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1.
Am J Hum Genet ; 111(9): 1970-1993, 2024 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-39106866

RESUMEN

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.


Asunto(s)
Proteína de Replicación C , Humanos , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Masculino , Células HeLa , Femenino , Fenotipo , Replicación del ADN/genética , Adulto , Mutación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Alelos
2.
Am J Hum Genet ; 110(4): 663-680, 2023 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-36965478

RESUMEN

The vast majority of human genes encode multiple isoforms through alternative splicing, and the temporal and spatial regulation of those isoforms is critical for organismal development and function. The spliceosome, which regulates and executes splicing reactions, is primarily composed of small nuclear ribonucleoproteins (snRNPs) that consist of small nuclear RNAs (snRNAs) and protein subunits. snRNA gene transcription is initiated by the snRNA-activating protein complex (SNAPc). Here, we report ten individuals, from eight families, with bi-allelic, deleterious SNAPC4 variants. SNAPC4 encoded one of the five SNAPc subunits that is critical for DNA binding. Most affected individuals presented with delayed motor development and developmental regression after the first year of life, followed by progressive spasticity that led to gait alterations, paraparesis, and oromotor dysfunction. Most individuals had cerebral, cerebellar, or basal ganglia volume loss by brain MRI. In the available cells from affected individuals, SNAPC4 abundance was decreased compared to unaffected controls, suggesting that the bi-allelic variants affect SNAPC4 accumulation. The depletion of SNAPC4 levels in HeLa cell lines via genomic editing led to decreased snRNA expression and global dysregulation of alternative splicing. Analysis of available fibroblasts from affected individuals showed decreased snRNA expression and global dysregulation of alternative splicing compared to unaffected cells. Altogether, these data suggest that these bi-allelic SNAPC4 variants result in loss of function and underlie the neuroregression and progressive spasticity in these affected individuals.


Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN , Paraparesia Espástica , Factores de Transcripción , Paraparesia Espástica/genética , Humanos , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Células HeLa , Isoformas de Proteínas/genética , RNA-Seq , Masculino , Femenino , Linaje , Alelos , Lactante , Preescolar , Niño , Adolescente , Estructura Secundaria de Proteína , ARN Nuclear Pequeño/genética
3.
Hum Mol Genet ; 31(24): 4121-4130, 2022 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-35913762

RESUMEN

The high-affinity copper transporter CTR1 is encoded by CTR1 (SLC31A1), a gene locus for which no detailed genotype-phenotype correlations have previously been reported. We describe identical twin male infants homozygous for a novel missense variant NM_001859.4:c.284 G > A (p.Arg95His) in CTR1 with a distinctive autosomal recessive syndrome of infantile seizures and neurodegeneration, consistent with profound central nervous system copper deficiency. We used clinical, biochemical and molecular methods to delineate the first recognized examples of human CTR1 deficiency. These included clinical phenotyping, brain imaging, assays for copper, cytochrome c oxidase (CCO), and mitochondrial respiration, western blotting, cell transfection experiments, confocal and electron microscopy, protein structure modeling and fetal brain and cerebral organoid CTR1 transcriptome analyses. Comparison with two other critical mediators of cellular copper homeostasis, ATP7A and ATP7B, genes associated with Menkes disease and Wilson disease, respectively, revealed that expression of CTR1 was highest. Transcriptome analyses identified excitatory neurons and radial glia as brain cell types particularly enriched for copper transporter transcripts. We also assessed the effects of Copper Histidinate in the patients' cultured cells and in the patients, under a formal clinical protocol. Treatment normalized CCO activity and enhanced mitochondrial respiration in vitro, and was associated with modest clinical improvements. In combination with present and prior studies, these infants' clinical, biochemical and molecular phenotypes establish the impact of this novel variant on copper metabolism and cellular homeostasis and illuminate a crucial role for CTR1 in human brain development. CTR1 deficiency represents a newly defined inherited disorder of brain copper metabolism.


Asunto(s)
Transportador de Cobre 1 , Cobre , Enfermedades Neurodegenerativas , Convulsiones , Humanos , Masculino , Cobre/metabolismo , Transportador de Cobre 1/genética , Gemelos , Lactante , Mutación Missense , Síndrome , Enfermedades Neurodegenerativas/diagnóstico , Enfermedades Neurodegenerativas/genética , Convulsiones/diagnóstico , Convulsiones/genética
4.
Int J Mol Sci ; 25(11)2024 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-38891937

RESUMEN

Modular supramolecular complexes, where different proteins are assembled to gather targeting capability and photofunctional properties within the same structures, are of special interest for bacterial photodynamic inactivation, given their inherent biocompatibility and flexibility. We have recently proposed one such structure, exploiting the tetrameric bacterial protein streptavidin as the main building block, to target S. aureus protein A. To expand the palette of targets, we have linked biotinylated Concanavalin A, a sugar-binding protein, to a methylene blue-labelled streptavidin. By applying a combination of spectroscopy and microscopy, we demonstrate the binding of Concanavalin A to the walls of Gram-positive S. aureus and Gram-negative E. coli. Photoinactivation is observed for both bacterial strains in the low micromolar range, although the moderate affinity for the molecular targets and the low singlet oxygen yields limit the overall efficiency. Finally, we apply a maximum entropy method to the analysis of autocorrelation traces, which proves particularly useful when interpreting signals measured for diffusing systems heterogeneous in size, such as fluorescent species bound to bacteria.


Asunto(s)
Pared Celular , Concanavalina A , Escherichia coli , Staphylococcus aureus , Concanavalina A/química , Concanavalina A/metabolismo , Escherichia coli/metabolismo , Staphylococcus aureus/metabolismo , Pared Celular/metabolismo , Estreptavidina/química , Estreptavidina/metabolismo , Proteínas Bacterianas/metabolismo , Unión Proteica
5.
Proc Natl Acad Sci U S A ; 117(1): 552-562, 2020 01 07.
Artículo en Inglés | MEDLINE | ID: mdl-31871193

RESUMEN

Systemic sclerosis (SSc) is a clinically heterogeneous autoimmune disease characterized by mutually exclusive autoantibodies directed against distinct nuclear antigens. We examined HLA associations in SSc and its autoantibody subsets in a large, newly recruited African American (AA) cohort and among European Americans (EA). In the AA population, the African ancestry-predominant HLA-DRB1*08:04 and HLA-DRB1*11:02 alleles were associated with overall SSc risk, and the HLA-DRB1*08:04 allele was strongly associated with the severe antifibrillarin (AFA) antibody subset of SSc (odds ratio = 7.4). These African ancestry-predominant alleles may help explain the increased frequency and severity of SSc among the AA population. In the EA population, the HLA-DPB1*13:01 and HLA-DRB1*07:01 alleles were more strongly associated with antitopoisomerase (ATA) and anticentromere antibody-positive subsets of SSc, respectively, than with overall SSc risk, emphasizing the importance of HLA in defining autoantibody subtypes. The association of the HLA-DPB1*13:01 allele with the ATA+ subset of SSc in both AA and EA patients demonstrated a transancestry effect. A direct correlation between SSc prevalence and HLA-DPB1*13:01 allele frequency in multiple populations was observed (r = 0.98, P = 3 × 10-6). Conditional analysis in the autoantibody subsets of SSc revealed several associated amino acid residues, mostly in the peptide-binding groove of the class II HLA molecules. Using HLA α/ß allelic heterodimers, we bioinformatically predicted immunodominant peptides of topoisomerase 1, fibrillarin, and centromere protein A and discovered that they are homologous to viral protein sequences from the Mimiviridae and Phycodnaviridae families. Taken together, these data suggest a possible link between HLA alleles, autoantibodies, and environmental triggers in the pathogenesis of SSc.


Asunto(s)
Autoanticuerpos/inmunología , Autoantígenos/genética , Antígenos HLA/genética , Imitación Molecular/inmunología , Esclerodermia Sistémica/genética , Negro o Afroamericano/genética , Alelos , Secuencia de Aminoácidos/genética , Antígenos Virales/genética , Antígenos Virales/inmunología , Autoantígenos/inmunología , Biología Computacional , Conjuntos de Datos como Asunto , Femenino , Predisposición Genética a la Enfermedad , Antígenos HLA/inmunología , Humanos , Masculino , Mimiviridae/inmunología , Phycodnaviridae/inmunología , Estructura Secundaria de Proteína/genética , Medición de Riesgo , Esclerodermia Sistémica/epidemiología , Esclerodermia Sistémica/inmunología , Homología de Secuencia de Aminoácido , Población Blanca/genética
6.
Nucleic Acids Res ; 48(14): 7856-7863, 2020 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-32619224

RESUMEN

The Fragile X-related disorders (FXDs) are Repeat Expansion Diseases, genetic disorders that result from the expansion of a disease-specific microsatellite. In those Repeat Expansion Disease models where it has been examined, expansion is dependent on functional mismatch repair (MMR) factors, including MutLγ, a heterodimer of MLH1/MLH3, one of the three MutL complexes found in mammals and a minor player in MMR. In contrast, MutLα, a much more abundant MutL complex that is the major contributor to MMR, is either not required for expansion or plays a limited role in expansion in many model systems. How MutLγ acts to generate expansions is unclear given its normal role in protecting against microsatellite instability and while MLH3 does have an associated endonuclease activity, whether that contributes to repeat expansion is uncertain. We show here, using a gene-editing approach, that a point mutation that eliminates the endonuclease activity of MLH3 eliminates expansions in an FXD mouse embryonic stem cell model. This restricts the number of possible models for repeat expansion and supports the idea that MutLγ may be a useful druggable target to reduce somatic expansion in those disorders where it contributes to disease pathology.


Asunto(s)
Síndrome del Cromosoma X Frágil/genética , Proteínas MutL/genética , Expansión de Repetición de Trinucleótido , Alelos , Animales , Línea Celular , Modelos Animales de Enfermedad , Masculino , Ratones , Mutación Puntual , Dominios Proteicos/genética , Células Madre
7.
PLoS Genet ; 15(5): e1008143, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31125343

RESUMEN

Maintenance of the correct redox status of iron is functionally important for critical biological processes. Multicopper ferroxidases play an important role in oxidizing ferrous iron, released from the cells, into ferric iron, which is subsequently distributed by transferrin. Two well-characterized ferroxidases, ceruloplasmin (CP) and hephaestin (HEPH) facilitate this reaction in different tissues. Recently, a novel ferroxidase, Hephaestin like 1 (HEPHL1), also known as zyklopen, was identified. Here we report a child with compound heterozygous mutations in HEPHL1 (NM_001098672) who presented with abnormal hair (pili torti and trichorrhexis nodosa) and cognitive dysfunction. The maternal missense mutation affected mRNA splicing, leading to skipping of exon 5 and causing an in-frame deletion of 85 amino acids (c.809_1063del; p.Leu271_ala355del). The paternal mutation (c.3176T>C; p.Met1059Thr) changed a highly conserved methionine that is part of a typical type I copper binding site in HEPHL1. We demonstrated that HEPHL1 has ferroxidase activity and that the patient's two mutations exhibited loss of this ferroxidase activity. Consistent with these findings, the patient's fibroblasts accumulated intracellular iron and exhibited reduced activity of the copper-dependent enzyme, lysyl oxidase. These results suggest that the patient's biallelic variants are loss-of-function mutations. Hence, we generated a Hephl1 knockout mouse model that was viable and had curly whiskers, consistent with the hair phenotype in our patient. These results enhance our understanding of the function of HEPHL1 and implicate altered ferroxidase activity in hair growth and hair disorders.


Asunto(s)
Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Adulto , Alelos , Animales , Sitios de Unión , Ceruloplasmina/metabolismo , Preescolar , Cobre/metabolismo , Femenino , Regulación de la Expresión Génica/genética , Variación Genética/genética , Células HEK293 , Cabello , Humanos , Hierro/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Oxidación-Reducción , Fenotipo
8.
J Biol Phys ; 48(2): 151-166, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35419659

RESUMEN

Computational design of antimicrobial peptides (AMPs) is a promising area of research for developing novel agents against drug-resistant bacteria. AMPs are present naturally in many organisms, from bacteria to humans, a time-tested mechanism that makes them attractive as effective antibiotics. Depending on the environment, AMPs can exhibit α-helical or ß-sheet conformations, a mix of both, or lack secondary structure; they can be linear or cyclic. Prediction of their structures is challenging but critical for rational design. Promising AMP leads can be developed using essentially two approaches: traditional modeling of the physicochemical mechanisms that determine peptide behavior in aqueous and membrane environments and knowledge-based, e.g., machine learning (ML) techniques, that exploit ever-growing AMP databases. Here, we explore the conformational landscapes of two recently ML-designed AMPs, characterize the dependence of these landscapes on the medium conditions, and identify features in peptide and membrane landscapes that mediate protein-membrane association. For both peptides, we observe greater conformational diversity in an aqueous solvent than in a less polar solvent, and one peptide is seen to alter its conformation more dramatically than the other upon the change of solvent. Our results support the view that structural rearrangement in response to environmental changes is central to the mechanism of membrane-structure disruption by linear peptides. We expect that the design of AMPs by ML will benefit from the incorporation of peptide conformational substates as quantified here with molecular simulations.


Asunto(s)
Péptidos Catiónicos Antimicrobianos , Péptidos Antimicrobianos , Humanos , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Solventes
9.
Biophys J ; 118(5): 1109-1118, 2020 03 10.
Artículo en Inglés | MEDLINE | ID: mdl-32023434

RESUMEN

Human glucokinase (GCK) is the prototypic example of an emerging class of proteins with allosteric-like behavior that originates from intrinsic polypeptide dynamics. High-resolution NMR investigations of GCK have elucidated millisecond-timescale dynamics underlying allostery. In contrast, faster motions have remained underexplored, hindering the development of a comprehensive model of cooperativity. Here, we map nanosecond-timescale dynamics and structural heterogeneity in GCK using a combination of unnatural amino acid incorporation, time-resolved fluorescence, and 19F nuclear magnetic resonance spectroscopy. We find that a probe inserted within the enzyme's intrinsically disordered loop samples multiple conformations in the unliganded state. Glucose binding and disease-associated mutations that suppress cooperativity alter the number and/or relative population of these states. Together, the nanosecond kinetics characterized here and the millisecond motions known to be essential for cooperativity provide a dynamical framework with which we address the origins of cooperativity and the mechanism of activated, hyperinsulinemia-associated, noncooperative variants.


Asunto(s)
Glucoquinasa , Glucoquinasa/genética , Glucoquinasa/metabolismo , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Conformación Molecular , Mutación
10.
Nucleic Acids Res ; 46(3): 1240-1255, 2018 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-29267981

RESUMEN

Rad4/XPC recognizes diverse DNA lesions including ultraviolet-photolesions and carcinogen-DNA adducts, initiating nucleotide excision repair. Studies have suggested that Rad4/XPC senses lesion-induced helix-destabilization to flip out nucleotides from damaged DNA sites. However, characterizing how DNA deformability and/or distortions impact recognition has been challenging. Here, using fluorescence lifetime measurements empowered by a maximum entropy algorithm, we mapped the conformational heterogeneities of artificially destabilized mismatched DNA substrates of varying Rad4-binding specificities. The conformational distributions, as probed by FRET between a cytosine-analog pair exquisitely sensitive to DNA twisting/bending, reveal a direct connection between intrinsic DNA deformability and Rad4 recognition. High-specificity CCC/CCC mismatch, free in solution, sampled a strikingly broad range of conformations from B-DNA-like to highly distorted conformations that resembled those observed with Rad4 bound; the extent of these distortions increased with bound Rad4 and with temperature. Conversely, the non-specific TAT/TAT mismatch had a homogeneous, B-DNA-like conformation. Molecular dynamics simulations also revealed a wide distribution of conformations for CCC/CCC, complementing experimental findings. We propose that intrinsic deformability promotes Rad4 damage recognition, perhaps by stalling a diffusing protein and/or facilitating 'conformational capture' of pre-distorted damaged sites. Surprisingly, even mismatched DNA specifically bound to Rad4 remains highly dynamic, a feature that may reflect the versatility of Rad4/XPC to recognize many structurally dissimilar lesions.


Asunto(s)
Reparación del ADN , ADN de Hongos/química , Proteínas de Unión al ADN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sitios de Unión , Daño del ADN , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Colorantes Fluorescentes/química , Expresión Génica , Cinética , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos/síntesis química , Oligodesoxirribonucleótidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Fluorescencia/métodos , Espectrometría de Fluorescencia/estadística & datos numéricos , Especificidad por Sustrato
11.
Hum Genet ; 137(4): 293-303, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29691655

RESUMEN

Progressive microcephaly and neurodegeneration are genetically heterogenous conditions, largely associated with genes that are essential for the survival of neurons. In this study, we interrogate the genetic etiology of two siblings from a non-consanguineous family with severe early onset of neurological manifestations. Whole exome sequencing identified novel compound heterozygous mutations in VARS that segregated with the proband: a missense (c.3192G>A; p.Met1064Ile) and a splice site mutation (c.1577-2A>G). The VARS gene encodes cytoplasmic valyl-tRNA synthetase (ValRS), an enzyme that is essential during eukaryotic translation. cDNA analysis on patient derived fibroblasts revealed that the splice site acceptor variant allele led to nonsense mediated decay, thus resulting in a null allele. Three-dimensional modeling of ValRS predicts that the missense mutation lies in a highly conserved region and could alter side chain packing, thus affecting tRNA binding or destabilizing the interface between the catalytic and tRNA binding domains. Further quantitation of the expression of VARS showed remarkably reduced levels of mRNA and protein in skin derived fibroblasts. Aminoacylation experiments on patient derived cells showed markedly reduced enzyme activity of ValRS suggesting the mutations to be loss of function. Bi-allelic mutations in cytoplasmic amino acyl tRNA synthetases are well-known for their role in neurodegenerative disorders, yet human disorders associated with VARS mutations have not yet been clinically well characterized. Our study describes the phenotype associated with recessive VARS mutations and further functional delineation of the pathogenicity of novel variants identified, which widens the clinical and genetic spectrum of patients with progressive microcephaly.


Asunto(s)
Atrofia/genética , Microcefalia/genética , Convulsiones/genética , Valina-ARNt Ligasa/genética , Alelos , Aminoacil-ARNt Sintetasas/genética , Atrofia/fisiopatología , Preescolar , Regulación Enzimológica de la Expresión Génica , Humanos , Lactante , Mutación con Pérdida de Función/genética , Masculino , Microcefalia/fisiopatología , Linaje , ARN de Transferencia/genética , Proteínas de Unión al ARN/genética , Convulsiones/fisiopatología , Aminoacilación de ARN de Transferencia/genética , Secuenciación del Exoma
12.
Blood ; 128(17): 2135-2143, 2016 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-27557945

RESUMEN

Cell motility, division, and structural integrity depend on dynamic remodeling of the cellular cytoskeleton, which is regulated in part by actin polymerization and depolymerization. In 3 families, we identified 4 children with recurrent infections and varying clinical manifestations including mild neutropenia, impaired wound healing, severe stomatitis with oral stenosis, and death. All patients studied had similar distinctive neutrophil herniation of the nuclear lobes and agranular regions within the cytosol. Chemotaxis and chemokinesis were markedly impaired, but staphylococcal killing was normal, and neutrophil oxidative burst was increased both basally and on stimulation. Neutrophil spreading on glass and cell polarization were also impaired. Neutrophil F-actin was elevated fourfold, suggesting an abnormality in F-actin regulation. Two-dimensional differential in-gel electrophoresis identified abnormal actin-interacting protein 1 (Aip1), encoded by WDR1, in patient samples. Biallelic mutations in WDR1 affecting distinct antiparallel ß-strands of Aip1 were identified in all patients. It has been previously reported that Aip1 regulates cofilin-mediated actin depolymerization, which is required for normal neutrophil function. Heterozygous mutations in clinically normal relatives confirmed that WDR1 deficiency is autosomal recessive. Allogeneic stem cell transplantation corrected the immunologic defect in 1 patient. Mutations in WDR1 affect neutrophil morphology, motility, and function, causing a novel primary immunodeficiency.


Asunto(s)
Citoesqueleto de Actina/patología , Síndromes de Inmunodeficiencia/patología , Trastornos Leucocíticos/genética , Proteínas de Microfilamentos/genética , Neutrófilos/patología , Niño , Electroforesis en Gel Bidimensional , Femenino , Predisposición Genética a la Enfermedad , Humanos , Immunoblotting , Síndromes de Inmunodeficiencia/inmunología , Trastornos Leucocíticos/inmunología , Trastornos Leucocíticos/patología , Masculino , Espectrometría de Masas , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/inmunología , Microscopía Confocal , Mutación , Neutrófilos/inmunología , Linaje
13.
Am J Med Genet A ; 173(3): 661-666, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28052552

RESUMEN

Joubert syndrome is a ciliopathy characterized by a specific constellation of central nervous system malformations that result in the pathognomonic "molar tooth sign" on imaging. More than 27 genes are associated with Joubert syndrome, but some patients do not have mutations in any of these genes. Celsr1, Celsr2, and Celsr3 are the mammalian orthologues of the drosophila planar cell polarity protein, flamingo; they play important roles in neural development, including axon guidance, neuronal migration, and cilium polarity. Here, we report bi-allelic mutations in CELSR2 in a Joubert patient with cortical heterotopia, microophthalmia, and growth hormone deficiency. © 2017 Wiley Periodicals, Inc.


Asunto(s)
Anomalías Múltiples/diagnóstico , Anomalías Múltiples/genética , Cadherinas/genética , Cerebelo/anomalías , Anomalías del Ojo/diagnóstico , Anomalías del Ojo/genética , Hormona de Crecimiento Humana/deficiencia , Enfermedades Renales Quísticas/diagnóstico , Enfermedades Renales Quísticas/genética , Microftalmía/genética , Mutación , Retina/anomalías , Alelos , Cadherinas/química , Niño , Facies , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Imagen por Resonancia Magnética , Modelos Moleculares , Polimorfismo de Nucleótido Simple , Conformación Proteica
14.
Proc Natl Acad Sci U S A ; 111(1): 314-9, 2014 Jan 07.
Artículo en Inglés | MEDLINE | ID: mdl-24351931

RESUMEN

Melatonin (N-acetyl-5-methoxytrypamine) is the vertebrate hormone of the night: circulating levels at night are markedly higher than day levels. This increase is driven by precisely regulated increases in acetylation of serotonin in the pineal gland by arylalkylamine N-acetyltransferase (AANAT), the penultimate enzyme in the synthesis of melatonin. This unique essential role of AANAT in vertebrate timekeeping is recognized by the moniker the timezyme. AANAT is also found in the retina, where melatonin is thought to play a paracrine role. Here, we focused on the evolution of AANAT in early vertebrates. AANATs from Agnathans (lamprey) and Chondrichthyes (catshark and elephant shark) were cloned, and it was found that pineal glands and retinas from these groups express a form of AANAT that is compositionally, biochemically, and kinetically similar to AANATs found in bony vertebrates (VT-AANAT). Examination of the available genomes indicates that VT-AANAT is absent from other forms of life, including the Cephalochordate amphioxus. Phylogenetic analysis and evolutionary rate estimation indicate that VT-AANAT evolved from the nonvertebrate form of AANAT after the Cephalochordate-Vertebrate split over one-half billion years ago. The emergence of VT-AANAT apparently involved a dramatic acceleration of evolution that accompanied neofunctionalization after a duplication of the nonvertebrate AANAT gene. This scenario is consistent with the hypotheses that the advent of VT-AANAT contributed to the evolution of the pineal gland and lateral eyes from a common ancestral photodetector and that it was not a posthoc recruitment.


Asunto(s)
N-Acetiltransferasa de Arilalquilamina/genética , Evolución Molecular , Regulación Enzimológica de la Expresión Génica , Melatonina/química , Secuencia de Aminoácidos , Animales , Biblioteca de Genes , Humanos , Lampreas , Funciones de Verosimilitud , Datos de Secuencia Molecular , Células Fotorreceptoras de Vertebrados/fisiología , Filogenia , Glándula Pineal/fisiología , Conformación Proteica , Retina/fisiología , Homología de Secuencia de Aminoácido , Tiburones , Ovinos , Factores de Tiempo , Vertebrados
15.
Mol Pharmacol ; 88(2): 238-44, 2015 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26013543

RESUMEN

6-Alkoxy-5-aryl-3-pyridincarboxamides, including the brain-penetrant compound 14G: [5-(4-chlorophenyl)-6-(cyclopropylmethoxy)-N-[(1R,2R)-2-hydroxy-cyclohexyl]-3-pyridinecarboxamide] and its peripherally restricted analog 14H: [5-(4-chlorophenyl)-N-[(1R,2R)-2-hydroxycyclohexyl]-6-(2-methoxyethoxy)-3-pyridinecarboxamide], have been recently introduced as selective, high-affinity antagonists of the human cannabinoid-1 receptor (hCB1R). Binding analyses revealed two orders of magnitude lower affinity of these compounds for mouse and rat versus human CB1R, whereas the affinity of rimonabant is comparable for all three CB1Rs. Modeling of ligand binding to CB1R and binding assays with native and mutant (Ile105Met) hCB1Rs indicate that the Ile105 to Met mutation in rodent CB1Rs accounts for the species-dependent affinity of 14G: and 14H: . Our work identifies Ile105 as a new pharmacophore component for developing better hCB1R antagonists and invalidates rodent models for assessing the antiobesity efficacy of 14G: and 14H: .


Asunto(s)
Encéfalo/metabolismo , Antagonistas de Receptores de Cannabinoides/síntesis química , Antagonistas de Receptores de Cannabinoides/farmacología , Niacinamida/análogos & derivados , Piperidinas/farmacología , Pirazoles/farmacología , Receptor Cannabinoide CB1/genética , Animales , Antagonistas de Receptores de Cannabinoides/química , Células HEK293 , Humanos , Isoleucina/metabolismo , Ratones , Modelos Moleculares , Niacinamida/síntesis química , Niacinamida/química , Niacinamida/farmacología , Piperidinas/química , Pirazoles/química , Ratas , Receptor Cannabinoide CB1/metabolismo , Rimonabant , Especificidad de la Especie , Relación Estructura-Actividad , Difracción de Rayos X
16.
Hum Mutat ; 33(8): 1207-15, 2012 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-22508683

RESUMEN

Copper (Cu) is a trace metal that readily gains and donates electrons, a property that renders it desirable as an enzyme cofactor but dangerous as a source of free radicals. To regulate cellular Cu metabolism, an elaborate system of chaperones and transporters has evolved, although no human Cu chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution of a highly conserved arginine residue at position 163, with tryptophan in domain II of CCS, which interacts directly with superoxide dismutase 1 (SOD1). Biochemical analyses of the patient's fibroblasts, mammalian cell transfections, immunoprecipitation assays, and Lys7Δ (CCS homolog) yeast complementation support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation in a gene coding for a Cu chaperone.


Asunto(s)
Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Western Blotting , Células Cultivadas , Células HeLa , Humanos , Inmunoprecipitación , Mutación , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1
17.
Am J Physiol Cell Physiol ; 303(7): C715-27, 2012 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-22723110

RESUMEN

A general question in molecular physiology is how to identify candidate protein kinases corresponding to a known or hypothetical phosphorylation site in a protein of interest. It is generally recognized that the amino acid sequence surrounding the phosphorylation site provides information that is relevant to identification of the cognate protein kinase. Here, we present a mass spectrometry-based method for profiling the target specificity of a given protein kinase as well as a computational tool for the calculation and visualization of the target preferences. The mass spectrometry-based method identifies sites phosphorylated in response to in vitro incubation of protein mixtures with active recombinant protein kinases followed by standard phosphoproteomic methodologies. The computational tool, called "PhosphoLogo," uses an information-theoretic algorithm to calculate position-specific amino acid preferences and anti-preferences from the mass-spectrometry data (http://helixweb.nih.gov/PhosphoLogo/). The method was tested using protein kinase A (catalytic subunit α), revealing the well-known preference for basic amino acids in positions -2 and -3 relative to the phosphorylated amino acid. It also provides evidence for a preference for amino acids with a branched aliphatic side chain in position +1, a finding compatible with known crystal structures of protein kinase A. The method was also employed to profile target preferences and anti-preferences for 15 additional protein kinases with potential roles in regulation of epithelial transport: CK2, p38, AKT1, SGK1, PKCδ, CaMK2δ, DAPK1, MAPKAPK2, PKD3, PIM1, OSR1, STK39/SPAK, GSK3ß, Wnk1, and Wnk4.


Asunto(s)
Marcación de Gen/métodos , Espectrometría de Masas/métodos , Proteínas Quinasas/química , Proteínas Quinasas/genética , Secuencia de Aminoácidos , Animales , Cromatografía Liquida/métodos , Datos de Secuencia Molecular , Fosforilación/fisiología , Proteínas Quinasas/metabolismo , Transporte de Proteínas/fisiología , Ratas
18.
Anal Biochem ; 427(1): 102-5, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22504734

RESUMEN

The maximum entropy method (MEM) has been used in many studies to reliably recover effective lifetimes from kinetics, whether measured experimentally or simulated computationally. Here, recent claims made by Mulligan et al. regarding MEM analyses of kinetics (Anal. Biochem. 421 (2012) 181-190) are shown to be unfounded. Their assertion that their software allows "analysis of datasets too noisy to process by existing iterative search algorithms" is refuted with a MEM analysis of their triexponential test case with increased noise. In addition, it is shown that lifetime distributions recovered from noisy kinetics data with the MEM can be improved by using a simple filter when bootstrapping the prior model. When deriving the bootstrapped model from the lifetime distribution obtained using a uniform model, only the slower processes are represented as Gaussians in the bootstrapped model. Using this new approach, results are clearly superior to those of Mulligan et al. despite the presence of increased noise. In a second example, ambiguity in the interpretation of Poisson kinetics in the presence of scattered excitation light is resolved by filtering the prior model.


Asunto(s)
Entropía , Modelos Químicos , Artefactos , Simulación por Computador , Cinética , Programas Informáticos
19.
Hum Mutat ; 30(4): 537-47, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19241467

RESUMEN

Autosomal recessive congenital ichthyosis (ARCI) is a heterogeneous group of rare cornification diseases. Germline mutations in TGM1 are the most common cause of ARCI in the United States. TGM1 encodes for the TGase-1 enzyme that functions in the formation of the cornified cell envelope. Structurally defective or attenuated cornified cell envelop have been shown in epidermal scales and appendages of ARCI patients with TGM1 mutations. We review the clinical manifestations as well as the molecular genetics of ARCI. In addition, we characterized 115 TGM1 mutations reported in 234 patients from diverse racial and ethnic backgrounds (Caucasion Americans, Norwegians, Swedish, Finnish, German, Swiss, French, Italian, Dutch, Portuguese, Hispanics, Iranian, Tunisian, Moroccan, Egyptian, Afghani, Hungarian, African Americans, Korean, Japanese and South African). We report 23 novel mutations: 71 (62%) missense; 20 (17%) nonsense; 9 (8%) deletion; 8 (7%) splice-site, and 7 (6%) insertion. The c.877-2A>G was the most commonly reported TGM1 mutation accounting for 34% (147 of 435) of all TGM1 mutant alleles reported to date. It had been shown that this mutation is common among North American and Norwegian patients due to a founder effect. Thirty-one percent (36 of 115) of all mutations and 41% (29 of 71) of missense mutations occurred in arginine residues in TGase-1. Forty-nine percent (35 of 71) of missense mutations were within CpG dinucleotides, and 74% (26/35) of these mutations were C>T or G>A transitions. We constructed a model of human TGase-1 and showed that all mutated arginines that reside in the two beta-barrel domains and two (R142 and R143) in the beta-sandwich are located at domain interfaces. In conclusion, this study expands the TGM1 mutation spectrum and summarizes the current knowledge of TGM1 mutations. The high frequency of mutated arginine codons in TGM1 may be due to the deamination of 5' methylated CpG dinucleotides.


Asunto(s)
Eritrodermia Ictiosiforme Congénita/genética , Mutación , Transglutaminasas/genética , Animales , Modelos Animales de Enfermedad , Genes Recesivos , Humanos , Eritrodermia Ictiosiforme Congénita/patología , Modelos Moleculares , Polimorfismo Genético , Estructura Terciaria de Proteína , Transglutaminasas/química
20.
J Phys Chem B ; 122(49): 11355-11362, 2018 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-30230838

RESUMEN

A continuum solvent model based on screened Coulomb potentials has been simplified and parametrized to sample native-like structures in replica-exchange simulations of each of six different peptides and miniproteins. Low-energy, native, and non-native structures were used to iteratively refine 11 parameter values. The centroid of the largest cluster of structures sampled in simulations initiated from an extended conformation represents the predicted structure. The main-chain rms deviation of this prediction from the experimental structure was 0.47 Å for the 12-residue Trp-zip2, 0.86 Å for the 14-residue MBH12, 2.53 Å for the 17-residue U(1-17)T9D, 2.03 Å for the 20-residue BS1, 1.08 Å for the 20-residue Trp-cage, and 3.64 Å for the 35-residue villin headpiece subdomain HP35. The centroid of the sixth largest cluster sampled for HP35 deviated by 0.91 Å. The CHARMM22/CMAP force field was used, with an additional ψ torsion term for residues other than glycine and proline. Six parameters govern the dielectric response of the continuum solvent, and four values of surface tension approximate nonpolar effects. An atom's self-energy and interaction energies are screened independently, each depending on whether the atom is part of a charged group, a neutral hydrogen-bonding main-chain group, or any other neutral group. The parameters inferred result in strong main-chain hydrogen bonds, consistent with the view that protein folding is dominated by the formation of these bonds. (1,2) Conformations of MBH12 and BS1 were excluded from the energy-function refinement, suggesting the parameters, referred to as SCP18, are transferable. An efficient estimate of solvent-accessible surface area is also described.


Asunto(s)
Péptidos/química , Proteínas/química , Secuencia de Aminoácidos , Simulación por Computador , Enlace de Hidrógeno , Modelos Químicos , Modelos Moleculares , Conformación Proteica , Solventes/química , Electricidad Estática , Termodinámica
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