RESUMEN
Cyclic nucleotide phosphodiesterase type 4 (PDE4) is a cAMP-specific phosphodiesterase that is found as four distinct genes in the mammalian genome (PDE4A, 4B, 4C, and 4D). Mutation analysis was done to identify the amino acids involved in activity and inhibitor selectivity. Mutations at Asp333 were made in HSPDE4D3 based on mutations that affect rolipram sensitivity in RNPDE4B1. The PDE4D3 Asp-Asn mutant was resistant to inhibition by rolipram as well as several other PDE4 inhibitors tested. These results suggest that this residue is near the inhibitor binding pocket in PDE4D3. Sequence comparison of PDE4 with cGMP-specific PDE proteins shows a conserved aspartic acid at position 333 in PDE4D3 and a conserved asparagine at this position in PDE enzymes that hydrolyze cGMP. Therefore, cGMP hydrolysis by PDE4D3 Asp-Asn was measured. PDE4D3 Asp-Asn hydrolyzes cGMP with kinetic constants similar to those observed for this protein with cAMP (K(m) approximately 20 microM, V(max) approximately 2 micromol AMP/min/mg recombinant protein). Under identical conditions, the K(m) value for cAMP hydrolysis by wild-type PDE4D3 is 3 microM and the V(max) value is 1 micromol AMP/min/mg recombinant protein. In addition, the PDE4D3 Asp-Ala mutant protein could hydrolyze cGMP. Finally, the analogous mutation in HSPDE4B1 (Asp413Asn) also allows hydrolysis of cGMP. These results show that this aspartic acid residue is important in inhibitor binding and nucleotide discrimination and suggest this residue is in the active site of PDE4.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/genética , 3',5'-AMP Cíclico Fosfodiesterasas/metabolismo , Mutagénesis , Nucleótidos/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células Cultivadas , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Humanos , Insectos , Cinética , Datos de Secuencia Molecular , Inhibidores de Fosfodiesterasa/farmacología , Purinonas/farmacología , Ratas , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Endothelin converting enzyme-1 (ECE-1) is a type II integral membrane protein and a zinc metalloendopeptidase. ECE-1 generates endothelin-1 (ET-1), the most potent vasoconstrictor yet discovered, by specific proteolytic processing of a precursor peptide, big ET-1. An insect cell expression system, which generates up to 4.3 mg of a secreted, soluble form of ECE-1 (solECE-1) per liter culture medium, has been established and solECE-1 was purified to homogeneity using five chromatographic steps. SolECE-1 expressed in insect cells could be suitable for X-ray structure determination as it is much less glycosylated than solECE-1 from mammalian cells. SolECE-1 from both sources, nonetheless, has comparable enzymatic properties. Despite apparent structural similarities, ECE-1 cleaves big ET-1 exclusively between Trp(21) and Val(22), in contrast to neprilysin, which cleaves big ET-1 at various sites. However, when linear big ET-1, in which the formation of disulfide bonds has been prevented by alkylation of the four cysteines, was used as substrate, it was cleaved by solECE-1 at multiple sites. This result indicates that secondary/tertiary structure of big ET-1 induced by disulfide bonds is essential for the specific cleavage of the Trp(21)-Val(22) bond by ECE-1. A continuous, fluorescent ECE-1 assay has been developed using a novel substrate, 2-aminobenzoyl-Arg-Pro-Pro-Gly-Phe-Ser-Pro-(p-nitro-Phe(8))-Arg. This simple and rapid assay can greatly facilitate discovery of novel ECE inhibitors useful as pharmaceutical agents.
Asunto(s)
Ácido Aspártico Endopeptidasas/química , Disulfuros/química , Endotelinas/química , Precursores de Proteínas/química , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/genética , Baculoviridae/genética , Bradiquinina/química , Cromatografía Líquida de Alta Presión , Endotelina-1 , Enzimas Convertidoras de Endotelina , Inhibidores Enzimáticos/farmacología , Glicopéptidos/farmacología , Glicosilación , Humanos , Cinética , Metaloendopeptidasas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Quinazolinas/farmacología , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , Spodoptera/genéticaRESUMEN
Endothelin-converting enzyme-1 (ECE-1) is a type II integral membrane protein that belongs to a family of metalloproteases which includes ECE-2, neprilysin (neutral endopeptidase 24.11, EC 3.4.24. 11), and Kell blood group protein. ECE-1 cleaves its biologically inactive native substrate, big endothelin-1, to generate a powerful vasoactive 21-amino acid peptide, endothelin-1. ECE-1 consists of a short N-terminal cytoplasmic tail, a transmembrane hydrophobic domain, and a large extracellular domain containing the catalytic site with a conserved Zn-binding motif. We have constructed a secreted, soluble form of ECE-1 (solECE-1) by fusing the cleavable N-terminal signal sequence of human alkaline phosphatase in frame with the entire extracellular domain of ECE-1. Stable transfectant CHO cell lines expressing up to 6.1 mg of solECE-1 per liter culture medium were established and solECE-1 was purified to homogeneity using three chromatographic steps with a 24% yield. SolECE-1 behaves as a dimer of 110-kDa subunits. SolECE-1 has a sharp pH optimum, similar to the native form, ECE-1a, but has a slightly more acidic pH optimum of 6.1-6.4 than that of 6.7-6.9 for ECE-1a. At its optimal pH of 6.4, solECE-1 cleaved big ET-1:big ET-2:big ET-3 in a ratio of 8.1:1:1.4, was inhibited by phosphoramidon with an IC50 value of 0.35 +/- 0.05 microM, had a Km value of 4.65 +/- 0.78 microM for big ET-1, and had a kcat value of 5.82 +/- 0.21 min-1, all values comparable to those for ECE-1a at its optimal pH of 6.8. Phosphoramidon inhibition of both ECE-1a and solECE-1 is highly pH-dependent. At pH 5.8, phosphoramidon inhibited ECE-1a and solECE-1 with IC50 values of 14 and 33 nM, respectively, which are 49- and 1224-fold more potent than at pH 7.2. SolECE-1 is highly glycosylated, similar to ECE-1a. Deglycosylation of solECE-1 by peptide N-glycosidase F shifted the apparent molecular weight of solECE-1 to approximately 80 kDa and the deglycosylated form(s) of solECE-1 preserved at least 72% of the activity of the glycosylated form.
Asunto(s)
Ácido Aspártico Endopeptidasas/biosíntesis , Ácido Aspártico Endopeptidasas/aislamiento & purificación , Animales , Ácido Aspártico Endopeptidasas/química , Células CHO , Línea Celular , Cricetinae , Enzimas Convertidoras de Endotelina , Activación Enzimática/efectos de los fármacos , Glicopéptidos/farmacología , Glicosilación , Humanos , Concentración de Iones de Hidrógeno , Cinética , Metaloendopeptidasas , Inhibidores de Proteasas/farmacología , Solubilidad , Especificidad por Sustrato , TransfecciónRESUMEN
OBJECTIVE: This study aimed to update a large kindred with juvenile-onset primary open-angle glaucoma (POAG) first described in 1940 and to identify the underlying genetic cause of the disease. DESIGN: Molecular genetic study of a single kindred, including clinical examination, retrospective review of clinical and family history records, linkage analysis, and mutation screening. PARTICIPANTS: The retrospective review included 957 members of a single large family. The linkage study included 40 members of 1 branch of the family in which juvenile-onset POAG is segregating in an autosomal-dominant pattern. Mutation screening included 15 at-risk family members with juvenile-onset POAG, probands of 40 families with adult-onset POAG, probands of 11 additional unrelated juvenile-onset POAG families, and 43 unrelated normal control subjects. INTERVENTION: Clinical and family history records were obtained, ophthalmologic examinations were performed, and blood samples were drawn for use in genotyping. MAIN OUTCOME MEASURES: Allele sizes of microsatellite repeat genetic markers from the vicinity of the GLC1A glaucoma gene on chromosome 1q were assigned based on size fractionation of DNA fragments generated by polymerase chain reaction (PCR). Linkage was established by the method of lod scores. Mutations were identified by determination of the DNA sequence of PCR products amplified from the trabecular meshwork inducible glucocorticoid response (TIGR) gene. Glaucoma status for purposes of linkage and mutation analysis was based on a combination of ophthalmologic examination, clinical records, family history, and previously published information. For some individuals reported in the pedigree, but not included in the genotyping studies, less information was available as presented in the text and tables. RESULTS: Autosomal-dominant POAG was confirmed or reported for 78 members of an 8-generation family. Linkage analysis showed significant evidence for linkage of juvenile-onset POAG in one branch of the family to D1S452 (maximum lod score of 6.42 at a recombination fraction of 0.00) and other markers in the vicinity of the GLC1A gene on chromosome 1q. Screening of the TIGR gene identified a mutation that results in substitution of asparagine for isoleucine at codon 477 near the carboxyterminal end of the protein. CONCLUSIONS: The authors' findings strongly suggest that the juvenile-onset POAG locus in this family is the GLC1A locus and that the underlying cause of the disease is the IIe477Asn TIGR mutation that cosegregates with juvenile-onset POAG in one branch of this large family. Lack of samples from deceased individuals prevented the authors from determining whether reported adult-onset cases in this family could also be attributed to the IIe477Asn TIGR mutation. Absence of the IIe477Asn TIGR mutation from other juvenile- and adult-onset POAG families implies that this TIGR mutation is not a common cause of glaucoma.
Asunto(s)
Proteínas del Ojo/genética , Glaucoma de Ángulo Abierto/genética , Glicoproteínas/genética , Malla Trabecular/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Cromosomas Humanos Par 1/genética , Proteínas del Citoesqueleto , ADN/análisis , Cartilla de ADN/química , Femenino , Ligamiento Genético , Genotipo , Glaucoma de Ángulo Abierto/patología , Humanos , Escala de Lod , Masculino , Persona de Mediana Edad , Linaje , Mutación Puntual/genética , Estudios RetrospectivosRESUMEN
PD 069185 is a highly selective and structurally novel inhibitor of endothelin converting enzyme-1 (ECE-1). PD 069185 is a trisubstituted quinazoline with an IC50 value of 0.9 +/- 0.1 microns for inhibition of human ECE-1 from the solubilized membrane fraction of CHO cells stably transfected with human ECE-1 cDNA. Kinetic analysis revealed that PD 069185 is best fit with a competitive inhibition model with a Ki value of 1.1 +/- 0.1 microns and binds in a reversible manner. The closely related enzyme, ECE-2, is not inhibited at up to 100 microns PD 069185. In addition, PD 069185 at 200-300 microns has little effect on other metalloproteases, such as neutral endopeptidase 24.11, stromelysin, gelatinase A, and collagenase, showing a high ECE-1 specificity. Data are also presented to show that this series of inhibitors are effective in inhibiting ECE-1 in intact cells and in attenuating the increase in perfusion pressure induced by big ET-1 in isolated rat mesentery. These non-peptidic ECE-1 inhibitors should serve as a valuable tool to study the pathophysiological role of endothelin and the therapeutic potential of ECE-1 inhibitors.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Quinazolinas/farmacología , Animales , Ácido Aspártico Endopeptidasas/genética , Células CHO , Cricetinae , Endotelina-1 , Enzimas Convertidoras de Endotelina , Endotelinas/metabolismo , Inhibidores Enzimáticos/química , Humanos , Técnicas In Vitro , Cinética , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Metaloendopeptidasas , Perfusión , Presión , Precursores de Proteínas/metabolismo , Quinazolinas/química , Ratas , Ratas Sprague-Dawley , TransfecciónRESUMEN
A series of bi-aryl pyridine carboxylic acids has been prepared and evaluated as inhibitors of ECE-1. The analogs were prepared by Pd catalyzed cross couplings of halogenated pyridines with heteroaryl organo-boranes, -tinate or -zincate derivatives.
Asunto(s)
Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores de Proteasas/síntesis química , Piridinas/síntesis química , Animales , Células CHO , Cricetinae , Enzimas Convertidoras de Endotelina , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Indicadores y Reactivos , Cinética , Metaloendopeptidasas/antagonistas & inhibidores , Paladio , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Piridinas/química , Piridinas/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Relación Estructura-Actividad , TransfecciónRESUMEN
PURPOSE: Recent reports have suggested that a gene responsible for juvenile-onset primary open-angle glaucoma exists on the long arm of chromosome 1 (1q). This report describes a previously unpublished family (UM:JG3) in which juvenile-onset glaucoma is segregating in an autosomal dominant manner. The clinical features in this family were compared with those seen in other pedigrees with this condition. Linkage analysis was performed to evaluate whether a glaucoma-causing gene in UM:JG3 is linked to genetic markers on chromosome 1q. METHODS: Affected family members, their siblings, children, and spouses were examined to identify the presence of glaucoma. Linkage studies were performed using short tandem repeat polymorphisms from chromosome 1q. Results of these studies were compared with those found for other families in which juvenile-onset primary open-angle glaucoma is linked genetically to the same chromosome 1q region. RESULTS: The UM:JG3 family includes 22 affected individuals over five generations, including 12 still living. The average age at diagnosis for living affected individuals was 26 years. An association between myopia and glaucoma was observed in this family, but the glaucoma was not associated with iris processes or other structural anomalies. The clinical course of disease and response to treatment were similar to other families with this disease. The disease phenotype in this family is linked to markers on chromosome 1q with a maximum lod score of 3.52 at a recombination fraction of 0.00 for marker D1S433. Haplotype analysis suggests the gene responsible for glaucoma in this family is located in an 8-cM region between markers D1S445 and D1S218. CONCLUSIONS: The glaucoma in UM:JG3 is linked to markers on chromosome 1q, with a candidate interval smaller than that in previous reports. In individuals with juvenile-onset open-angle glaucoma linked to chromosome 1q, the phenotype can range from mild ocular hypertension to blindness, resulting from marked elevations in intraocular pressure, with age at diagnosis ranging from 6 to 62 years. However, most affected individuals display a characteristic phenotype that includes onset in the first three decades of life, unusually high intraocular pressures, and the need for surgical therapy to prevent loss of vision. Whether differences in expression among families is due to allelic heterogeneity remains to be determined.