RESUMEN
Neurotransmitters are released from synaptic vesicles, the membrane of which fuses with the plasma membrane upon calcium influx. This membrane fusion reaction is driven by the formation of a tight complex comprising the plasma membrane N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins syntaxin-1a and SNAP-25 with the vesicle SNARE protein synaptobrevin. The neuronal protein Munc18-1 forms a stable complex with syntaxin-1a. Biochemically, syntaxin-1a cannot escape the tight grip of Munc18-1, so formation of the SNARE complex is inhibited. However, Munc18-1 is essential for the release of neurotransmitters in vivo. It has therefore been assumed that Munc18-1 makes the bound syntaxin-1a available for SNARE complex formation. Exactly how this occurs is still unclear, but it is assumed that structural rearrangements occur. Here, we used a series of mutations to specifically weaken the complex at different positions in order to induce these rearrangements biochemically. Our approach was guided through sequence and structural analysis and supported by molecular dynamics simulations. Subsequently, we created a homology model showing the complex in an altered conformation. This conformation presumably represents a more open arrangement of syntaxin-1a that permits the formation of a SNARE complex to be initiated while still bound to Munc18-1. In the future, research should investigate how this central reaction for neuronal communication is controlled by other proteins.
RESUMEN
Intellectual developmental disorder with paroxysmal dyskinesia or seizures (IDDPADS, OMIM#619150) is an ultra-rare childhood-onset autosomal recessive movement disorder manifesting paroxysmal dyskinesia, global developmental delay, impaired cognition, progressive psychomotor deterioration and/or drug-refractory seizures. We investigated three consanguineous Pakistani families with six affected individuals presenting overlapping phenotypes partially consistent with the reported characteristics of IDDPADS. Whole exome sequencing identified a novel missense variant in Phosphodiesterase 2A (PDE2A): NM_002599.4: c.1514T > C p.(Phe505Ser) that segregated with the disease status of individuals in these families. Retrospectively, we performed haplotype analysis that revealed a 3.16 Mb shared haplotype at 11q13.4 among three families suggesting a founder effect in this region. Moreover, we also observed abnormal mitochondrial morphology in patient fibroblasts compared to controls. Belonging to diverse age groups (13 years-60 years), patients presented paroxysmal dyskinesia, developmental delay, cognitive abnormalities, speech impairment, and drug-refractory seizures with variable onset of disease (as early as 3 months of age to 7 years). Together with the previous reports, we observed that intellectual disability, progressive psychomotor deterioration, and drug-refractory seizures are consistent outcomes of the disease. However, permanent choreodystonia showed variability. We also noticed that the later onset of paroxysmal dyskinesia manifests severe attacks in terms of duration. Being the first report from Pakistan, we add to the clinical and mutation spectrum of PDE2A-related recessive disease raising the total number of patients from six to 12 and variants from five to six. Together, with our findings, the role of PDE2A is strengthened in critical physio-neurological processes.
Asunto(s)
Corea , Discapacidad Intelectual , Humanos , Discapacidad Intelectual/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 2/genética , Corea/genética , Estudios Retrospectivos , Linaje , Mutación/genética , Consanguinidad , ConvulsionesRESUMEN
Influenza A virus (IAV) enters host cells mostly through clathrin-dependent receptor-mediated endocytosis. A single bona fide entry receptor protein supporting this entry mechanism remains elusive. Here we performed proximity ligation of biotin to host cell surface proteins in the vicinity of attached trimeric hemagglutinin-HRP and characterized biotinylated targets using mass spectrometry. This approach identified transferrin receptor 1 (TfR1) as a candidate entry protein. Genetic gain-of-function and loss-of-function experiments, as well as in vitro and in vivo chemical inhibition, confirmed the functional involvement of TfR1 in IAV entry. Recycling deficient mutants of TfR1 do not support entry, indicating that TfR1 recycling is essential for this function. The binding of virions to TfR1 via sialic acids confirmed its role as a directly acting entry factor, but unexpectedly even headless TfR1 promoted IAV particle uptake in trans. TIRF microscopy localized the entering virus-like particles in the vicinity of TfR1. Our data identify TfR1 recycling as a revolving door mechanism exploited by IAV to enter host cells.
Asunto(s)
Virus de la Influenza A , Transferrina , Virus de la Influenza A/fisiología , Internalización del Virus , Endocitosis/fisiología , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismoRESUMEN
Craniofacial microsomia (CFM; also known as Goldenhar syndrome), is a craniofacial developmental disorder of variable expressivity and severity with a recognizable set of abnormalities. These birth defects are associated with structures derived from the first and second pharyngeal arches, can occur unilaterally and include ear dysplasia, microtia, preauricular tags and pits, facial asymmetry and other malformations. The inheritance pattern is controversial, and the molecular etiology of this syndrome is largely unknown. A total of 670 patients belonging to unrelated pedigrees with European and Chinese ancestry with CFM, are investigated. We identify 18 likely pathogenic variants in 21 probands (3.1%) in FOXI3. Biochemical experiments on transcriptional activity and subcellular localization of the likely pathogenic FOXI3 variants, and knock-in mouse studies strongly support the involvement of FOXI3 in CFM. Our findings indicate autosomal dominant inheritance with reduced penetrance, and/or autosomal recessive inheritance. The phenotypic expression of the FOXI3 variants is variable. The penetrance of the likely pathogenic variants in the seemingly dominant form is reduced, since a considerable number of such variants in affected individuals were inherited from non-affected parents. Here we provide suggestive evidence that common variation in the FOXI3 allele in trans with the pathogenic variant could modify the phenotypic severity and accounts for the incomplete penetrance.
Asunto(s)
Síndrome de Goldenhar , Animales , Ratones , Síndrome de Goldenhar/patología , Asimetría Facial , Linaje , Factores de Transcripción ForkheadRESUMEN
Telomere maintenance 2 (TELO2), Tel2 interacting protein 2 (TTI2), and Tel2 interacting protein 1 (TTI1) are the three components of the conserved Triple T (TTT) complex that modulates activity of phosphatidylinositol 3-kinase-related protein kinases (PIKKs), including mTOR, ATM, and ATR, by regulating the assembly of mTOR complex 1 (mTORC1). The TTT complex is essential for the expression, maturation, and stability of ATM and ATR in response to DNA damage. TELO2- and TTI2-related bi-allelic autosomal-recessive (AR) encephalopathies have been described in individuals with moderate to severe intellectual disability (ID), short stature, postnatal microcephaly, and a movement disorder (in the case of variants within TELO2). We present clinical, genomic, and functional data from 11 individuals in 9 unrelated families with bi-allelic variants in TTI1. All present with ID, and most with microcephaly, short stature, and a movement disorder. Functional studies performed in HEK293T cell lines and fibroblasts and lymphoblastoid cells derived from 4 unrelated individuals showed impairment of the TTT complex and of mTOR pathway activity which is improved by treatment with Rapamycin. Our data delineate a TTI1-related neurodevelopmental disorder and expand the group of disorders related to the TTT complex.
Asunto(s)
Microcefalia , Trastornos del Movimiento , Trastornos del Neurodesarrollo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Células HEK293 , Serina-Treonina Quinasas TORRESUMEN
In formin-family proteins, actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, with reaction rates that vary by at least 20-fold between formins. Each cell expresses distinct formins that assemble one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates in vivo. Fus1 assembles the actin fusion focus, necessary for gamete fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on fusion focus architecture, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in Fus1 FH2 that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional FH2 domain property. Thus, rates of actin assembly are tailored for assembly of specific actin structures.
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Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Forminas , Proteínas de Microfilamentos/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMEN
PURPOSE: CTR9 is a subunit of the PAF1 complex (PAF1C) that plays a crucial role in transcription regulation by binding CTR9 to RNA polymerase II. It is involved in transcription-coupled histone modification through promoting H3K4 and H3K36 methylation. We describe the clinical and molecular studies in 13 probands, harboring likely pathogenic CTR9 missense variants, collected through GeneMatcher. METHODS: Exome sequencing was performed in all individuals. CTR9 variants were assessed through 3-dimensional modeling of the activated human transcription complex Pol II-DSIF-PAF-SPT6 and the PAF1/CTR9 complex. H3K4/H3K36 methylation analysis, mitophagy assessment based on tetramethylrhodamine ethyl ester perchlorate immunofluorescence, and RNA-sequencing in skin fibroblasts from 4 patients was performed. RESULTS: Common clinical findings were variable degrees of intellectual disability, hypotonia, joint hyperlaxity, speech delay, coordination problems, tremor, and autism spectrum disorder. Mild dysmorphism and cardiac anomalies were less frequent. For 11 CTR9 variants, de novo occurrence was shown. Three-dimensional modeling predicted a likely disruptive effect of the variants on local CTR9 structure and protein interaction. Additional studies in fibroblasts did not unveil the downstream functional consequences of the identified variants. CONCLUSION: We describe a neurodevelopmental disorder caused by (mainly) de novo variants in CTR9, likely affecting PAF1C function.
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Trastorno del Espectro Autista , Discapacidad Intelectual , Trastornos del Neurodesarrollo , Fosfoproteínas , Factores de Transcripción , Regulación de la Expresión Génica , Heterocigoto , Humanos , Discapacidad Intelectual/genética , Trastornos del Neurodesarrollo/genética , Fosfoproteínas/genética , Factores de Transcripción/genéticaRESUMEN
Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.
Asunto(s)
Estrés del Retículo Endoplásmico/fisiología , Virus de la Influenza A/genética , Neuraminidasa/metabolismo , Células A549 , Retículo Endoplásmico/metabolismo , Células HEK293 , Interacciones Huésped-Patógeno/fisiología , Humanos , Virus de la Influenza A/metabolismo , Virus de la Influenza A/patogenicidad , Respuesta de Proteína Desplegada/genética , Respuesta de Proteína Desplegada/fisiología , Proteínas no Estructurales Virales/genética , Replicación Viral/genéticaRESUMEN
Knobloch syndrome is an autosomal recessive phenotype mainly characterized by retinal detachment and encephalocele caused by biallelic pathogenic variants in the COL18A1 gene. However, there are patients clinically diagnosed as Knobloch syndrome with unknown molecular etiology not linked to COL18A1. We studied an historical pedigree (published in 1998) designated as KNO2 (Knobloch type 2 syndrome with intellectual disability, autistic behavior, retinal degeneration, encephalocele). Whole exome sequencing of the two affected siblings and the normal parents resulted in the identification of a PAK2 non-synonymous substitution p.(Glu435Lys) as a causative variant. The variant was monoallelic and apparently de novo in both siblings indicating a likely germ-line mosaicism in one of the parents; the mosaicism, however, could not be observed after deep sequencing of blood parental DNA. PAK2 encodes a member of a small group of serine/threonine kinases; these P21-activating kinases (PAKs) are essential in signal transduction and cellular regulation (cytoskeletal dynamics, cell motility, death and survival signaling and cell cycle progression). Structural analysis of the PAK2 p.(Glu435Lys) variant that is located in the kinase domain of the protein predicts a possible compromise in the kinase activity. Functional analysis of the p.(Glu435Lys) PAK2 variant in transfected HEK293T cells results in a partial loss of the kinase activity. PAK2 has been previously suggested as an autism-related gene. Our results show that PAK2-induced phenotypic spectrum is broad and not fully understood. We conclude that the KNO2 syndrome in the studied family is dominant and caused by a deleterious variant in the PAK2 gene.
Asunto(s)
Degeneración Retiniana , Desprendimiento de Retina , Encefalocele/diagnóstico , Encefalocele/genética , Encefalocele/patología , Células HEK293 , Humanos , Mutación , Degeneración Retiniana/genética , Degeneración Retiniana/patología , Desprendimiento de Retina/congénito , Desprendimiento de Retina/genética , Quinasas p21 Activadas/genéticaRESUMEN
Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.
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Aspartato-ARNt Ligasa/genética , Mutación con Ganancia de Función/genética , Mutación con Pérdida de Función/genética , Trastornos del Neurodesarrollo/genética , Aminoacil-ARN de Transferencia/genética , Alelos , Aminoacil-ARNt Sintetasas/genética , Línea Celular , Femenino , Predisposición Genética a la Enfermedad/genética , Humanos , Masculino , Linaje , ARN de Transferencia/genética , Células Madre/fisiologíaRESUMEN
The molecular cause of the majority of rare autosomal recessive disorders remains unknown. Consanguinity due to extensive homozygosity unravels many recessive phenotypes and facilitates the detection of novel gene-disease links. Here, we report two siblings with phenotypic signs, including intellectual disability (ID), developmental delay and microcephaly from a Pakistani consanguineous family in which we have identified homozygosity for p(Tyr103His) in the PSMB1 gene (Genbank NM_002793) that segregated with the disease phenotype. PSMB1 encodes a ß-type proteasome subunit (i.e. ß6). Modeling of the p(Tyr103His) variant indicates that this variant weakens the interactions between PSMB1/ß6 and PSMA5/α5 proteasome subunits and thus destabilizes the 20S proteasome complex. Biochemical experiments in human SHSY5Y cells revealed that the p(Tyr103His) variant affects both the processing of PSMB1/ß6 and its incorporation into proteasome, thus impairing proteasome activity. CRISPR/Cas9 mutagenesis or morpholino knock-down of the single psmb1 zebrafish orthologue resulted in microcephaly, microphthalmia and reduced brain size. Genetic evidence in the family and functional experiments in human cells and zebrafish indicates that PSMB1/ß6 pathogenic variants are the cause of a recessive disease with ID, microcephaly and developmental delay due to abnormal proteasome assembly.
Asunto(s)
Enanismo/genética , Microcefalia/genética , Complejo de la Endopetidasa Proteasomal/genética , Alelos , Animales , Niño , Consanguinidad , Discapacidades del Desarrollo/complicaciones , Discapacidades del Desarrollo/genética , Discapacidades del Desarrollo/patología , Enanismo/complicaciones , Femenino , Homocigoto , Humanos , Discapacidad Intelectual/complicaciones , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Masculino , Microcefalia/complicaciones , Microcefalia/patología , Modelos Moleculares , Linaje , Fenotipo , Pez Cebra/genéticaRESUMEN
In a consanguineous Pakistani family with two affected individuals, a homozygous variant Gly399Val in the eighth transmembrane domain of the taurine transporter SLC6A6 was identified resulting in a hypomorph transporting capacity of ~15% compared with normal. Three-dimensional modeling of this variant has indicated that it likely causes displacement of the Tyr138 (TM3) side chain, important for transport of taurine. The affected individuals presented with rapidly progressive childhood retinal degeneration, cardiomyopathy and almost undetectable plasma taurine levels. Oral taurine supplementation of 100 mg/kg/day resulted in maintenance of normal blood taurine levels. Following approval by the ethics committee, a long-term supplementation treatment was introduced. Remarkably, after 24-months, the cardiomyopathy was corrected in both affected siblings, and in the 6-years-old, the retinal degeneration was arrested, and the vision was clinically improved. Similar therapeutic approaches could be employed in Mendelian phenotypes caused by the dysfunction of the hundreds of other molecular transporters.
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Cardiomiopatías/tratamiento farmacológico , Glicoproteínas de Membrana/deficiencia , Proteínas de Transporte de Membrana/deficiencia , Degeneración Retiniana/tratamiento farmacológico , Taurina/uso terapéutico , Adolescente , Transporte Biológico , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Niño , Femenino , Humanos , Masculino , Linaje , Degeneración Retiniana/metabolismo , Degeneración Retiniana/patologíaRESUMEN
The catalytic activity of the protease MALT1 is required for adaptive immune responses and regulatory T (Treg)-cell development, while dysregulated MALT1 activity can lead to lymphoma. MALT1 activation requires its monoubiquitination on lysine 644 (K644) within the Ig3 domain, localized adjacent to the protease domain. The molecular requirements for MALT1 monoubiquitination and the mechanism by which monoubiquitination activates MALT1 had remained elusive. Here, we show that the Ig3 domain interacts directly with ubiquitin and that an intact Ig3-ubiquitin interaction surface is required for the conjugation of ubiquitin to K644. Moreover, by generating constitutively active MALT1 mutants that overcome the need for monoubiquitination, we reveal an allosteric communication between the ubiquitination site K644, the Ig3-protease interaction surface, and the active site of the protease domain. Finally, we show that MALT1 mutants that alter the Ig3-ubiquitin interface impact the biological response of T cells. Thus, ubiquitin binding by the Ig3 domain promotes MALT1 activation by an allosteric mechanism that is essential for its biological function.
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Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , Ubiquitina , Ubiquitinación , Regulación Alostérica , Células HEK293 , Humanos , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/química , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/genética , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas/metabolismo , Mutación , Unión Proteica , Dominios Proteicos , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitinación/genética , Ubiquitinación/fisiologíaRESUMEN
Immune checkpoints are crucial in the maintenance of antitumor immune responses. The activation or blockade of immune checkpoints is dependent on the interactions between receptors and ligands; such interactions can provide inhibitory or stimulatory signals, including the enhancement or suppression of T-cell proliferation, differentiation, and/or cytokine secretion. B-and T-lymphocyte attenuator (BTLA) is a lymphoid-specific cell surface receptor which is present on T-cells and interacts with herpes virus entry mediator (HVEM), which is present on tumor cells. The binding of HVEM to BTLA triggers an inhibitory signal which attenuates the immune response. This feature is interesting for studying the molecular interactions between HVEM and BTLA, as they may be targeted for novel immunotherapies. This work was based on the crystal structure of the BTLA/HVEM complex showing that BTLA binds the N-terminal cysteine-rich domain of HVEM. We investigated the amino acid sequence of HVEM and used molecular modeling methods to develop inhibitors of the BTLA/HVEM interaction. We synthesized novel compounds and determined their ability to interact with the BTLA protein and inhibit the formation of the BTLA/HVEM complex. Our results suggest that the HVEM (14-39) peptide is a potent inhibitor of the formation of the BTLA/HVEM protein complex.
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Disulfuros/química , Péptidos/farmacología , Receptores Inmunológicos/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral/metabolismo , Sitios de Unión/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Péptidos/síntesis química , Péptidos/química , Unión Proteica/efectos de los fármacos , Conformación Proteica , Receptores Inmunológicos/química , Miembro 14 de Receptores del Factor de Necrosis Tumoral/químicaRESUMEN
Cargo transport along the cytoplasmic microtubular network is essential for neuronal function, and cytoplasmic dynein-1 is an established molecular motor that is critical for neurogenesis and homeostasis. We performed whole-exome sequencing, homozygosity mapping, and chromosomal microarray studies in five individuals from three independent pedigrees and identified likely-pathogenic variants in DYNC1I2 (Dynein Cytoplasmic 1 Intermediate Chain 2), encoding a component of the cytoplasmic dynein 1 complex. In a consanguineous Pakistani family with three affected individuals presenting with microcephaly, severe intellectual disability, simplification of cerebral gyration, corpus callosum hypoplasia, and dysmorphic facial features, we identified a homozygous splice donor site variant (GenBank: NM_001378.2:c.607+1G>A). We report two additional individuals who have similar neurodevelopmental deficits and craniofacial features and harbor deleterious variants; one individual bears a c.740A>G (p.Tyr247Cys) change in trans with a 374 kb deletion encompassing DYNC1I2, and an unrelated individual harbors the compound-heterozygous variants c.868C>T (p.Gln290∗) and c.740A>G (p.Tyr247Cys). Zebrafish larvae subjected to CRISPR-Cas9 gene disruption or transient suppression of dync1i2a displayed significantly altered craniofacial patterning with concomitant reduction in head size. We monitored cell death and cell cycle progression in dync1i2a zebrafish models and observed significantly increased apoptosis, likely due to prolonged mitosis caused by abnormal spindle morphology, and this finding offers initial insights into the cellular basis of microcephaly. Additionally, complementation studies in zebrafish demonstrate that p.Tyr247Cys attenuates gene function, consistent with protein structural analysis. Our genetic and functional data indicate that DYNC1I2 dysfunction probably causes an autosomal-recessive microcephaly syndrome and highlight further the critical roles of the dynein-1 complex in neurodevelopment.
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Anomalías Craneofaciales/etiología , Dineínas/genética , Discapacidad Intelectual/etiología , Malformaciones Arteriovenosas Intracraneales/etiología , Microcefalia/etiología , Mutación , Pez Cebra/crecimiento & desarrollo , Adulto , Alelos , Secuencia de Aminoácidos , Animales , Preescolar , Anomalías Craneofaciales/patología , Dineínas/química , Dineínas/metabolismo , Exoma , Femenino , Homocigoto , Humanos , Lactante , Discapacidad Intelectual/patología , Malformaciones Arteriovenosas Intracraneales/patología , Masculino , Microcefalia/patología , Linaje , Fenotipo , Conformación Proteica , Homología de Secuencia , Secuenciación del Exoma , Adulto Joven , Pez Cebra/genética , Pez Cebra/metabolismoRESUMEN
Modulation by neuropeptides enhances several functions of acid-sensing ion channels (ASICs), such as pain sensation and acid-induced neuronal injury. The acid-induced opening of ASICs is transient, because of a rapid desensitization. Neuropeptides containing an Arg-Phe-amide motif affect ASIC desensitization and allow continuous activity of ASICs. In spite of the importance of the sustained ASIC activity during prolonged acidification, the molecular mechanisms of ASIC modulation by neuropeptides is only poorly understood. To identify the FRRFa (Phe-Arg-Arg-Phe-amide) binding site on ASIC1a, we carried out an in silico docking analysis and verified functionally the docking predictions. The docking experiments indicated three possible binding pockets, located (1) in the acidic pocket between the thumb, finger, ß-ball and palm domains, (2) in a pocket at the bottom of the thumb domain, and (3) in the central vestibule along with the connected side cavities. Functional measurements of mutant ASIC1a confirmed the importance of residues of the lower palm, which encloses the central vestibule and its side cavities, for the FRRFa effects. The combined docking and functional experiments strongly suggest that FRRFa binds to the central vestibule and its side cavities to change ASIC desensitization.
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Canales Iónicos Sensibles al Ácido , Dipéptidos/metabolismo , Neuropéptidos/metabolismo , Canales Iónicos Sensibles al Ácido/química , Canales Iónicos Sensibles al Ácido/metabolismo , Animales , Sitios de Unión , Simulación del Acoplamiento Molecular/métodos , Oocitos , Unión Proteica , Dominios Proteicos , Xenopus laevisRESUMEN
FBXL3 (F-Box and Leucine Rich Repeat Protein 3) encodes a protein that contains an F-box and several tandem leucine-rich repeats (LRR) domains. FBXL3 is part of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complex that binds and leads to phosphorylation-dependent degradation of the central clock protein cryptochromes (CRY1 and CRY2) by the proteasome and its absence causes circadian phenotypes in mice and behavioral problems. No FBXL3-related phenotypes have been described in humans. By a combination of exome sequencing and homozygosity mapping, we analyzed two consanguineous families with intellectual disability and identified homozygous loss-of-function (LoF) variants in FBXL3. In the first family, from Pakistan, an FBXL3 frameshift variant [NM_012158.2:c.885delT:p.(Leu295Phefs*25)] was the onlysegregating variant in five affected individuals in two family loops (LOD score: 3.12). In the second family, from Lebanon, we identified a nonsense variant [NM_012158.2:c.445C>T:p.(Arg149*)]. In a third patient from Italy, a likely deleterious non-synonymous variant [NM_012158.2:c.1072T>C:p.(Cys358Arg)] was identified in homozygosity. Protein 3D modeling predicted that the Cys358Arg change influences the binding with CRY2 by destabilizing the structure of the FBXL3, suggesting that this variant is also likely to be LoF. The eight affected individuals from the three families presented with a similar phenotype that included intellectual disability, developmental delay, short stature and mild facial dysmorphism, mainly large nose with a bulbous tip. The phenotypic similarity and the segregation analysis suggest that FBXL3 biallelic, LoF variants link this gene with syndromic autosomal recessive developmental delay/intellectual disability.
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Alelos , Discapacidades del Desarrollo/genética , Enanismo/genética , Proteínas F-Box/genética , Variación Genética , Discapacidad Intelectual/genética , Adulto , Consanguinidad , Análisis Mutacional de ADN , Discapacidades del Desarrollo/diagnóstico , Enanismo/diagnóstico , Proteínas F-Box/química , Facies , Femenino , Homocigoto , Humanos , Discapacidad Intelectual/diagnóstico , Masculino , Persona de Mediana Edad , Modelos Moleculares , Linaje , Fenotipo , Conformación Proteica , Relación Estructura-Actividad , Adulto JovenRESUMEN
Kinesin proteins are critical for various cellular functions such as intracellular transport and cell division, and many members of the family have been linked to monogenic disorders and cancer. We report eight individuals with intellectual disability and microcephaly from four unrelated families with parental consanguinity. In the affected individuals of each family, homozygosity for likely pathogenic variants in KIF14 were detected; two loss-of-function (p.Asn83Ilefs*3 and p.Ser1478fs), and two missense substitutions (p.Ser841Phe and p.Gly459Arg). KIF14 is a mitotic motor protein that is required for spindle localization of the mitotic citron rho-interacting kinase, CIT, also mutated in microcephaly. Our results demonstrate the involvement of KIF14 in development and reveal a wide phenotypic variability ranging from fetal lethality to moderate developmental delay and microcephaly.
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Discapacidad Intelectual/genética , Cinesinas/genética , Microcefalia/genética , Proteínas Oncogénicas/genética , Niño , Preescolar , Femenino , Humanos , Discapacidad Intelectual/patología , Cinesinas/química , Cinesinas/metabolismo , Mutación con Pérdida de Función , Microcefalia/patología , Mutación Missense , Proteínas Oncogénicas/química , Proteínas Oncogénicas/metabolismo , Linaje , Fenotipo , Dominios Proteicos , SíndromeRESUMEN
PURPOSE: To elucidate the novel molecular cause in two unrelated consanguineous families with autosomal recessive intellectual disability. METHODS: A combination of homozygosity mapping and exome sequencing was used to locate the plausible genetic defect in family F162, while only exome sequencing was followed in the family PKMR65. The protein 3D structure was visualized with the University of California-San Francisco Chimera software. RESULTS: All five patients from both families presented with severe intellectual disability, aggressive behavior, and speech and motor delay. Four of the five patients had microcephaly. We identified homozygous missense variants in LINGO1, p.(Arg290His) in family F162 and p.(Tyr288Cys) in family PKMR65. Both variants were predicted to be pathogenic, and segregated with the phenotype in the respective families. Molecular modeling of LINGO1 suggests that both variants interfere with the glycosylation of the protein. CONCLUSION: LINGO1 is a transmembrane receptor, predominantly found in the central nervous system. Published loss-of-function studies in mouse and zebrafish have established a crucial role of LINGO1 in normal neuronal development and central nervous system myelination by negatively regulating oligodendrocyte differentiation and neuronal survival. Taken together, our results indicate that biallelic LINGO1 missense variants cause autosomal recessive intellectual disability in humans.
Asunto(s)
Discapacidad Intelectual/genética , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Alelos , Mapeo Cromosómico/métodos , Familia , Femenino , Frecuencia de los Genes/genética , Genotipo , Homocigoto , Humanos , Trastornos del Desarrollo del Lenguaje/genética , Masculino , Proteínas de la Membrana/fisiología , Microcefalia/genética , Actividad Motora/genética , Mutación Missense/genética , Proteínas del Tejido Nervioso/fisiología , Pakistán , Linaje , Fenotipo , Análisis de Secuencia de Proteína , Secuenciación del ExomaRESUMEN
Antibody based immune-checkpoint blockade therapy is a major breakthrough in oncology, leading to clinical benefit for cancer patients. Among the growing family of inhibitory receptors, the B and T lymphocyte attenuator (BTLA), which interacts with herpes virus entry mediator (HVEM), is a promising target for immunotherapy. Indeed, BTLA inhibits T-cell proliferation and cytokine production. The crystal structure of the BTLA/HVEM complex has shown that the HVEM(26-38) fragment is directly involved in protein binding. We designed and analyzed the capacity of several analogs of this fragment to block the ligation between BTLA and HVEM, using competitive ELISA and cellular assay. We found that the HVEM(23-39) peptide can block BTLA/HVEM ligation. However, the blocking ability was due to the Cys encompassed in this peptide and that even free cysteine targeted the BTLA protein and blocked its interaction with HVEM. These data highlight a Cys-related artefact in vitro, which should be taken in consideration for future development of BTLA/HVEM blocking compounds.