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1.
Biochim Biophys Acta Proteins Proteom ; 1872(6): 141043, 2024 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-39128657

RESUMO

Canavan disease is caused by mutations in the ASPA gene, leading to diminished catalytic activity of aspartoacylase in the brain. Clinical missense mutations are found throughout the enzyme structure, with many of these mutated enzymes having not only decreased activity but also compromised stability. High-throughput screening of a small molecule library has identified several compounds that significantly increase the thermal stability of the E285A mutant enzyme, the most predominant clinical mutation in Canavan disease, while having a negligible effect on the native enzyme. Based on the initial successes, some structural analogs of these initial hits were selected for further examination. Glutathione, NAAG and patulin were each confirmed to be competitive inhibitors, indicating the binding of these compounds at the dimer interface or near the active site of the E285A enzyme. The experimental results were theoretically examined with the help of the docking analysis method. The structure activity-guided optimization of these compounds can potentially lead to potential pharmacological chaperones that could alleviate the detrimental effect of ASPA mutations in Canavan patients.


Assuntos
Amidoidrolases , Doença de Canavan , Doença de Canavan/tratamento farmacológico , Doença de Canavan/genética , Doença de Canavan/enzimologia , Amidoidrolases/genética , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Amidoidrolases/química , Humanos , Simulação de Acoplamento Molecular , Estabilidade Enzimática/efeitos dos fármacos , Mutação de Sentido Incorreto , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/química , Domínio Catalítico , Mutação , Ensaios de Triagem em Larga Escala
2.
J Mol Graph Model ; 74: 44-53, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28349879

RESUMO

The disruption of aspartoacylase enzyme's catalytic activity causes fatal neurodegenerative Canavan disease. By molecular dynamics and docking methods, here we studied two deleterious mutations that have been identified in the Canavan patients' genotype E285A, F295S, and revealed the possible cause for the enzyme inhibition due to the drastic changes in active site dynamics, loss of interactions among Arg 71, Arg 168 and the substrate and pKa value of critical Glu178 residue. In addition to changes in the enzyme dynamics, free energy calculations show that the binding energy of substrate decreases dramatically up on mutations.


Assuntos
Amidoidrolases/química , Doença de Canavan/enzimologia , Amidoidrolases/genética , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Doença de Canavan/genética , Domínio Catalítico , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação de Sentido Incorreto , Ligação Proteica , Termodinâmica
3.
Hum Mutat ; 38(5): 524-531, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28101991

RESUMO

We describe 14 patients with 12 novel missense mutations in ASPA, the gene causing Canavan disease (CD). We developed a method to study the effect of these 12 variants on the function of aspartoacylase-the hydrolysis of N-acetyl-l-aspartic acid (NAA) to aspartate and acetate. The wild-type ASPA open reading frame (ORF) and the ORFs containing each of the variants were transfected into HEK293 cells. Enzyme activity was determined by incubating cell lysates with NAA and measuring the released aspartic acid by LC-MS/MS. Clinical data were obtained for 11 patients by means of questionnaires. Four patients presented with a non-typical clinical picture or with the milder form of CD, whereas seven presented with severe CD. The mutations found in the mild patients corresponded to the variants with the highest residual enzyme activities, suggesting that this assay can help evaluate unknown variants found in patients with atypical presentation. We have detected a correlation between clinical presentation, enzyme activity, and genotype for CD.


Assuntos
Amidoidrolases/metabolismo , Doença de Canavan/diagnóstico , Doença de Canavan/enzimologia , Fenótipo , Adolescente , Alelos , Amidoidrolases/química , Criança , Pré-Escolar , Análise Mutacional de DNA , Ativação Enzimática , Genótipo , Humanos , Lactente , Masculino , Modelos Moleculares , Mutação , Conformação Proteica
4.
Biochemistry ; 53(30): 4970-8, 2014 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-25003821

RESUMO

Canavan disease (CD) is a fatal, childhood neurological disorder caused by mutations in the ASPA gene, leading to catalytic deficiencies in the aspartoacylase (ASPA) enzyme and impaired N-acetyl-l-aspartic acid metabolism in the brain. To study the possible structural defects triggered by these mutations, four ASPA missense mutations associated with different disease severities have been structurally characterized. These mutant enzymes each have overall structures similar to that of the native ASPA enzyme, but with varying degrees of alterations that offer explanations for the respective loss of catalytic activity. The K213E mutant, a nonconservative mutant associated with a mild disease phenotype, has minimal structural differences compared to the native enzyme. In contrast, the loss of van der Waals contacts in the F295S mutant and the loss of hydrophobic and hydrogen bonding interactions in the Y231C mutant lead to a local collapse of the hydrophobic core structure in the carboxyl-terminal domain, contributing to a decrease in protein stability. The structure of the E285A mutant, the most common clinical mutant, reveals that the loss of hydrogen bonding interactions with the carboxylate side chain of Glu285 disturbs the active site architecture, leading to altered substrate binding and lower catalytic activity. Our improved understanding of the nature of these structural defects provides a basis for the development of treatment therapies for CD.


Assuntos
Amidoidrolases/química , Amidoidrolases/deficiência , Doença de Canavan/enzimologia , Domínio Catalítico/genética , Mutação de Sentido Incorreto/genética , Amidoidrolases/genética , Doença de Canavan/genética , Linhagem Celular , Cristalografia por Raios X , Humanos , Relação Estrutura-Atividade
5.
Arch Biochem Biophys ; 548: 66-73, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24632142

RESUMO

Aspartoacylase catalyzes the metabolism of an important amino acid in the brain, with the release acetate serving as the source for fatty acid biosynthesis. Defects in this enzyme lead to a loss of activity and the symptoms of a fatal neurological disorder called Canavan disease. Extensive evidence, including deglycosylation studies, differential activity upon eukaryotic host expression and site directed mutagenesis, have supported the presence of a glycan that plays an essential role in the stability and catalytic activity of mammalian aspartoacylase. However, the structure of this enzyme did not show the presence of any non-amino acid components at the putative glycosylation site. A more extensive study specifically designed to resolve this discrepancy has now shown that recombinantly-expressed human aspartoacylase is not glycosylated, but is still fully functional and stable even when produced from a bacterial expression system. Alternative interpretations of the prior experiments now present a consistent picture of the structural components of this essential brain enzyme.


Assuntos
Amidoidrolases/metabolismo , Glicoproteínas/metabolismo , Amidoidrolases/química , Amidoidrolases/genética , Sequência de Aminoácidos , Doença de Canavan/enzimologia , Clonagem Molecular , Glicoproteínas/química , Glicoproteínas/genética , Glicosilação , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/análise , Pichia/genética , Mutação Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
6.
Clin Biochem ; 46(18): 1902-4, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24036223

RESUMO

OBJECTIVE: Canavan disease (OMIM 271900) is a severe autosomal recessive neurodegenerative disorder characterized by spongy degeneration of the brain and caused by mutations in the gene encoding for aspartoacylase (ASPA). The enzyme is responsible for the catalyses of the brain-specific compound N-acetylaspartate (NAA). DESIGN AND METHODS: We report the case of two Egyptian sibling patients suspected of Canavan disease (CD) showing clinical deterioration, white matter degeneration, megalencephaly and severe intellectual impairment. The patients underwent magnetic resonance imaging (MRI) and biochemical analysis of NAA in biological fluid samples (serum and urine). Subsequently, in order to determine the mutation responsible for CD in these two sibs, a molecular biological examination was performed. RESULTS: MRI findings and quantification of high NAA excretion (1378.5 and 680.1µmolNAA/mmolcreatinine in urine of 4months and 4years old patients, respectively) confirmed the diagnosis of CD and prompted a search for the responsible mutation. The molecular biological analysis revealed homozygosity for the substitution T530C (Ile177Thr) in the exon 4 of the ASPA gene in both sibs. A total loss of enzymatic activity was also recorded. CONCLUSIONS: The substitution T530C (Ile177Thr) results in a novel missense mutation causing a CD phenotype with severe clinical characteristics. This mutation was not previously described in the literature. In these two sibs, urinary concentration of NAA appears to correlate inversely to symptom severity and CD progression.


Assuntos
Amidoidrolases/genética , Ácido Aspártico/análogos & derivados , Doença de Canavan/enzimologia , Doença de Canavan/etiologia , Mutação de Sentido Incorreto , Ácido Aspártico/sangue , Ácido Aspártico/urina , Doença de Canavan/genética , Pré-Escolar , Homozigoto , Humanos , Lactente , Imageamento por Ressonância Magnética
7.
J Inherit Metab Dis ; 36(1): 1-6, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22850825

RESUMO

Canavan disease (CD) is a fatal neurological disorder caused by defects in the gene that encodes for a critical metabolic enzyme. The enzyme aspartoacylase catalyzes the deacetylation of N-acetylaspartate to produce acetate required for fatty acid biosynthesis in the brain. The loss of aspartoacylase activity leads to the demyelination and disrupted brain development that is found in CD patients. Sixteen different clinical mutants of aspartoacylase have been cloned, expressed and purified to examine their properties and the relationship between enzyme properties and disease phenotype. In contrast to numerous cell culture studies that reported virtually complete loss of function, each of these purified mutant enzymes was found to have measureable catalytic activity. However, the activities of these mutants are diminished, by as little as three-fold to greater than 100-fold when compared to the native enzyme. Many of these mutated enzyme forms show decreased thermal stability and an increased propensity for denaturation upon exposure to urea, but only four of the 16 mutants examined showed both diminished thermal and diminished conformational stability. Significantly, each of these lower stability mutants are responsible for the more severe phenotypes of CD, while patients with milder forms of CD have aspartoacylase mutants with generally high catalytic activity and with either good thermal or good conformational stability. These results suggest that the loss of catalytic function and the accumulation of N-acetylaspartate in Canavan disease is at least partially a consequence of the decreased protein stability caused by these mutations.


Assuntos
Amidoidrolases/metabolismo , Ácido Aspártico/análogos & derivados , Doença de Canavan/enzimologia , Doença de Canavan/patologia , Amidoidrolases/genética , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Encéfalo/enzimologia , Encéfalo/metabolismo , Encéfalo/patologia , Doença de Canavan/genética , Doença de Canavan/metabolismo , Catálise , Progressão da Doença , Humanos , Mutação , Fenótipo
8.
Sci China Life Sci ; 55(12): 1109-19, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-23233226

RESUMO

In this work, the most detrimental missense mutations of aspartoacylase that cause Canavan's disease were identified computationally and the substrate binding efficiencies of those missense mutations were analyzed. Out of 30 missense mutations, I-Mutant 2.0, SIFT and PolyPhen programs identified 22 variants that were less stable, deleterious and damaging respectively. Subsequently, modeling of these 22 variants was performed to understand the change in their conformations with respect to the native aspartoacylase by computing their root mean squared deviation (RMSD). Furthermore, the native protein and the 22 mutants were docked with the substrate NAA (N-Acetyl-Aspartic acid) to explain the substrate binding efficiencies of those detrimental missense mutations. Among the 22 mutants, the docking studies identified that 15 mutants caused lower binding affinity for NAA than the native protein. Finally, normal mode analysis determined that the loss of binding affinity of these 15 mutants was caused by altered flexibility in the amino acids that bind to NAA compared with the native protein. Thus, the present study showed that the majority of the substrate-binding amino acids in those 15 mutants displayed loss of flexibility, which could be the theoretical explanation of decreased binding affinity between the mutant aspartoacylases and NAA.


Assuntos
Amidoidrolases/genética , Doença de Canavan/genética , Mutação de Sentido Incorreto , Amidoidrolases/química , Doença de Canavan/enzimologia , Humanos , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato
9.
J Cereb Blood Flow Metab ; 32(9): 1725-36, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22617649

RESUMO

The inherited leukodystrophy Canavan disease arises due to a loss of the ability to catabolize N-acetylaspartic acid (NAA) in the brain and constitutes a major point of focus for efforts to define NAA function. Accumulation of noncatabolized NAA is diagnostic for Canavan disease, but contrasts with the abnormally low NAA associated with compromised neuronal integrity in a broad spectrum of other clinical conditions. Experimental evidence for NAA function supports a role in white matter lipid synthesis, but does not explain how both elevated and lowered NAA can be associated with pathology in the brain. We have undertaken a systematic analysis of postnatal development in a mouse model of Canavan disease that delineates development and pathology by identifying markers of oxidative stress preceding oligodendrocyte loss and dysmyelination. These data suggest a role for NAA in the maintenance of metabolic integrity in oligodendrocytes that may be of relevance to the strong association between NAA and neuronal viability. N-acetylaspartic acid is proposed here to support lipid synthesis and energy metabolism via the provision of substrate for both cellular processes during early postnatal development.


Assuntos
Amidoidrolases/metabolismo , Metabolismo Energético/fisiologia , Bainha de Mielina/fisiologia , Animais , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Biomarcadores , Doença de Canavan/enzimologia , Doença de Canavan/metabolismo , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Doenças Desmielinizantes/metabolismo , Doenças Desmielinizantes/patologia , Dependovirus/genética , Glucose/deficiência , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Mutação/fisiologia , Oligodendroglia/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia
10.
Mol Biol Rep ; 39(5): 6197-201, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22219087

RESUMO

Canavan disease (OMIM 271900) is an autosomal recessive lethal neurodegenerative disorder characterized by spongy degeneration of the brain. A highly consanguineous Pakistani family with Canavan disease was enrolled on the basis of diagnosis. All the affected individuals have mental retardation, megalocephaly and degradation of motor skills, poor head control, partial vision loss, weakness of the muscles and raised urinary concentration of N-acetyl aspartic acid in the urine. Blood samples were collected from affected as well as normal siblings and processed for DNA purification. Linkage analysis was performed by typing three short tandem repeat markers D17S1583 (7.19 cM), D17S1828 (10.02 cM) and D17S919 (14.69 cM) for an already-reported gene/locus ASPA at chromosome 17p13.2 causing Canavan disease. During linkage analysis, all the affected individuals were homozygous for short tandem repeat markers while the normal siblings were heterozygous showing co-segregation of the disease. Gene ASPA (NM_000049) was undertaken to sequence for mutation analysis. As a result of sequence analysis, we found missense substitution 740A→G (p.G274R) in exon 6 of gene ASPA. To our knowledge, this is the first report about Canavan disease on a Pakistani family.


Assuntos
Amidoidrolases/genética , Doença de Canavan/enzimologia , Doença de Canavan/genética , Mutação de Sentido Incorreto/genética , Adolescente , Sequência de Bases , Criança , Análise Mutacional de DNA , Família , Feminino , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Paquistão , Linhagem , Adulto Jovem
11.
Mol Genet Metab ; 102(2): 176-80, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21095151

RESUMO

Canavan disease is a fatal neurological disease without any effective treatments to slow the relentless progress of this disorder. Enzyme replacement therapy has been used effectively to treat a number of metabolic disorders, but the presence of the blood-brain-barrier presents an additional challenge in the treatment of neurological disorders. Studies have begun with the aim of establishing a treatment protocol that can effectively replace the defective enzyme in Canavan disease patients. The human enzyme, aspartoacylase, has been cloned, expressed and purified, and the surface lysyl groups modified through PEGylation. Fully active modified enzymes were administered to mice that are defective in this enzyme and that show many of the symptoms of Canavan disease. Statistically significant increases in brain enzyme activity levels have been achieved in this animal model, as well as decreases in the elevated substrate levels that mimic those found in Canavan disease patients. These results demonstrate that the modified enzyme is gaining access to the brain and functions to correct this metabolic defect. The stage is now set for a long term study to optimize this enzyme replacement approach for the development of a treatment protocol.


Assuntos
Amidoidrolases/química , Amidoidrolases/uso terapêutico , Doença de Canavan/terapia , Terapia de Reposição de Enzimas , Animais , Doença de Canavan/enzimologia , Modelos Animais de Doenças , Portadores de Fármacos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Modelos Moleculares , Polietilenoglicóis/farmacologia , Estrutura Terciária de Proteína , Resultado do Tratamento
12.
Genet Med ; 12(4 Suppl): S5-S14, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20393311

RESUMO

Genetic testing for Tay-Sachs and Canavan disease is particularly important for Ashkenazi Jews, because both conditions are more frequent in that population. This comparative case study was possible because of different patenting and licensing practices. The role of DNA testing differs between Tay-Sachs and Canavan diseases. The first-line screening test for Tay-Sachs remains an enzyme activity test rather than genotyping. Genotyping is used for preimplantation diagnosis and confirmatory testing. In contrast, DNA-based testing is the basis for Canavan screening and diagnosis. The HEXA gene for Tay-Sachs was cloned at the National Institutes of Health, and the gene was patented but has not been licensed. The ASPA gene for Canavan disease was cloned and patented by Miami Children's Hospital. Miami Children's Hospital did not inform family members and patient groups that had contributed to the gene discovery that it was applying for a patent, and pursued restrictive licensing practices when a patent issued in 1997. This led to intense controversy, litigation, and a sealed, nonpublic 2003 settlement that apparently allowed for nonexclusive licensing. A survey of laboratories revealed a possible price premium for ASPA testing, with per-unit costs higher than for other genetic tests in the Secretary's Advisory Committee on Genetics, Health, and Society case studies. The main conclusion from comparing genetic testing for Tay-Sachs and Canavan diseases, however, is that patenting and licensing conducted without communication with patients and advocates cause mistrust and can lead to controversy and litigation, a negative model to contrast with the positive model of patenting and licensing for genetic testing of cystic fibrosis.


Assuntos
Doença de Canavan/diagnóstico , Triagem de Portadores Genéticos , Testes Genéticos/ética , Licenciamento em Medicina/ética , Patentes como Assunto/ética , Doença de Tay-Sachs/diagnóstico , Amidoidrolases/genética , Doença de Canavan/enzimologia , Doença de Canavan/genética , Genes , Testes Genéticos/economia , Humanos , Judeus/genética , Licenciamento em Medicina/economia , Doença de Tay-Sachs/enzimologia , Doença de Tay-Sachs/genética , Cadeia alfa da beta-Hexosaminidase/genética
13.
Brain Dev ; 32(10): 879-82, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20129749

RESUMO

We herein describe the first Chinese case of Canavan disease diagnosed by biochemical analysis and confirmed by DNA studies. We report two novel mutations: c.2T>C/M1T, an initiation codon mutation, and c.209A>G/N70S, which is located at the enzyme-substrate binding site. The combination of these two mutations resulted in a congenital form of Canavan disease.


Assuntos
Amidoidrolases/genética , Doença de Canavan/genética , Mutação de Sentido Incorreto/genética , Amidoidrolases/metabolismo , Povo Asiático/genética , Sítios de Ligação/genética , Encéfalo/patologia , Doença de Canavan/enzimologia , Doença de Canavan/patologia , China , Códon/genética , DNA/genética , Éxons/genética , Cabeça/anatomia & histologia , Humanos , Lactente , Íntrons/genética , Imageamento por Ressonância Magnética , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa
15.
J Neurosci Res ; 87(15): 3415-27, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19739253

RESUMO

Loss of the oligodendrocyte (OL)-specific enzyme aspartoacylase (ASPA) from gene mutation results in the sponginess and loss of white matter (WM) in Canavan disease (CD). This study addresses the fate of OLs during the pathophysiology of CD in an adult ASPA knockout (KO) mouse strain. Massive arrays of neural stem/progenitor cells, immunopositive for PSA-NCAM, nestin, vimentin, and NG2, were observed within the severely affected spongy WM of the KO mouse brain. In these mice, G1-->S cell cycle progression was confirmed by an increase in cdk2-kinase activity, a reduction in mitotic inhibitors p21(Cip1) and p27(Kip1), and an increase in bromodeoxyuridine (BrdU) incorporation. Highly acetylated nuclear histones H2B and H3 were detected in adult KO mouse WM, suggesting the existence of noncompact chromatin as seen during early development. Costaining for BrdU- or Ki67-positive cells with markers for neural progenitors confirmed a continuous generation of OL lineage cells in KO WM. We observed a severe reduction in 21.5- and 18.5-kDa myelin basic protein and PLP/DM20 proteolipid proteins combined with a decrease in myelinated fibers and a perinuclear retention of myelin protein staining, indicating impairment in protein trafficking. Death of OLs, neurons, and astrocytes was identified in every region of the KO brain. Immature OLs constituted the largest population of dying cells, particularly in WM. We also report an early expression of full-length ASPA mRNA in normal mouse brain at embryonic day 12.5, when OL progenitors first appear during development. These findings support involvement of ASPA in CNS development and function.


Assuntos
Amidoidrolases/genética , Encéfalo/anormalidades , Encéfalo/enzimologia , Doença de Canavan/enzimologia , Oligodendroglia/enzimologia , Células-Tronco/enzimologia , Animais , Biomarcadores/metabolismo , Encéfalo/fisiopatologia , Doença de Canavan/genética , Doença de Canavan/fisiopatologia , Ciclo Celular/genética , Morte Celular/genética , Diferenciação Celular/genética , Sobrevivência Celular/genética , Quinase 2 Dependente de Ciclina/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação Enzimológica da Expressão Gênica/genética , Histonas/metabolismo , Camundongos , Camundongos Knockout , Proteínas da Mielina/metabolismo , Degeneração Neural/enzimologia , Degeneração Neural/genética , Degeneração Neural/fisiopatologia , Oligodendroglia/patologia , Transporte Proteico/genética , RNA Mensageiro/metabolismo , Células-Tronco/patologia
16.
Zhonghua Yi Xue Za Zhi ; 88(25): 1742-5, 2008 Jul 01.
Artigo em Chinês | MEDLINE | ID: mdl-19035082

RESUMO

OBJECTIVE: To investigate the regulation of cyclooxygenase (Cox)-2/2', 3'-cyclic nucleotide3' phosphohydrolase (CNPase) on the oligodendrocyte apoptosis in the pathogenesis of the heroin-induced spongiform leucoencephalopathy (HSLE). METHODS: Samples of frontal lobe, cerebellum, and corpus callosum were obtained from the brains during autopsy of 4 HSLE patients and 5 patients who died of diseases other than cerebral diseases (controls) and underwent light microscopy and electron microscopy. Immunocytochemistry was carried out to detect the expression of myelin basic protein (MBP), caspase-3, COX-2, and CNPase protein. Apoptosis was examined by TUNEL staining. RESULTS: Widespread demyelination was seen in the white matter of the frontal lobe, cerebellum, and corpus callosum of the HSLE cases, most severely in cerebellum. In he HSLE group, the levels of caspase-3 and COX-2 expression were significantly higher, and the level of CNPase was significantly lower than those of the control group (all P < 0.05). CONCLUSION: Widespread demyelination in the white matter is a prevailing pathological change of HSLE. Oligodendrocyte apoptosis is one of the causes of HSLE. The upregulation of COX-2 and downregulation of CNPase may contribute to the pathogenesis.


Assuntos
2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Apoptose , Doença de Canavan/enzimologia , Ciclo-Oxigenase 2/metabolismo , Oligodendroglia/enzimologia , Adulto , Idoso , Doença de Canavan/induzido quimicamente , Doença de Canavan/patologia , Caspase 3/metabolismo , Feminino , Heroína , Humanos , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Oligodendroglia/patologia , Oligodendroglia/ultraestrutura
17.
Clin Biochem ; 41(7-8): 611-5, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18280251

RESUMO

OBJECTIVE: To verify the effect of and to date the unknown T677C mutation of the human N-acetylaspartoacylase (hASPA) gene on the function of the mutated enzyme. DESIGN AND METHODS: Wild type and I226T-mutated proteins were expressed and purified from a transformed Escherichia coli colony. Enzymatic activities were measured in the presence of varying substrate concentrations. RESULTS: Whilst kinetic parameters of wild type hASPA were in line with data in literature, I226T-mutated hASPA showed no enzymatic activity. CONCLUSION: Data indicated that this new mutation might be responsible in homozygosis for the phenotype corresponding to Canavan disease.


Assuntos
Amidoidrolases/genética , Mutação/genética , Amidoidrolases/biossíntese , Amidoidrolases/isolamento & purificação , Substituição de Aminoácidos/genética , Doença de Canavan/diagnóstico , Doença de Canavan/enzimologia , Doença de Canavan/genética , Pré-Escolar , Citosina , Ativação Enzimática/genética , Feminino , Humanos , Masculino , Mutagênese Sítio-Dirigida , Timina
19.
J Trop Pediatr ; 54(3): 208-10, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17999961

RESUMO

Canavan disease is a neurodegenerative disease with autosomal recessive inheritance. Although this disease is prevalant among Ashkenazi Jewish population, several cases have been reported from all over the world. Canavan disease is caused by a genetic mutation in aspartoacylase gene. We have identified a novel mutation, a homozygous C432+1G>A mutation, in a 10-month-old boy who has a typical Canavan phenotype (without macrocephaly) accompanied by typical brain magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS) and diffusion magnetic resonance findings. The patient's mother was found to be heterozygous for this mutation. We believe that future studies of aspartoacylase gene in various ethnic groups could lead to a better understanding of Canavan's pathophysiology and gene therapy.


Assuntos
Amidoidrolases/genética , Doença de Canavan/genética , Doença de Canavan/enzimologia , Humanos , Lactente , Masculino , Mutação , Turquia
20.
Brain Res ; 1148: 1-14, 2007 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-17391648

RESUMO

Mutations that result in near undetectable activity of aspartoacylase, which catalyzes the deacetylation of N-acetyl-l-aspartate, correlate with Canavan Disease, a neurodegenerative disorder usually fatal during childhood. The underlying biochemical mechanisms of how these mutations ablate activity are poorly understood. Therefore, we developed and tested a three-dimensional homology model of aspartoacylase based on zinc dependent carboxypeptidase A. Mutations of the putative zinc-binding residues (H21G, E24D/G, and H116G), the general proton donor (E178A), and mutants designed to switch the order of the zinc-binding residues (H21E/E24H and E24H/H116E) yielded wild-type aspartoacylase protein levels and undetectable ASPA activity. Mutations that affect substrate carboxyl binding (R71N) and transition state stabilization (R63N) also yielded wild-type aspartoacylase protein levels and undetectable aspartoacylase activity. Alanine substitutions of Cys124 and Cys152, residues indicated by homology modeling to be in close proximity and in the proper orientation for disulfide bonding, yielded reduced ASPA protein and activity levels. Finally, expression of several previously tested (E24G, D68A, C152W, E214X, D249V, E285A, and A305E) and untested (H21P, A57T, I143T, P183H, M195R, K213E/G274R, G274R, and F295S) Canavan Disease mutations resulted in undetectable enzyme activity, and only E285A and P183H showed wild-type aspartoacylase protein levels. These results show that aspartoacylase is a member of the caboxypeptidase A family and offer novel explanations for most loss-of-function aspartoacylase mutations associated with Canavan Disease.


Assuntos
Amidoidrolases/química , Amidoidrolases/genética , Química Encefálica/genética , Doença de Canavan/enzimologia , Doença de Canavan/genética , Mutação/genética , Sequência de Aminoácidos/genética , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Carboxipeptidases A/química , Carboxipeptidases A/genética , Análise Mutacional de DNA/métodos , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Predisposição Genética para Doença/genética , Humanos , Modelos Moleculares , Filogenia , Estrutura Terciária de Proteína/genética , Homologia de Sequência de Aminoácidos
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