RESUMO
Mucopolysaccharidosis type II (MPS II: also called as Hunter syndrome) is an X-linked recessive lysosomal storage disorder characterized by the accumulation of extracellular glycosaminoglycans due to the deficiency of the enzyme iduronate-2-sulfatase (IDS). Previous observations suggested that MPS II can be classified into two distinct disease subtypes: (1) severe type of MPS II involves a decline in the cognitive ability of a patient and (2) attenuated type of MPS II exhibits no such intellectual phenotype. To determine whether such disease subtypes of MPS II could be explained by genetic diagnosis, we analyzed mutations in the IDS gene of 65 patients suffering from MPS II among the Japanese population who were diagnosed with both the accumulation of urinary glycosaminoglycans and a decrease in their IDS enzyme activity between 2004 and 2014. Among the specimens examined, we identified the following mutations: 33 missense, 8 nonsense, 7 frameshift, 4 intronic changes affecting splicing, 8 recombinations involving IDS-IDS2, and 7 other mutations including 4 large deletions. Consistent with the previous data, the results of our study showed that most of the attenuated phenotype was derived from the missense mutations of the IDS gene, whereas mutations associated with a large structural alteration including recombination, splicing, frameshift, and nonsense mutations were linked to the severe phenotype of MPS II. Furthermore, we conducted a homology modeling study of IDS P120R and N534I mutant as representatives of the causative mutation of the severe and attenuated type of MPS II, respectively. We found that the substitution of P120R of the IDS enzyme was predicted to deform the α-helix generated by I119-F123, leading to the major structural alteration of the wild-type IDS enzyme. In sharp contrast, the effect of the structural alteration of N534I was marginal; thus, this mutation was pathogenically predicted to be associated with the attenuated type of MPS II. These results suggest that a combination of the genomic diagnosis of the IDS gene and the structural prediction of the IDS enzyme could enable the prediction of a phenotype more effectively.
Assuntos
Glicoproteínas/química , Glicoproteínas/genética , Mucopolissacaridose II/genética , Mucopolissacaridose II/patologia , Mutação , Povo Asiático/genética , Feminino , Predisposição Genética para Doença , Glicosaminoglicanos/urina , Humanos , Japão , Masculino , Modelos Moleculares , Linhagem , Estrutura Secundária de Proteína , Homologia Estrutural de ProteínaRESUMO
N-glycosylation is a major posttranslational modification that endows proteins with various functions. It is established that N-glycans are essential for the correct folding and stability of some enzymes; however, the actual effects of N-glycans on their activities are poorly understood. Here, we show that human α-l-iduronidase (hIDUA), of which a dysfunction causes accumulation of dermatan/heparan sulfate leading to mucopolysaccharidosis type I, uses its own N-glycan as a substrate binding and catalytic module. Structural analysis revealed that the mannose residue of the N-glycan attached to N372 constituted a part of the substrate-binding pocket and interacted directly with a substrate. A deglycosylation study showed that enzyme activity was highly correlated with the N-glycan attached to N372. The kinetics of native and deglycosylated hIDUA suggested that the N-glycan is also involved in catalytic processes. Our study demonstrates a previously unrecognized function of N-glycans.
Assuntos
Iduronidase/química , Iduronidase/metabolismo , Modelos Moleculares , Polissacarídeos/química , Polissacarídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Biocatálise , Dicroísmo Circular , Cristalografia por Raios X , Dermatan Sulfato/metabolismo , Eletroforese em Gel de Poliacrilamida , Heparitina Sulfato/metabolismo , Humanos , Iduronidase/genética , Cinética , Manose/química , Manose/metabolismo , Dados de Sequência Molecular , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/metabolismo , Mutação , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
Hydrogen sulfide (H2S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H2S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H2S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine ß-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H2S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H2S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.
Assuntos
Sulfeto de Hidrogênio/química , Óxido Nítrico/química , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/química , Enxofre/química , Biotina/química , Linhagem Celular Tumoral , Cistationina beta-Sintase/química , Cisteína/química , Humanos , Mutagênese Sítio-Dirigida , Neuroblastoma/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Plasmídeos/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Krabbe disease is an autosomal recessive leukodystrophy caused by a deficiency of the galactocerebrosidase (GALC) enzyme. Hematopoietic stem cells transplantation is the only available treatment option for pre-symptomatic patients. We have previously reported the chaperone effect of N-octyl-4-epi-ß-valienamine (NOEV) on mutant GM1 ß-galactosidase proteins, and in a murine GM1-gangliosidosis model. In this study, we examined its chaperone effect on mutant GALC proteins. We found that NOEV strongly inhibited GALC activity in cell lysates of GALC-transfected COS1 cells. In vitro NOEV treatment stabilized GALC activity under heat denaturation conditions. We also examined the effect of NOEV on cultured COS1 cells expressing mutant GALC activity and human skin fibroblasts from Krabbe disease patients: NOEV significantly increased the enzyme activity of mutants of late-onset forms. Moreover, we confirmed that NOEV could enhance the maturation of GALC precursor to its mature active form. Model structural analysis showed NOEV binds to the active site of human GALC protein. These results, for the first time, provide clear evidence that NOEV is a chaperone with promising potential for patients with Krabbe disease resulting from the late-onset mutations.
Assuntos
Galactosilceramidase/genética , Hexosaminas/uso terapêutico , Leucodistrofia de Células Globoides/tratamento farmacológico , Leucodistrofia de Células Globoides/genética , Adulto , Idade de Início , Animais , Células COS , Células Cultivadas , Criança , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos , Galactosilceramidase/antagonistas & inibidores , Galactosilceramidase/química , Humanos , Lactente , Leucodistrofia de Células Globoides/patologia , Chaperonas Moleculares/uso terapêuticoRESUMO
We report the case of a 42-yearold woman diagnosed with heterozygous Fabry disease (FD) due to a novel α-galactosidase A Pro210Ser mutation and exhibiting a unique distribution of synaptopodin within podocytes. The patient was referred to our hospital with moderate proteinuria, and a renal biopsy was performed. Light microscopic examination of the specimen revealed diffuse global enlargement of podocytes, which also showed foamy changes. Electron microscopy revealed abundant myeloid bodies in podocytes and focal mitochondrial abnormalities within the tubules. The patient exhibited none of the characteristic symptoms of FD except hypohidrosis and had no obvious family history. Genetic analysis revealed a novel missense mutation (Pro210Ser) in the α-galactosidase A gene. She was ultimately diagnosed with FD based on immunohistochemical staining indicating large amounts of accumulated globotriaosylceramide in her podocytes, detection of urinary globotriaosylceramide secretion using high-performance thin-layer chromatography/ immunostaining, and structural modeling of the mutated α-galactosidase A (Pro210Ser). Immunostaining of the swollen and foamy podocytes using podocyte-associated antibodies (against podocalyxin, Wilms tumor-1, vimentin, and synaptopodin) revealed a unique distribution of synaptopodin surrounding globotriaosylceramide. To our knowledge, this is the first report of immunohistologically detected synaptopodin upregulation in foamy podocytes in a patient with FD.
Assuntos
Doença de Fabry/genética , Heterozigoto , Proteínas dos Microfilamentos/análise , Mutação de Sentido Incorreto , Podócitos/química , Vacúolos/química , alfa-Galactosidase/genética , Adulto , Biópsia , Análise Mutacional de DNA , Terapia de Reposição de Enzimas , Doença de Fabry/diagnóstico , Doença de Fabry/tratamento farmacológico , Doença de Fabry/enzimologia , Feminino , Predisposição Genética para Doença , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Moleculares , Fenótipo , Podócitos/ultraestrutura , Triexosilceramidas/análise , Vacúolos/ultraestrutura , alfa-Galactosidase/uso terapêuticoRESUMO
Allelic mutations, predominantly missense ones, of the α-l-iduronidase (IDUA) gene cause mucopolysaccharidosis type I (MPS I), which exhibits heterogeneous phenotypes. These phenotypes are basically classified into severe, intermediate, and attenuated types. We previously examined the structural changes in IDUA due to MPS I by homology modeling, but the reliability was limited because of the low sequence identity. In this study, we built new structural models of mutant IDUAs due to 57 amino acid substitutions that had been identified in 27 severe, 1 severe-intermediate, 13 intermediate, 1 attenuated-intermediate and 15 attenuated type MPS I patients based on the crystal structure of human IDUA, which was recently determined by us. The structural changes were examined by calculating the root-mean-square distances (RMSD) and the number of atoms influenced by the amino acid replacements. The results revealed that the structural changes of the enzyme protein tended to be correlated with the severity of the disease. Then we focused on the structural changes resulting from amino acid replacements in the immunoglobulin-like domain and adjacent region, of which the structure had been missing in the IDUA model previously built. Coloring of atoms influenced by an amino acid substitution was performed in each case and the results revealed that the structural changes occurred in a region far from the active site of IDUA, suggesting that they affected protein folding. Structural analysis is thus useful for elucidation of the basis of MPS I.
Assuntos
Substituição de Aminoácidos , Iduronidase/química , Modelos Moleculares , Mucopolissacaridose I/genética , Mutação , Domínio Catalítico , Expressão Gênica , Humanos , Iduronidase/genética , Iduronidase/isolamento & purificação , Mucopolissacaridose I/enzimologia , Mucopolissacaridose I/patologia , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Índice de Gravidade de Doença , Homologia Estrutural de Proteína , Relação Estrutura-AtividadeRESUMO
Nitric oxide (NO) physiologically regulates numerous cellular responses through S-nitrosylation of protein cysteine residues. We performed antibody-array screening in conjunction with biotin-switch assays to look for S-nitrosylated proteins. Using this combination of techniques, we found that phosphatase with sequence homology to tensin (PTEN) is selectively S-nitrosylated by low concentrations of NO at a specific cysteine residue (Cys-83). S-nitrosylation of PTEN (forming SNO-PTEN) inhibits enzymatic activity and consequently stimulates the downstream Akt cascade, indicating that Cys-83 is a critical site for redox regulation of PTEN function. In ischemic mouse brain, we observed SNO-PTEN in the core and penumbra regions but found SNO-Akt, which is known to inhibit Akt activity, only in the ischemic core. These findings suggest that low concentrations of NO, as found in the penumbra, preferentially S-nitrosylate PTEN, whereas higher concentrations of NO, known to exist in the ischemic core, also S-nitrosylate Akt. In the penumbra, inhibition of PTEN (but not Akt) activity by S-nitrosylation would be expected to contribute to cell survival by means of enhanced Akt signaling. In contrast, in the ischemic core, SNO-Akt formation would inhibit this neuroprotective pathway. In vitro model systems support this notion. Thus, we identify unique sites of PTEN and Akt regulation by means of S-nitrosylation, resulting in an "on-off" pattern of control of Akt signaling.
Assuntos
PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Homologia de Sequência , Transdução de Sinais/fisiologia , Animais , Apoptose , Ativação Enzimática , Células HEK293 , Humanos , Camundongos , Neurônios/citologia , Neurônios/fisiologia , Óxido Nítrico/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genéticaRESUMO
In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure. Molecular dynamics (MD) simulation is a method for predicting protein dynamics and can detect potential ensembles of protein binding sites and hidden sites unobservable in a single-point structure. In this study, to demonstrate the utility of virtual screening with protein dynamics, MD simulations were performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding sites with a high probability of existence. The structure of the binding site obtained through MD simulation revealed pockets in addition to the active site that was present in the initial structure. Using the obtained binding site structures, virtual screening of 4.8 million compounds by docking simulation, in vitro assays, and X-ray analysis was conducted, successfully identifying two hit compounds.
RESUMO
A modified alpha-N-acetylgalactosaminidase (NAGA) with alpha-galactosidase A (GLA)-like substrate specificity was designed on the basis of structural studies and was produced in Chinese hamster ovary cells. The enzyme acquired the ability to catalyze the degradation of 4-methylumbelliferyl-alpha-D-galactopyranoside. It retained the original NAGA's stability in plasma and N-glycans containing many mannose 6-phosphate (M6P) residues, which are advantageous for uptake by cells via M6P receptors. There was no immunological cross-reactivity between the modified NAGA and GLA, and the modified NAGA did not react to serum from a patient with Fabry disease recurrently treated with a recombinant GLA. The enzyme cleaved globotriaosylceramide (Gb3) accumulated in cultured fibroblasts from a patient with Fabry disease. Furthermore, like recombinant GLA proteins presently used for enzyme replacement therapy (ERT) for Fabry disease, the enzyme intravenously injected into Fabry model mice prevented Gb3 storage in the liver, kidneys, and heart and improved the pathological changes in these organs. Because this modified NAGA is hardly expected to cause an allergic reaction in Fabry disease patients, it is highly promising as a new and safe enzyme for ERT for Fabry disease.
Assuntos
Terapia de Reposição de Enzimas/métodos , Doença de Fabry/tratamento farmacológico , alfa-N-Acetilgalactosaminidase/química , alfa-N-Acetilgalactosaminidase/uso terapêutico , Substituição de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Catálise , Células Cultivadas , Cricetinae , Cricetulus , Meios de Cultivo Condicionados/química , DNA Complementar/metabolismo , Modelos Animais de Doenças , Estabilidade de Medicamentos , Doença de Fabry/enzimologia , Doença de Fabry/metabolismo , Fibroblastos/efeitos dos fármacos , Corantes Fluorescentes/metabolismo , Galactosídeos/metabolismo , Vetores Genéticos , Humanos , Concentração de Íons de Hidrogênio , Himecromona/análogos & derivados , Himecromona/metabolismo , Imuno-Histoquímica , Rim/efeitos dos fármacos , Rim/patologia , Rim/ultraestrutura , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/ultraestrutura , Camundongos , Camundongos Knockout , Modelos Moleculares , Peso Molecular , Miocárdio/patologia , Miocárdio/ultraestrutura , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Retroviridae/genética , Transfecção , Triexosilceramidas/metabolismo , alfa-N-Acetilgalactosaminidase/genética , alfa-N-Acetilgalactosaminidase/isolamento & purificaçãoRESUMO
To elucidate the bases of Wolman disease (WD) and cholesteryl ester storage disease (CESD) from the viewpoint of enzyme structure, we constructed a structural model of human lysosomal acid lipase (LAL) using molecular modeling software Modeller. The results revealed that the residues responsible for WD/CESD tend to be less solvent-accessible than others. Then, we examined the structural changes in the LAL protein caused by the WD/CESD mutations, using molecular modeling software TINKER. The results indicated that conformational changes of the functionally important residues and/or large conformational changes tend to cause the severe clinical phenotype (WD), whereas small conformational changes tend to cause the mild clinical phenotype (CESD), although there have been several exceptions. Further structural analysis is required to clarify the relationship between the three-dimensional structural changes and clinical phenotypes.
Assuntos
Doença do Armazenamento de Colesterol Éster/genética , Modelos Moleculares , Mutação , Esterol Esterase/química , Doença de Wolman/genética , Doença do Armazenamento de Colesterol Éster/metabolismo , Humanos , Conformação Proteica , Software , Esterol Esterase/genética , Relação Estrutura-Atividade , Doença de Wolman/metabolismoRESUMO
Recently, male subjects harboring the c.196G>C nucleotide change which leads to the E66Q enzyme having low α-galactosidase A (GLA) activity have been identified at an unexpectedly high frequency on Japanese and Korean screening for Fabry disease involving dry blood spots and plasma/serum samples. Individuals with the E66Q enzyme have been suspected to have the later-onset Fabry disease phenotype leading to renal and cardiac disease. However, there has been no convincing evidence for this. To determine whether c.196G>C (E66Q) is disease-causing or not, we performed biochemical, pathological and structural studies. It was predicted that the E66Q amino acid substitution causes a small conformational change on the molecular surface of GLA, which leads to instability of the enzyme protein. However, biochemical studies revealed that subjects harboring the E66Q enzyme exhibited relatively high residual enzyme activity in white blood cells, and that there was no accumulation of globotriaosylceramide in cultured fibroblasts or an increased level of plasma globotriaosylsphingosine in these subjects. An electron microscopic examination did not reveal any pathological changes specific to Fabry disease in biopsied skin tissues from a male subject with the E66Q enzyme. These results strongly suggest that the c.196G>C is not a pathogenic mutation but is a functional polymorphism.
Assuntos
Doença de Fabry/enzimologia , Doença de Fabry/genética , Mutação/genética , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Povo Asiático , Células Cultivadas , Pré-Escolar , Análise Mutacional de DNA , Doença de Fabry/patologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Heterozigoto , Humanos , Immunoblotting , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Modelos Moleculares , Fenótipo , Reação em Cadeia da Polimerase em Tempo Real , Pele/citologia , Pele/enzimologia , Triexosilceramidas/sangue , alfa-Galactosidase/químicaRESUMO
Recently, plasma globotriaosylsphingosine (lyso-Gb3) has attracted attention as a biomarker of Fabry disease. However, we found a subset of Fabry disease patients who did not show any increase in the plasma lyso-Gb3 concentration, although other patients exhibited apparent enhancement of it. This subset predominantly exhibited the clinical phenotype of later-onset Fabry disease, and gene analysis revealed that the patients harbored the M296I mutation common to Japanese Fabry patients. This amino acid substitution is predicted to cause a small conformational change on the surface of the α-galactosidase A molecule, resulting in residual enzyme activity. Plasma lyso-Gb3 is a good biomarker of Fabry disease but care should be taken when it is used for a definitive diagnosis.
Assuntos
Doença de Fabry/diagnóstico , Glicolipídeos/sangue , Esfingolipídeos/sangue , alfa-Galactosidase/genética , Adulto , Substituição de Aminoácidos , Povo Asiático , Biomarcadores/sangue , Criança , Pré-Escolar , Doença de Fabry/enzimologia , Doença de Fabry/genética , Feminino , Humanos , Isoleucina/química , Isoleucina/genética , Masculino , Metionina/química , Metionina/genética , Pessoa de Meia-Idade , Mutação , Fenótipo , alfa-Galactosidase/química , alfa-Galactosidase/metabolismoRESUMO
Mucopolysaccharidosis type VI (MPS VI) is a genetic disorder caused by a deficiency of arylsulfatase B (ARSB). In our previous study, we investigated the structural changes in ARSB caused by amino acid substitutions associated with MPS VI, and revealed that such structural changes in ARSB were correlated with the clinical phenotypes. To the best of our knowledge, there is no database containing the structures of mutant ARSBs. Here, we built a database of clinical phenotypes, genotypes and structures of mutant ARSBs (http://mps6-database.org). This database can be accessed via the Internet, and is user friendly being equipped with powerful computational tools. This database will be useful for a better understanding of MPS VI.
Assuntos
Bases de Dados Genéticas , Mucopolissacaridose VI/genética , N-Acetilgalactosamina-4-Sulfatase/genética , Interface Usuário-Computador , Genótipo , Humanos , Internet , Mutação , N-Acetilgalactosamina-4-Sulfatase/química , FenótipoRESUMO
To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of ß-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human ß-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.
Assuntos
Gangliosídeo G(M2)/metabolismo , Cadeia beta da beta-Hexosaminidase/química , Cadeia beta da beta-Hexosaminidase/metabolismo , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Humanos , Immunoblotting , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Doença de Sandhoff/tratamento farmacológico , Doença de Tay-Sachs/tratamento farmacológico , Cadeia beta da beta-Hexosaminidase/genética , Cadeia beta da beta-Hexosaminidase/uso terapêuticoRESUMO
Angiotensin II type 1 receptor (AT1 receptor) blockers (ARBs) are one of the most popular anti-hypertensive agents. Control of blood pressure (BP) by ARBs is now a therapeutic target for the organ protection in patients with hypertension. Recent meta-analysis demonstrated the possibility that telmisartan was the strongest ARB for the reduction of BP in patients with essential hypertension. However, which molecular interactions of telmisartan with the AT1 receptor could explain its strongest BP lowering activity remains unclear. To address the issue, we constructed models for the interaction between commonly used ARBs and AT1 receptor and compared the docking model of telmisartan with that of other ARBs. Telmisartan has a unique binding mode to the AT1 receptor due to its distal benzimidazole portion. This unique portion could explain the highest molecular lipophilicity, the greatest volume distribution and the strongest binding affinity of telmisartan to AT1 receptor. Furthermore, telmisartan was found to firmly bind to the AT1 receptor through the unique "delta lock" structure. Our present study suggests that due to its "delta lock" structure, telmisartan may be superior to other ARBs in halting cardiovascular disease in patients with hypertension.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Anti-Hipertensivos/química , Benzimidazóis/química , Benzoatos/química , Receptor Tipo 1 de Angiotensina/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Benzimidazóis/farmacologia , Benzoatos/farmacologia , Bovinos , Cristalografia por Raios X , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/metabolismo , Modelos Moleculares , TelmisartanRESUMO
Small molecules including imino sugars are expected to act as chaperones for a mutant α-galactosidase A (GLA), which will be useful for pharmacological chaperone therapy for Fabry disease. However, there is little detailed information about the molecular mechanism. We paid attention to an M51I mutant GLA which had been reported to strongly react to an imino sugar. The predicted structural change caused by this amino acid substitution is very small and located on the surface of the molecule. We produced the mutant enzyme in yeast, and determined its enzymological characteristics. The enzymological parameter values are almost the same as those of the wild-type GLA, although the mutant enzyme is unstable not only under neutral pH conditions but also under acidic ones. Then, we directly examined the effect of imino sugars including 1-deoxygalactonojirimycin and galactostatin bisulfite on the purified mutant enzyme. The imino sugars apparently improved the stability of the mutant enzyme under both neutral and acidic pH conditions. The results of surface plasmon resonance biosensor assaying suggested that the imino sugars retained their binding activity as to the mutant enzyme under both neutral and acidic pH conditions. This information will facilitate improvement of pharmacological chaperone therapy for Fabry disease.
Assuntos
Substituição de Aminoácidos/genética , Imino Açúcares/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , alfa-Galactosidase/química , alfa-Galactosidase/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática/efeitos dos fármacos , Estabilidade Enzimática/genética , Imino Açúcares/farmacologia , Modelos Moleculares , Proteínas Mutantes/genética , Ligação Proteica/efeitos dos fármacos , alfa-Galactosidase/genéticaRESUMO
Fabry disease is a genetic disorder caused by a deficiency of α-galactosidase A (GLA). In our previous studies, we structurally investigated Fabry disease using a structural analysis system, and revealed that structural changes in GLA are very important for understanding the molecular basis of this disease. To the best of our knowledge, there is no database including the structures of mutant GLAs. Herein, we constructed a database of clinical phenotypes, genotypes and structures of mutant GLAs. This database can be accessed as 'fabry-database.org', and is user friendly, being equipped with powerful computational tools. This database will help researchers and clinicians who study Fabry disease.
Assuntos
Bases de Dados Genéticas , Doença de Fabry/genética , Mutação/genética , Fenótipo , alfa-Galactosidase/química , alfa-Galactosidase/genética , Genótipo , Humanos , Internet , Interface Usuário-ComputadorRESUMO
Recently, pharmacological chaperone therapy for Pompe disease with small molecules such as imino sugars has attracted interest. But mutant acid α-glucosidase (GAA) species responsive to imino sugars are limited. To elucidate the characteristics of a mutant GAA responsive to imino sugars, we performed biochemical and structural analyses. Among cultured fibroblast cell lines derived from Japanese Pompe patients, only one carrying p.S529V/p.S619R amino acid substitutions responded to 1-deoxynojirimycin (DNJ), and an expression study revealed that DNJ, N-butyl-deoxynojirimycin and nojirimycin-1-sulfonic acid increased the enzyme activity of the S529V mutant GAA expressed in Chinese hamster ovary cells. The results of western blotting analysis suggested that these imino sugars facilitated the intracellular transportation of the mutant GAA and stabilized it. Among these imino sugars, DNJ exhibited the strongest action on the mutant GAA. Structural analysis revealed that DNJ almost completely occupied the active site pocket, and interacted with amino acid residues comprising it through van der Waals contacts and hydrogen bonds. This information will be useful for improvement of pharmacological chaperone therapy for Pompe disease.
Assuntos
Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/metabolismo , Substituição de Aminoácidos/genética , Animais , Células CHO , Domínio Catalítico/genética , Células Cultivadas , Cricetinae , Cricetulus , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Doença de Depósito de Glicogênio Tipo II/enzimologia , Doença de Depósito de Glicogênio Tipo II/genética , Humanos , Imino Açúcares/metabolismo , Imino Açúcares/farmacologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica , alfa-Glucosidases/genéticaRESUMO
Fabry disease is a genetic disorder caused by a deficiency of alpha-galactosidase, exhibiting a wide clinical spectrum, from the early-onset severe 'classic' form to the late-onset mild 'variant' one. Recent screening of newborns revealed that the incidence of Fabry disease is unexpectedly high, and that the genotypes of patients with this disease are quite heterogeneous and many novel mutations have been identified in them. This suggests that a lot of Fabry patients will be found in an early clinical stage when the prognosis is obscure and a proper therapeutic schedule for them cannot be determined. Thus, it is significant to predict the clinical phenotype of this disease resulting from a novel mutation. Herein, we proposed a phenotype prediction model based on sequential and structural information. As far as we know, this is the first report of phenotype prediction for Fabry disease. First, we investigated the sequential and structural changes in the alpha-galactosidase molecule responsible for Fabry disease. The results showed that there are quite large differences in several properties between the classic and variant groups. We then developed a phenotype prediction model involving the decision tree technique. The accuracy of this prediction model is high (86%), and Matthew's correlation coefficient is also high (0.49). The phenotype predictor proposed in this paper may be useful for determining a proper therapeutic schedule for this disease.
Assuntos
Doença de Fabry/diagnóstico , Doença de Fabry/enzimologia , Análise de Sequência de Proteína , alfa-Galactosidase/química , Bases de Dados Genéticas , Árvores de Decisões , Doença de Fabry/patologia , Humanos , Modelos Genéticos , Mutação/genética , FenótipoRESUMO
We developed a new method that uses a set of drug-like compounds to select reliable in silico drug screening results. If some active compounds are known, the screening results that rank these active compounds at the top should be reliable. If no active compound is known, how to select the result is in question. We propose a concept of a set of "universal active probes" (UAPs), which is a set of small active compounds that bind to different kinds of proteins. We found that the hit ratio of the true active compounds in in silico screening shows positive correlation to that of the UAPs, probably because UAPs form a set of drug-like compounds. Thus, if the UAPs were added to the compound library, the screening result that shows a high hit ratio of the UAPs could give reliable actual hit compounds for the target protein. We examined this method for several targets and found this idea useful.