domain FXa inhibitor, O. moubata TAP, and in soft tick-derived platelet antiaggregants (e.g., O. moubata disagregin). Altogether, these antihemostatic factors illustrate the divergence between hard and soft ticks. The unsurpassed versatility of tick-derived Kunitz inhibitors establishes them as valuable tools for biochemical investigations, but also as lead compounds for the development of novel antithrombotics.
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http://dx.doi.org/10.1016/j.ibmb.2009.07.003DOI Listing
September 2009

Plasminogen substrate recognition by the streptokinase-plasminogen catalytic complex is facilitated by Arg253, Lys256, and Lys257 in the streptokinase beta-domain and kringle 5 of the substrate.

J Biol Chem 2009 Jul 27;284(29):19511-21. Epub 2009 May 27.

Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

Streptokinase (SK) conformationally activates the central zymogen of the fibrinolytic system, plasminogen (Pg). The SK.Pg* catalytic complex binds Pg as a specific substrate and cleaves it into plasmin (Pm), which binds SK to form the SK.Pm complex that propagates Pm generation. Catalytic complex formation is dependent on lysine-binding site (LBS) interactions between a Pg/Pm kringle and the SK COOH-terminal Lys(414). Pg substrate recognition is also LBS-dependent, but the kringle and SK structural element(s) responsible have not been identified. SK mutants lacking Lys(414) with Ala substitutions of charged residues in the SK beta-domain 250-loop were evaluated in kinetic studies that resolved conformational and proteolytic Pg activation. Activation of [Lys]Pg and mini-Pg (containing only kringle 5 of Pg) by SK with Ala substitutions of Arg(253), Lys(256), and Lys(257) showed decreases in the bimolecular rate constant for Pm generation, with nearly total inhibition for the SK Lys(256)/Lys(257) double mutant. Binding of bovine Pg (BPg) to the SK.Pm complex containing fluorescently labeled Pm demonstrated LBS-dependent assembly of a SK.labeled Pm.BPg ternary complex, whereas BPg did not bind to the complex containing the SK Lys(256)/Lys(257) mutant. BPg was activated by SK.Pm with a K(m) indistinguishable from the K(D) for BPg binding to form the ternary complex, whereas the SK Lys(256)/Lys(257) mutant did not support BPg activation. We conclude that SK residues Arg(253), Lys(256), and Lys(257) mediate Pg substrate recognition through kringle 5 of the [Lys]Pg and mini-Pg substrates. A molecular model of the SK.kringle 5 complex identifies the putative interactions involved in LBS-dependent Pg substrate recognition.
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http://dx.doi.org/10.1074/jbc.M109.005512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740577PMC
July 2009

Potent phagocytic activity with impaired antigen presentation identifying lipopolysaccharide-tolerant human monocytes: demonstration in isolated monocytes from cystic fibrosis patients.

J Immunol 2009 May;182(10):6494-507

Research Unit, Laboratory of Tumor Immunology, La Paz Hospital, Madrid, Spain.

Monocyte exposure to LPS induces a transient state in which these cells are refractory to further endotoxin stimulation. This phenomenon, termed endotoxin tolerance (ET), is characterized by a decreased production of cytokines in response to the proinflammatory stimulus. We have established a robust model of ET and have determined the time frame and features of LPS unresponsiveness in cultured human monocytes. A large number of genes transcribed in tolerant monocytes were classified as either "tolerizable" or "nontolerizable" depending on their expression levels during the ET phase. Tolerant monocytes exhibit rapid IL-1R-associated kinase-M (IRAK-M) overexpression, high levels of triggering receptor expressed on myeloid cells-1 (TREM-1) and CD64, and a marked down-regulation of MHC molecules and NF-kappaB2. These cells combine potent phagocytic activity with impaired capability for Ag presentation. We also show that circulating monocytes isolated from cystic fibrosis patients share all the determinants that characterize cells locked in an ET state. These findings identify a new mechanism that contributes to impaired inflammation in cystic fibrosis patients despite a high frequency of infections. Our results indicate that a tolerant phenotype interferes with timing, efficiency, and outcome of the innate immune responses against bacterial infections.
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http://dx.doi.org/10.4049/jimmunol.0803350DOI Listing
May 2009

Contribution of globular death domains and unstructured linkers to MyD88.IRAK-4 heterodimer formation: an explanation for the antagonistic activity of MyD88s.

Biochem Biophys Res Commun 2009 Feb 22;380(1):183-7. Epub 2009 Jan 22.

Cardiovascular Research Center and Institut de Recerca, Hospital de la Santa Creu i Sant Pau, Sant Antoni Maria Claret 167, 08025 Barcelona, Spain.

Homotypic interactions of death domains (DD) mediate complex formation between MyD88 and IL-1 receptor-associated kinases (IRAKs). A truncated splice variant of MyD88, MyD88s, cannot recruit IRAK-4 and fails to elicit inflammatory responses. We have generated recombinant DD of MyD88 and IRAK-4, both alone and extended by the linkers to TIR or kinase domains. We show that both MyD88 DD variants bind to the linker-extended IRAK-4 DD and pull-down full-length IRAK-4 from monocyte extracts. By contrast, residues up to Glu(116) from the DD-kinase connector of IRAK-4 are needed for strong interactions with the adaptor. Our findings indicate that residues 110-120, which form a C-terminal extra helix in MyD88, but not the irregular linker between DD and TIR domains, are required for IRAK-4 recruitment, and provide a straightforward explanation for the negative regulation of innate immune responses mediated by MyD88s.
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http://dx.doi.org/10.1016/j.bbrc.2009.01.069DOI Listing
February 2009

Mutation update of spinal muscular atrophy in Spain: molecular characterization of 745 unrelated patients and identification of four novel mutations in the SMN1 gene.

Hum Genet 2009 Feb 3;125(1):29-39. Epub 2008 Dec 3.

Servicio de Genética, Hospital de la Santa Creu i Sant Pau, Sant Antoni Ma. Claret 167, 08025 Barcelona, Spain.

Spinal muscular atrophy (SMA) is caused by mutations in the SMN1 gene. We have studied the molecular pathology of SMA in 745 unrelated Spanish patients using PCR-RFLP, SMN gene dosage analysis, linkage studies, long-range PCR and direct sequencing. Our systematic approach allowed us to complete genetic testing and risk assessment in 736 SMA patients (98.8%). Females were more frequently affected by the acute form of the disease (type I), whereas chronic forms (type II-III) predominated in males (p<0.008). Absence of the SMN1 gene was detected in 671 patients (90%), and hybrid SMN1-SMN2 genes were observed in 37 cases (5%). Furthermore, we detected 13 small mutations in 28 patients (3.8%), four of which were previously identified in other populations (c.91dupT; c.770_780dup11; p.Tyr272Cys and p.Thr274Ile), while five mutations were found to date only in Spanish patients (c.399_402delAGAG, p.Ile116Phe, p.Gln136Glu, c.740dupC and c.834+2T>G). The c.399_402delAGAG mutation accounted for 1.9% of all Spanish SMA patients. Finally, we discovered four novel mutations: c.312dupA, c.411delT, p.Trp190X and p.Met263Thr. Our results confirm that most SMA cases are due to large genetic rearrangements in the repetitive region of the SMA locus, resulting in absence-dysfunction of the SMN1 gene. By contrast, ancestrally inherited small mutations are responsible for only a small number of cases. Four prevalent changes in exons 3 and 6 (c.399_402delAGAG; c.770_780dup11; p.Tyr272Cys; p.Thr274Ile) accounted for almost 70% of our patients with these subtle mutations. An SMN-SMN dimer model featuring tight hydrophobic-aromatic interactions is proposed to explain the impact of mutations at the C-terminal end of the protein.
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http://dx.doi.org/10.1007/s00439-008-0598-1DOI Listing
February 2009