RESUMEN
For over a decade, folic acid (FA) supplementation has been widely prescribed to pregnant women to prevent neural tube closure defects in newborns. Although neural tube closure occurs within the first trimester, high doses of FA are given throughout pregnancy, the physiological consequences of which are unknown. FA can cause epigenetic modification of the cytosine residues in the CpG dinucleotide, thereby affecting gene expression. Dysregulation of crucial gene expression during gestational development may have lifelong adverse effects or lead to neurodevelopmental defects, such as autism. We have investigated the effect of FA supplementation on gene expression in lymphoblastoid cells by whole-genome expression microarrays. The results showed that high FA caused dysregulation by ≥ four-fold up or down to more than 1000 genes, including many imprinted genes. The aberrant expression of three genes (FMR1, GPR37L1, TSSK3) was confirmed by Western blot analyses. The level of altered gene expression changed in an FA concentration-dependent manner. We found significant dysregulation in gene expression at concentrations as low as 15 ng/ml, a level that is lower than what has been achieved in the blood through FA fortification guidelines. We found evidence of aberrant promoter methylation in the CpG island of the TSSK3 gene. Excessive FA supplementation may require careful monitoring in women who are planning for, or are in the early stages of pregnancy. Aberrant expression of genes during early brain development may have an impact on behavioural characteristics.
Asunto(s)
Dieta , Suplementos Dietéticos , Ácido Fólico/administración & dosificación , Expresión Génica/efectos de los fármacos , Impresión Genómica/efectos de los fármacos , Línea Celular , Metilación de ADN/efectos de los fármacos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Ataxia telangiectasia (A-T) is a progressive neurodegenerative disorder caused by disruption of the gene, ataxia telangiectasia mutated (ATM). Present study was aimed at identifying proteins that are present in abnormal levels in A-T brain that may identify alternative targets for therapeutic interventions. Proteomic and Western blot analysis have shown massive expression of the small heat shock protein 27 (Hsp27) in frontal cortices of A-T brains compared to negligible levels in controls. The expression of other stress proteins, Hsp70, alphaB-crystallin, and prohibitin remained unchanged in the A-T and control brains. Significant decreases in reactive oxygen species, protein carbonyl groups and lipid peroxidation products were observed in the A-T brains. There is no evidence of caspase 3 activation or DAXX mediated apoptosis. We propose that neurons in the frontal lobe are protected by the expression of Hsp27, which scavenges the oxidative stress molecules formed consequent to the primary loss of ATM function.
Asunto(s)
Ataxia Telangiectasia/metabolismo , Encéfalo/metabolismo , Proteínas de Choque Térmico HSP27/biosíntesis , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada , Western Blotting , Química Encefálica , Caspasa 3/metabolismo , Proteínas de Ciclo Celular , Niño , Proteínas de Unión al ADN , Femenino , Proteínas de Choque Térmico , Humanos , Masculino , Chaperonas Moleculares , Datos de Secuencia Molecular , Proteínas Serina-Treonina Quinasas , Especies Reactivas de Oxígeno/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas Supresoras de Tumor , Adulto JovenRESUMEN
Autism is a pervasive, heterogeneous, neurodevelopmental disability characterized by impairments in verbal communications, reciprocal social interactions, and restricted repetitive stereotyped behaviors. Evidence suggests the involvement of multiple genetic factors in the etiology of autism, and extensive genome-wide association studies have revealed several candidate genes that bear single nucleotide polymorphisms (SNPs) in non-coding and coding regions. We have shown that a non-conservative, non-synonymous SNP in the glyoxalase I gene, GLOI, may be an autism susceptibility factor. The GLOI rs2736654 SNP is a CâA change that causes an Ala111Glu change in the Glo1 enzyme. To identify the significance of the SNP, we have conducted functional assays for Glo1. We now present evidence that the presence of the A-allele at rs2736654 results in reduced enzyme activity. Glo1 activity is decreased in lymphoblastoid cells that are homozygous for the A allele. The Glu-isoform of Glo1 in these cells is hyperphosphorylated. Direct HPLC measurements of the glyoxalase I substrate, methylglyoxal (MG), show an increase in MG in these cells. Western blot analysis revealed elevated levels of the receptor for advanced glycation end products (RAGEs). We also show that MG is toxic to the developing neuronal cells. We suggest that accumulation of MG results in the formation of AGEs, which induce expression of the RAGE that during crucial neuronal development may be a factor in the pathology of autism.
Asunto(s)
Trastorno Autístico/enzimología , Trastorno Autístico/genética , Lactoilglutatión Liasa/genética , Polimorfismo de Nucleótido Simple/genética , Alelos , Animales , Trastorno Autístico/metabolismo , Western Blotting , Células Cultivadas , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Predisposición Genética a la Enfermedad/genética , Productos Finales de Glicación Avanzada/genética , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Lactoilglutatión Liasa/metabolismo , Piruvaldehído/metabolismo , Conejos , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismoRESUMEN
BACKGROUND: Tripeptidyl aminopeptidase I (TPPI) is a crucial lysosomal enzyme that is deficient in the fatal neurodegenerative disorder called classic late-infantile neuronal ceroid lipofuscinosis (LINCL). It is involved in the catabolism of proteins in the lysosomes. Recent X-ray crystallographic studies have provided insights into the structural/functional aspects of TPPI catalysis, and indicated presence of an octahedrally coordinated Ca(2+). METHODOLOGY: Purified precursor and mature TPPI were used to study inhibition by NBS and EDTA using biochemical and immunological approaches. Site-directed mutagenesis with confocal imaging technique identified a critical W residue in TPPI activity, and the processing of precursor into mature enzyme. PRINCIPAL FINDINGS: NBS is a potent inhibitor of the purified TPPI. In mammalian TPPI, W542 is critical for tripeptidyl peptidase activity as well as autocatalysis. Transfection studies have indicated that mutants of the TPPI that harbor residues other than W at position 542 have delayed processing, and are retained in the ER rather than transported to lysosomes. EDTA inhibits the autocatalytic processing of the precursor TPPI. CONCLUSIONS/SIGNIFICANCE: We propose that W542 and Ca(2+) are critical for maintaining the proper tertiary structure of the precursor proprotein as well as the mature TPPI. Additionally, Ca(2+) is necessary for the autocatalytic processing of the precursor protein into the mature TPPI. We have identified NBS as a potent TPPI inhibitor, which led in delineating a critical role for W542 residue. Studies with such compounds will prove valuable in identifying the critical residues in the TPPI catalysis and its structure-function analysis.
Asunto(s)
Aminopeptidasas/química , Aminopeptidasas/metabolismo , Biocatálisis , Calcio/metabolismo , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Lipofuscinosis Ceroideas Neuronales/enzimología , Serina Proteasas/química , Serina Proteasas/metabolismo , Triptófano/metabolismo , Aminopeptidasas/deficiencia , Aminopeptidasas/genética , Animales , Secuencia de Bases , Bromosuccinimida/farmacología , Células CHO , Cricetinae , Cricetulus , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/deficiencia , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/metabolismo , Humanos , Indoles/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Oxidación-Reducción , Conformación Proteica , Transporte de Proteínas , Serina Proteasas/deficiencia , Serina Proteasas/genética , Tripeptidil Peptidasa 1RESUMEN
Autism is a neurodevelopmental disability characterized by deficits in verbal communications, impairments in social interactions, and repetitive behaviors. Several studies have indicated strong involvement of multigenic components in the etiology of autism. Linkage analyses and candidate gene search approaches so far have not identified any reliable susceptibility genes. We are using a proteomic approach to identify protein abnormalities due to aberrant gene expression in autopsied autism brains. In four of eight autism brains, we have found an increase in polarity (more acidic) of glyoxalase I (Glo1) by two-dimensional gel electrophoresis. To identify the molecular change resulting in the shift of Glo1 polarity, we undertook sequencing of GLO1 gene. Direct sequencing of GLO1 gene/mRNA in these brains, has identified a single nucleotide polymorphism (SNP), C419A. The SNP causes an Ala111Glu change in the protein sequence. Population genetics of GLO1 C419A SNP studied in autism (71 samples) and normal and neurological controls (49 samples) showed significantly higher frequency for the A419 (allele frequency 0.6 in autism and 0.4 in controls, one-tailed Fisher's test P < 0.0079). Biochemical measurements have revealed a 38% decrease in Glo1 enzyme activity in autism brains (one-tailed t-test P < 0.026). Western blot analysis has also shown accumulation of advanced glycation end products (AGE's) in autism brains. These data suggest that homozygosity for A419 GLO1 resulting in Glu111 is a predisposing factor in the etiology of autism.