RESUMEN
Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation to generate an inventory of potential RAB18 effectors. A restricted set of 28 RAB18 interactions were dependent on the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor complex. Twelve of these 28 interactions are supported by prior reports, and we have directly validated novel interactions with SEC22A, TMCO4, and INPP5B. Consistent with a role for RAB18 in regulating membrane contact sites, interactors included groups of microtubule/membrane-remodeling proteins, membrane-tethering and docking proteins, and lipid-modifying/transporting proteins. Two of the putative interactors, EBP and OSBPL2/ORP2, have sterol substrates. EBP is a Δ8-Δ7 sterol isomerase, and ORP2 is a lipid transport protein. This prompted us to investigate a role for RAB18 in cholesterol biosynthesis. We found that the cholesterol precursor and EBP-product lathosterol accumulates in both RAB18-null HeLa cells and RAB3GAP1-null fibroblasts derived from an affected individual. Furthermore, de novo cholesterol biosynthesis is impaired in cells in which RAB18 is absent or dysregulated or in which ORP2 expression is disrupted. Our data demonstrate that guanine nucleotide exchange factor-dependent Rab interactions are highly amenable to interrogation by proximity biotinylation and may suggest that Micro syndrome is a cholesterol biosynthesis disorder.
Asunto(s)
Biotinilación , Esteroles , Proteínas de Unión al GTP rab , Humanos , Colesterol/biosíntesis , Colesterol/metabolismo , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Células HeLa , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Esteroles/biosíntesis , Esteroles/metabolismo , Células Cultivadas , Técnicas de Silenciamiento del Gen , Transporte de Proteínas/genéticaRESUMEN
Inosine triphosphate pyrophosphatase (ITPase), encoded by the ITPA gene in humans, is an important enzyme that preserves the integrity of cellular nucleotide pools by hydrolyzing the noncanonical purine nucleotides (deoxy)inosine and (deoxy)xanthosine triphosphate into monophosphates and pyrophosphate. Variants in the ITPA gene can cause partial or complete ITPase deficiency. Partial ITPase deficiency is benign but clinically relevant as it is linked to altered drug responses. Complete ITPase deficiency causes a severe multisystem disorder characterized by seizures and encephalopathy that is frequently associated with fatal infantile dilated cardiomyopathy. In the absence of ITPase activity, its substrate noncanonical nucleotides have the potential to accumulate and become aberrantly incorporated into DNA and RNA. Hence, the pathophysiology of ITPase deficiency could arise from metabolic imbalance, altered DNA or RNA regulation, or from a combination of these factors. Here, we review the known functions of ITPase and highlight recent work aimed at determining the molecular basis for ITPA-associated pathogenesis which provides evidence for RNA dysfunction. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development.
Asunto(s)
Nucleótidos , ARN , Humanos , Nucleótidos/metabolismo , ARN/genética , Inosina , Inosina Trifosfato , Pirofosfatasas/genética , Pirofosfatasas/metabolismo , ADNRESUMEN
Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.
Asunto(s)
Inosina Trifosfato , ARN , Humanos , Animales , Ratones , Ratas , Inosina Trifosfato/metabolismo , Pirofosfatasas/genética , Inosina , NucleótidosRESUMEN
Typical Martsolf syndrome is characterized by congenital cataracts, postnatal microcephaly, developmental delay, hypotonia, short stature and biallelic hypomorphic mutations in either RAB3GAP1 or RAB3GAP2. Genetic analysis of 85 unrelated "mutation negative" probands with Martsolf or Martsolf-like syndromes identified two individuals with different homozygous null mutations in ITPA, the gene encoding inosine triphosphate pyrophosphatase (ITPase). Both probands were from multiplex families with a consistent, lethal and highly distinctive disorder; a Martsolf-like syndrome with infantile-onset dilated cardiomyopathy. Severe ITPase-deficiency has been previously reported with infantile epileptic encephalopathy (MIM 616647). ITPase acts to prevent incorporation of inosine bases (rI/dI) into RNA and DNA. In Itpa-null cells dI was undetectable in genomic DNA. dI could be identified at a low level in mtDNA without detectable mitochondrial genome instability, mtDNA depletion or biochemical dysfunction of the mitochondria. rI accumulation was detectable in proband-derived lymphoblastoid RNA. In Itpa-null mouse embryos rI was detectable in the brain and kidney with the highest level seen in the embryonic heart (rI at 1 in 385 bases). Transcriptome and proteome analysis in mutant cells revealed no major differences with controls. The rate of transcription and the total amount of cellular RNA also appeared normal. rI accumulation in RNA-and by implication rI production-correlates with the severity of organ dysfunction in ITPase deficiency but the basis of the cellulopathy remains cryptic. While we cannot exclude cumulative minor effects, there are no major anomalies in the production, processing, stability and/or translation of mRNA.
Asunto(s)
Cardiomiopatía Dilatada/enzimología , Cardiomiopatía Dilatada/genética , Catarata/enzimología , Catarata/genética , Hipogonadismo/enzimología , Hipogonadismo/genética , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Errores Innatos del Metabolismo/enzimología , Errores Innatos del Metabolismo/genética , Pirofosfatasas/deficiencia , Animales , Secuencia de Bases , Preescolar , Análisis Mutacional de ADN , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Femenino , Homocigoto , Humanos , Inosina/metabolismo , Masculino , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/enzimología , Mutación , Linaje , Pirofosfatasas/genética , ARN/genética , ARN/metabolismo , Secuenciación del ExomaRESUMEN
During neurotransmission, synaptic vesicles undergo multiple rounds of exo-endocytosis, involving recycling and/or degradation of synaptic proteins. While ubiquitin signaling at synapses is essential for neural function, it has been assumed that synaptic proteostasis requires the ubiquitin-proteasome system (UPS). We demonstrate here that turnover of synaptic membrane proteins via the endolysosomal pathway is essential for synaptic function. In both human and mouse, hypomorphic mutations in the ubiquitin adaptor protein PLAA cause an infantile-lethal neurodysfunction syndrome with seizures. Resulting from perturbed endolysosomal degradation, Plaa mutant neurons accumulate K63-polyubiquitylated proteins and synaptic membrane proteins, disrupting synaptic vesicle recycling and neurotransmission. Through characterization of this neurological intracellular trafficking disorder, we establish the importance of ubiquitin-mediated endolysosomal trafficking at the synapse.
Asunto(s)
Epilepsia/genética , Proteínas/genética , Espasmos Infantiles/genética , Transmisión Sináptica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Modelos Animales de Enfermedad , Epilepsia/diagnóstico , Fibroblastos/metabolismo , Técnicas de Genotipaje , Humanos , Lactante , Recién Nacido , Imagen por Resonancia Magnética , Ratones , Ratones Transgénicos , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Conformación Proteica , Proteínas/metabolismo , Células de Purkinje/metabolismo , Espasmos Infantiles/diagnóstico , Vesículas Sinápticas/metabolismo , Transcriptoma , Ubiquitina/genética , Ubiquitina/metabolismoRESUMEN
PURPOSE: To characterize features associated with de novo mutations affecting SATB2 function in individuals ascertained on the basis of intellectual disability. METHODS: Twenty previously unreported individuals with 19 different SATB2 mutations (11 loss-of-function and 8 missense variants) were studied. Fibroblasts were used to measure mutant protein production. Subcellular localization and mobility of wild-type and mutant SATB2 were assessed using fluorescently tagged protein. RESULTS: Recurrent clinical features included neurodevelopmental impairment (19/19), absent/near absent speech (16/19), normal somatic growth (17/19), cleft palate (9/19), drooling (12/19), and dental anomalies (8/19). Six of eight missense variants clustered in the first CUT domain. Sibling recurrence due to gonadal mosaicism was seen in one family. A nonsense mutation in the last exon resulted in production of a truncated protein retaining all three DNA-binding domains. SATB2 nuclear mobility was mutation-dependent; p.Arg389Cys in CUT1 increased mobility and both p.Gly515Ser in CUT2 and p.Gln566Lys between CUT2 and HOX reduced mobility. The clinical features in individuals with missense variants were indistinguishable from those with loss of function. CONCLUSION: SATB2 haploinsufficiency is a common cause of syndromic intellectual disability. When mutant SATB2 protein is produced, the protein appears functionally inactive with a disrupted pattern of chromatin or matrix association.Genet Med advance online publication 02 February 2017.
Asunto(s)
Discapacidad Intelectual/genética , Mutación con Pérdida de Función , Proteínas de Unión a la Región de Fijación a la Matriz/genética , Mutación Missense , Factores de Transcripción/genética , Línea Celular , Estudios de Cohortes , Estudios de Asociación Genética , Haploinsuficiencia/genética , Células HeLa , Humanos , Discapacidad Intelectual/fisiopatología , Proteínas de Unión a la Región de Fijación a la Matriz/fisiología , Unión Proteica/genética , Factores de Transcripción/fisiología , Secuenciación Completa del GenomaRESUMEN
Purpose: Peroxisomes perform complex metabolic and catabolic functions essential for normal growth and development. Mutations in 14 genes cause a spectrum of peroxisomal disease in humans. Most recently, PEX11B was associated with an atypical peroxisome biogenesis disorder (PBD) in a single individual. In this study, we identify further PEX11B cases and delineate associated phenotypes. Methods: Probands from three families underwent next generation sequencing (NGS) for diagnosis of a multisystem developmental disorder. Autozygosity mapping was conducted in one affected sibling pair. ExomeDepth was used to identify copy number variants from NGS data and confirmed by dosage analysis. Biochemical profiling was used to investigate the metabolic signature of the condition. Results: All patients presented with bilateral cataract at birth but the systemic phenotype was variable, including short stature, skeletal abnormalities, and dysmorphism-features not described in the original case. Next generation sequencing identified biallelic loss-of-function mutations in PEX11B as the underlying cause of disease in each case (PEX11B c.235C>T p.(Arg79Ter) homozygous; PEX11B c.136C>T p.(Arg46Ter) homozygous; PEX11B c.595C>T p.(Arg199Ter) heterozygous, PEX11B ex1-3 del heterozygous). Biochemical studies identified very low plasmalogens in one patient, whilst a mildly deranged very long chain fatty acid profile was found in another. Conclusions: Our findings expand the phenotypic spectrum of the condition and underscore congenital cataract as the consistent primary presenting feature. We also find that biochemical measurements of peroxisome function may be disturbed in some cases. Furthermore, diagnosis by NGS is proficient and may circumvent the requirement for an invasive skin biopsy for disease identification from fibroblast cells.
Asunto(s)
Catarata/genética , ADN/genética , Proteínas de la Membrana/genética , Mutación , Trastorno Peroxisomal/genética , Adolescente , Adulto , Catarata/congénito , Catarata/metabolismo , Niño , Preescolar , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN , Femenino , Estudios de Seguimiento , Heterocigoto , Humanos , Masculino , Proteínas de la Membrana/metabolismo , Linaje , Trastorno Peroxisomal/metabolismo , Fenotipo , Factores de Tiempo , Adulto JovenRESUMEN
Gillespie syndrome (GS) is characterized by bilateral iris hypoplasia, congenital hypotonia, non-progressive ataxia, and progressive cerebellar atrophy. Trio-based exome sequencing identified de novo mutations in ITPR1 in three unrelated individuals with GS recruited to the Deciphering Developmental Disorders study. Whole-exome or targeted sequence analysis identified plausible disease-causing ITPR1 mutations in 10/10 additional GS-affected individuals. These ultra-rare protein-altering variants affected only three residues in ITPR1: Glu2094 missense (one de novo, one co-segregating), Gly2539 missense (five de novo, one inheritance uncertain), and Lys2596 in-frame deletion (four de novo). No clinical or radiological differences were evident between individuals with different mutations. ITPR1 encodes an inositol 1,4,5-triphosphate-responsive calcium channel. The homo-tetrameric structure has been solved by cryoelectron microscopy. Using estimations of the degree of structural change induced by known recessive- and dominant-negative mutations in other disease-associated multimeric channels, we developed a generalizable computational approach to indicate the likely mutational mechanism. This analysis supports a dominant-negative mechanism for GS variants in ITPR1. In GS-derived lymphoblastoid cell lines (LCLs), the proportion of ITPR1-positive cells using immunofluorescence was significantly higher in mutant than control LCLs, consistent with an abnormality of nuclear calcium signaling feedback control. Super-resolution imaging supports the existence of an ITPR1-lined nucleoplasmic reticulum. Mice with Itpr1 heterozygous null mutations showed no major iris defects. Purkinje cells of the cerebellum appear to be the most sensitive to impaired ITPR1 function in humans. Iris hypoplasia is likely to result from either complete loss of ITPR1 activity or structure-specific disruption of multimeric interactions.
Asunto(s)
Aniridia/etiología , Aniridia/patología , Ataxia Cerebelosa/etiología , Ataxia Cerebelosa/patología , Genes Dominantes/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Discapacidad Intelectual/etiología , Discapacidad Intelectual/patología , Mutación/genética , Adolescente , Adulto , Animales , Células Cultivadas , Niño , Femenino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/química , Linfocitos/metabolismo , Linfocitos/patología , Masculino , Ratones , Microscopía Confocal , Persona de Mediana Edad , Linaje , Conformación ProteicaRESUMEN
RAB18, RAB3GAP1, RAB3GAP2 and TBC1D20 are each mutated in Warburg Micro syndrome, a rare autosomal recessive multisystem disorder. RAB3GAP1 and RAB3GAP2 form a binary 'RAB3GAP' complex that functions as a guanine-nucleotide exchange factor (GEF) for RAB18, whereas TBC1D20 shows modest RAB18 GTPase-activating (GAP) activity in vitro. Here, we show that in the absence of functional RAB3GAP or TBC1D20, the level, localization and dynamics of cellular RAB18 is altered. In cell lines where TBC1D20 is absent from the endoplasmic reticulum (ER), RAB18 becomes more stably ER-associated and less cytosolic than in control cells. These data suggest that RAB18 is a physiological substrate of TBC1D20 and contribute to a model in which a Rab-GAP can be essential for the activity of a target Rab. Together with previous reports, this indicates that Warburg Micro syndrome can be caused directly by loss of RAB18, or indirectly through loss of RAB18 regulators RAB3GAP or TBC1D20.
Asunto(s)
Anomalías Múltiples/etiología , Anomalías Múltiples/patología , Catarata/congénito , Córnea/anomalías , Regulación de la Expresión Génica , Hipogonadismo/etiología , Hipogonadismo/patología , Discapacidad Intelectual/etiología , Discapacidad Intelectual/patología , Microcefalia/etiología , Microcefalia/patología , Atrofia Óptica/etiología , Atrofia Óptica/patología , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab1/metabolismo , Proteínas de Unión al GTP rab3/metabolismo , Anomalías Múltiples/metabolismo , Animales , Western Blotting , Estudios de Casos y Controles , Catarata/etiología , Catarata/metabolismo , Catarata/patología , Células Cultivadas , Córnea/metabolismo , Córnea/patología , Citosol/metabolismo , Retículo Endoplásmico/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Hidrólisis , Hipogonadismo/metabolismo , Discapacidad Intelectual/metabolismo , Ratones , Ratones Noqueados , Microcefalia/metabolismo , Atrofia Óptica/metabolismo , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab1/genética , Proteínas de Unión al GTP rab3/genéticaRESUMEN
Autosomal recessive cutis laxa type 3A is caused by mutations in ALDH18A1, a gene encoding the mitochondrial enzyme Δ(1)-pyrroline-5-carboxylate synthase (P5CS). It is a rare disorder with only six pathogenic mutations and 10 affected individuals from five families previously described in the literature. Here we report the identification of novel compound heterozygous missense mutations in two affected siblings from a Lebanese family by whole-exome sequencing. The mutations alter a conserved C-terminal domain of the encoded protein and reduce protein stability as determined through Western blot analysis of patient fibroblasts. Patient fibroblasts exhibit a lipid droplet phenotype similar to that recently reported in Warburg Micro syndrome, a disorder with similar features but hitherto unrelated cellular etiology.
RESUMEN
The ancestral Rab GTPase Rab18 and both subunits of the Rab3GAP complex are mutated in the human neurological and developmental disorder Warburg Micro syndrome. Here, we demonstrate that the Rab3GAP complex is a specific Rab18 guanine nucleotide exchange factor (GEF). The Rab3GAP complex localizes to the endoplasmic reticulum (ER) and is necessary for ER targeting of Rab18. It is also sufficient to promote membrane recruitment of Rab18. Disease-associated point mutations of conserved residues in either the Rab3GAP1 (T18P and E24V) or Rab3GAP2 (R426C) subunits result in loss of the Rab18 GEF and membrane-targeting activities. Supporting the view that Rab18 activity is important for ER structure, in the absence of either Rab3GAP subunit or Rab18 function, ER tubular networks marked by reticulon 4 were disrupted, and ER sheets defined by CLIMP-63 spread out into the cell periphery. Micro syndrome is therefore a disease characterized by direct loss of Rab18 function or loss of Rab18 activation at the ER by its GEF Rab3GAP.
Asunto(s)
Retículo Endoplásmico/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Unión al GTP rab/fisiología , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , GTP Fosfohidrolasas/metabolismo , Células HEK293 , Células HeLa , Humanos , Lípidos/química , Proteínas de la Membrana/metabolismo , Mutación , Fenotipo , Mutación Puntual , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.
Asunto(s)
Anomalías Múltiples/fisiopatología , Catarata/congénito , Córnea/anomalías , Citoesqueleto/fisiología , Modelos Animales de Enfermedad , Ojo/crecimiento & desarrollo , Hipogonadismo/fisiopatología , Discapacidad Intelectual/fisiopatología , Microcefalia/fisiopatología , Neuronas/fisiología , Atrofia Óptica/fisiopatología , Proteínas de Unión al GTP rab/fisiología , Animales , Catarata/fisiopatología , Córnea/fisiopatología , Ratones , Ratones Noqueados , Proteínas de Unión al GTP rab/genéticaRESUMEN
blind sterile (bs) is a spontaneous autosomal-recessive mouse mutation discovered more than 30 years ago. Phenotypically, bs mice exhibit nuclear cataracts and male infertility; genetic analyses assigned the bs locus to mouse chromosome 2. In this study, we first positionally cloned the bs locus and identified a putative causative mutation in the Tbc1d20 gene. Functional analysis established the mouse TBC1D20 protein as a GTPase-activating protein (GAP) for RAB1 and RAB2, and bs as a TBC1D20 loss-of-function mutation. Evaluation of bs mouse embryonic fibroblasts (mEFs) identified enlarged Golgi morphology and aberrant lipid droplet (LD) formation. Based on the function of TBC1D20 as a RABGAP and the bs cataract and testicular phenotypes, we hypothesized that mutations in TBC1D20 may contribute to Warburg micro syndrome (WARBM); WARBM constitutes a spectrum of disorders characterized by eye, brain, and endocrine abnormalities caused by mutations in RAB3GAP1, RAB3GAP2, and RAB18. Sequence analysis of a cohort of 77 families affected by WARBM identified five distinct TBC1D20 loss-of-function mutations, thereby establishing these mutations as causative of WARBM. Evaluation of human fibroblasts deficient in TBC1D20 function identified aberrant LDs similar to those identified in the bs mEFs. Additionally, our results show that human fibroblasts deficient in RAB18 and RAB3GAP1 function also exhibit aberrant LD formation. These findings collectively indicate that a defect in LD formation/metabolism may be a common cellular abnormality associated with WARBM, although it remains unclear whether abnormalities in LD metabolism are contributing to WARBM disease pathology.
Asunto(s)
Anomalías Múltiples/genética , Catarata/congénito , Catarata/genética , Córnea/anomalías , Hipogonadismo/genética , Infertilidad Masculina/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Mutación , Atrofia Óptica/genética , Proteínas de Unión al GTP rab1/genética , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/patología , Catarata/diagnóstico , Catarata/metabolismo , Línea Celular , Córnea/metabolismo , Análisis Mutacional de ADN , Facies , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Hipogonadismo/diagnóstico , Hipogonadismo/metabolismo , Discapacidad Intelectual/diagnóstico , Discapacidad Intelectual/metabolismo , Cristalino/patología , Imagen por Resonancia Magnética , Masculino , Ratones , Microcefalia/diagnóstico , Microcefalia/metabolismo , Atrofia Óptica/diagnóstico , Atrofia Óptica/metabolismo , Linaje , Fenotipo , Alineación de Secuencia , Testículo/patología , Proteínas de Unión al GTP rab1/metabolismoRESUMEN
Warburg Micro syndrome and Martsolf syndrome (MS) are heterogeneous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Causative biallelic germline mutations have been identified in RAB3GAP1, RAB3GAP2, or RAB18, each of which encode proteins involved in membrane trafficking. This report provides an up to date overview of all known disease variants identified in 29 previously published families and 52 new families. One-hundred and forty-four Micro and nine Martsolf families were investigated, identifying mutations in RAB3GAP1 in 41% of cases, mutations in RAB3GAP2 in 7% of cases, and mutations in RAB18 in 5% of cases. These are listed in Leiden Open source Variation Databases, which was created by us for all three genes. Genotype-phenotype correlations for these genes have now established that the clinical phenotypes in Micro syndrome and MS represent a phenotypic continuum related to the nature and severity of the mutations present in the disease genes, with more deleterious mutations causing Micro syndrome and milder mutations causing MS. RAB18 has not yet been linked to the RAB3 pathways, but mutations in all three genes cause an indistinguishable phenotype, making it likely that there is some overlap. There is considerable genetic heterogeneity for these disorders and further gene identification will help delineate these pathways.
Asunto(s)
Catarata/genética , Genotipo , Hipogonadismo/genética , Discapacidad Intelectual/genética , Mutación Missense , Fenotipo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab3/genética , Secuencia de Aminoácidos , Animales , Catarata/patología , Niño , Preescolar , Humanos , Hipogonadismo/patología , Lactante , Discapacidad Intelectual/patología , Imagen por Resonancia Magnética , Masculino , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab3/químicaRESUMEN
Micro syndrome (OMIM 60018) and Martsolf syndrome (OMIM 21270) are related rare autosomal recessive disorders characterized by ocular and neurological abnormalities and hypothalamic hypogonadism. Micro syndrome has been associated with causative mutations in three disease genes: RAB3GAP1, RAB3GAP2 and RAB18. Martsolf syndrome has been associated with a mutation in RAB3GAP2. The present review summarizes the current literature on these genes and the proteins they encode.
Asunto(s)
Anomalías Múltiples/genética , Catarata/congénito , Hipogonadismo/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Atrofia Óptica/genética , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab3/genética , Anomalías Múltiples/enzimología , Animales , Catarata/enzimología , Catarata/genética , Córnea/anomalías , Córnea/enzimología , Humanos , Hipogonadismo/enzimología , Discapacidad Intelectual/enzimología , Microcefalia/enzimología , Mutación , Atrofia Óptica/enzimología , Proteínas de Unión al GTP rab/fisiología , Proteínas de Unión al GTP rab3/fisiologíaRESUMEN
Warburg Micro syndrome and Martsolf syndrome are heterogenous autosomal-recessive developmental disorders characterized by brain, eye, and endocrine abnormalities. Previously, identification of mutations in RAB3GAP1 and RAB3GAP2 in both these syndromes implicated dysregulation of the RAB3 cycle (which controls calcium-mediated exocytosis of neurotransmitters and hormones) in disease pathogenesis. RAB3GAP1 and RAB3GAP2 encode the catalytic and noncatalytic subunits of the hetrodimeric enzyme RAB3GAP (RAB3GTPase-activating protein), a key regulator of the RAB3 cycle. We performed autozygosity mapping in five consanguineous families without RAB3GAP1/2 mutations and identified loss-of-function mutations in RAB18. A c.71T > A (p.Leu24Gln) founder mutation was identified in four Pakistani families, and a homozygous exon 2 deletion (predicted to result in a frameshift) was found in the fifth family. A single family whose members were compound heterozygotes for an anti-termination mutation of the stop codon c.619T > C (p.X207QextX20) and an inframe arginine deletion c.277_279 del (p.Arg93 del) were identified after direct gene sequencing and multiplex ligation-dependent probe amplification (MLPA) of a further 58 families. Nucleotide binding assays for RAB18(Leu24Gln) and RAB18(Arg93del) showed that these mutant proteins were functionally null in that they were unable to bind guanine. The clinical features of Warburg Micro syndrome patients with RAB3GAP1 or RAB3GAP2 mutations and RAB18 mutations are indistinguishable, although the role of RAB18 in trafficking is still emerging, and it has not been linked previously to the RAB3 pathway. Knockdown of rab18 in zebrafish suggests that it might have a conserved developmental role. Our findings imply that RAB18 has a critical role in human brain and eye development and neurodegeneration.
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Mutación , Proteínas de Unión al GTP rab/genética , Anomalías Múltiples/genética , Anomalías Múltiples/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Catarata/congénito , Catarata/genética , Catarata/metabolismo , Codón de Terminación , Consanguinidad , Córnea/anomalías , Córnea/metabolismo , Análisis Mutacional de ADN , Femenino , Efecto Fundador , Haplotipos , Humanos , Hipogonadismo/genética , Hipogonadismo/metabolismo , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Masculino , Microcefalia/genética , Microcefalia/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Atrofia Óptica/genética , Atrofia Óptica/metabolismo , Linaje , Fenotipo , Unión Proteica , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Unión al GTP rab/química , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab3/genéticaRESUMEN
Neuronal calcium sensor-1 (NCS-1) is a Ca(2+) sensor protein that has been implicated in the regulation of various aspects of neuronal development and neurotransmission. It exerts its effects through interactions with a range of target proteins one of which is interleukin receptor accessory protein like-1 (IL1RAPL1) protein. Mutations in IL1RAPL1 have recently been associated with autism spectrum disorders and a missense mutation (R102Q) on NCS-1 has been found in one individual with autism. We have examined the effect of this mutation on the structure and function of NCS-1. From use of NMR spectroscopy, it appeared that the R102Q affected the structure of the protein particularly with an increase in the extent of conformational exchange in the C-terminus of the protein. Despite this change NCS-1(R102Q) did not show changes in its affinity for Ca(2+) or binding to IL1RAPL1 and its intracellular localisation was unaffected. Assessment of NCS-1 dynamics indicated that it could rapidly cycle between cytosolic and membrane pools and that the cycling onto the plasma membrane was specifically changed in NCS-1(R102Q) with the loss of a Ca(2+) -dependent component. From these data we speculate that impairment of the normal cycling of NCS-1 by the R102Q mutation could have subtle effects on neuronal signalling and physiology in the developing and adult brain.
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Trastorno Autístico/genética , Mutación/genética , Proteínas Sensoras del Calcio Neuronal/química , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/química , Neuropéptidos/metabolismo , Sustitución de Aminoácidos/genética , Animales , Calcio/farmacología , Señalización del Calcio/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Proteína Accesoria del Receptor de Interleucina-1/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Proteínas Mutantes/metabolismo , Proteínas Sensoras del Calcio Neuronal/genética , Neuropéptidos/genética , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Relación Estructura-Actividad , Red trans-Golgi/efectos de los fármacos , Red trans-Golgi/metabolismoRESUMEN
The activation of regulated exocytosis occurs by a rise in cytosolic Ca(2+) concentration. Synaptotagmins act as the Ca(2+) sensors, whereas the machinery that allows fusion of secretory vesicles with the plasma membrane consists of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including syntaxin 1, SNAP-25, and VAMP. Within the pathway leading to exocytosis, there is an essential requirement for a member of the conserved Sec1/Munc18 (SM) protein family, which in neurotransmitter and neurohormone release in mammalian cells is Munc18-1. The exact role of Munc18-1 and the steps within exocytosis in which it acts have been intensively investigated. Current evidence suggests that Munc18-1 acts via distinct modes of interactions with syntaxin 1 and the other SNARE proteins and influences all of the steps leading to exocytosis, including vesicle recruitment, tethering, docking, priming, and membrane fusion.
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Exocitosis , Proteínas Munc18/metabolismo , Animales , Humanos , Proteínas Munc18/química , Proteínas Munc18/genética , Mutación/genética , Unión Proteica , Proteínas Qa-SNARE/metabolismo , Proteínas de Unión al GTP rab3/metabolismoRESUMEN
Rab proteins are GTPases that transit between GTP- and GDP-bound states. In the GTP-bound form they can recruit specific effector to membrane domains. It is possible that the exchange of Rab effectors between membranes and cytosol would be determined by the exchange of the particular Rab partner. We have compared the cycling of three Rab3/27 effectors, Granuphilin, Noc2, and Rabphilin, in PC12 cells using fluorescence recovery after photobleaching of EGFP-tagged proteins. All three effectors become localised to secretory granules. Granuphilin and Noc2 showed little or no exchange between secretory granules and cytosol whereas Rabphilin showed rapid and complete exchange. Both Noc2 and Rabphilin were found to be recruited to granules by Rab27 but the data suggest that Rabphilin did not form stable complexes with Rab27 on secretory granules and so Rab effector cycling between membranes and cytosol can be independent of that of the Rab protein.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citosol/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas/metabolismo , Vesículas Secretoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Péptidos y Proteínas de Señalización Intracelular , Proteínas del Tejido Nervioso/genética , Células PC12 , Transporte de Proteínas , Proteínas/genética , Ratas , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genética , Proteínas rab27 de Unión a GTP , Proteína de Unión al GTP rab3A/genética , Proteína de Unión al GTP rab3A/metabolismo , Rabfilina-3ARESUMEN
Munc18-1 plays a crucial role in regulated exocytosis in neurons and neuroendocrine cells through modulation of vesicle docking and membrane fusion. The molecular basis for Munc18 function is still unclear, as are the links with Rabs and SNARE [SNAP (soluble N-ethylmaleimide-sensitive factor-attachment protein) receptor] proteins that are also required. Munc18-1 can bind to SNAREs through at least three modes of interaction, including binding to the closed conformation of syntaxin 1. Using a gain-of-function mutant of Munc18-1 (E466K), which is based on a mutation in the related yeast protein Sly1p, we have identified a direct interaction of Munc18-1 with Rab3A, which is increased by the mutation. Expression of Munc18-1 with the E466K mutation increased exocytosis in adrenal chromaffin cells and PC12 cells (pheochromocytoma cells) and was found to increase the density of secretory granules at the periphery of PC12 cells, suggesting a stimulatory effect on granule recruitment through docking or tethering. Both the increase in exocytosis and changes in granule distribution appear to require Munc18-1 E466K binding to the closed form of syntaxin 1, suggesting a role for this interaction in bridging Rab- and SNARE-mediated events in exocytosis.