RESUMEN
OBJECTIVES: To determine the efficacy and safety of cyclosporine (CyA) in a large national registry-based population of patients with steroid-refractory (SR) acute severe ulcerative colitis (ASUC) and to establish predictors of efficacy and adverse events. METHODS: Multicenter study of SR-ASUC treated with CyA, based on data from the ENEIDA registry. SR-ASUC patients treated with infliximab (IFX) or sequential rescue therapy (CyA-IFX or IFX-CyA) were used as comparators. RESULTS: Of 740 SR-ASUC patients, 377 received CyA, 131 IFX and 63 sequential rescue therapy. The cumulative colectomy rate was higher in the CyA (24.1%) and sequential therapy (32.7%) than in the IFX group (14.5%; P=0.01) at 3 months and 5 years. There were no differences in early and late colectomy between CyA and IFX in patients treated after 2005. 62% of patients receiving CyA remained colectomy-free in the long term (median 71 months). There were no differences in mortality between CyA (2.4%), IFX (1.5%) and sequential therapy (0%; P=0.771). The proportion of patients with serious adverse events (SAEs) was lower in CyA (15.4%) than in IFX treated patients (26.5%) or sequential therapy (33.4%; P<0.001). This difference in favor of CyA was maintained when only patients treated after 2005 were analyzed. CONCLUSIONS: Treatment with CyA showed a lower rate of SAE and a similar efficacy to that of IFX thereby supporting the use of either CyA or IFX in SR-ASUC. In addition, the risk-benefit of sequential CyA-IFX for CyA non-responders is acceptable.
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Colitis Ulcerosa/tratamiento farmacológico , Ciclosporina/uso terapéutico , Inmunosupresores/uso terapéutico , Sistema de Registros , Enfermedad Aguda , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Niño , Colectomía/estadística & datos numéricos , Femenino , Fármacos Gastrointestinales/uso terapéutico , Humanos , Infecciones/inducido químicamente , Infliximab/uso terapéutico , Masculino , Persona de Mediana Edad , Mortalidad , Índice de Severidad de la Enfermedad , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Phenotypic traits of familial IBD relative to sporadic cases are controversial, probably related to limited statistical power of published evidence. AIM: To know if there are phenotype differences between familial and sporadic IBD, evaluating the prospective Spanish registry (ENEIDA) with 11,983 cases. METHODS: 5783 patients (48.3%) had ulcerative colitis (UC) and 6200 (51.7%) Crohn's disease (CD). Cases with one or more 1st, 2nd or 3rd degree relatives affected by UC/CD were defined as familial case. RESULTS: In UC and CD, familial cases compared with sporadic cases had an earlier disease onset (UC: 33 years [IQR 25-44] vs 37 years [IQR 27-49]; p<0.0001); (CD: 27 years [IQR 21-35] vs 29 years [IQR 22-40]; p<0.0001), higher prevalence of extraintestinal immune-related manifestations (EIMs) (UC: 17.2% vs 14%; p=0.04); (CD: 30.1% vs 23.6%; p<0.0001). Familial CD had higher percentage of ileocolic location (42.7% vs 51.8%; p=0.0001), penetrating behavior (21% vs 17.6%; p=0.01) and perianal disease (32% vs 27.1%; p=0.003). Differences are not influenced by degree of consanguinity. CONCLUSION: When a sufficiently powered cohort is evaluated, familial aggregation in IBD is associated to an earlier disease onset, more EIMs and more severe phenotype in CD. This feature should be taken into account at establishing predictors of disease course.
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Colitis Ulcerosa/genética , Colitis Ulcerosa/patología , Enfermedad de Crohn/genética , Enfermedad de Crohn/patología , Adulto , Edad de Inicio , Enfermedades del Ano/etiología , Colitis Ulcerosa/inmunología , Colon , Enfermedad de Crohn/inmunología , Femenino , Humanos , Íleon , Masculino , Fenotipo , Sistema de Registros , Índice de Severidad de la Enfermedad , España , Adulto JovenRESUMEN
AIM: The management of abdominal abscesses complicating Crohn's disease is complex and involves a difficult choice between medical, radiological and surgical procedures. The long-term outcome was compared for two strategies for the management of abdominal abscess: percutaneous drainage (PD) followed by rescue surgery in the case of failure vs direct immediate surgery (IS). We also compared the results of IS with surgery performed after PD failure. METHODS: We retrospectively identified 44 patients with Crohn's disease with an abdominal abscess from January 2000 to December 2009. Therapeutic success was defined as abscess resolution and no reappearance within 1 year of follow-up. RESULTS: The first therapeutic approach was PD in 22 cases and IS in the other 22 cases. IS had a higher therapeutic success rate than PD (95.5% vs 27.2% respectively; P < 0.001). PD was the only independent variable related to treatment failure in the multivariate analysis after adjustment for possible confounders such as abscess size, multilocularity, presence of fistula and corticosteroid use (OR 88.26, 95% CI 7.38-1055.36; P < 0.001). Surgery after failure of PD (n = 16) was associated with longer total hospitalization (56.12 ± 35.89 vs 27.52 ± 15.11 days; P = 0.017) and longer postoperative stay (44.0 ± 83.7 vs 14.3 ± 30 days; P = 0.179) and needed a second operation more often (5/16, 31% vs 1/22, 4.5%; P = 0.065) than IS. CONCLUSIONS: Percutaneous drainage provided durable abscess resolution in only one-third of the patients compared with more than 90% of those treated with IS. In addition, surgery performed after PD failure results in a poorer outcome than IS.
Asunto(s)
Absceso Abdominal/cirugía , Enfermedad de Crohn/complicaciones , Drenaje , Terapia Recuperativa , Absceso Abdominal/etiología , Adulto , Femenino , Humanos , Tiempo de Internación , Masculino , Persona de Mediana Edad , Reoperación , Estudios Retrospectivos , Factores de Tiempo , Insuficiencia del Tratamiento , Adulto JovenRESUMEN
BACKGROUND: Recently, the notion that smoking may adversely affect Crohn's disease (CD) outcomes has been challenged by the suggestion that the widespread use of immunosuppressants and anti-TNF drugs might offset the adverse effects of tobacco. AIM: To reassess the influence of tobacco smoking on disease phenotype and complications on a time-dependent analysis, taking into account the different therapeutic interventions. METHODS: We designed a retrospective cohort study of 3224 patients with Crohn's disease. The data were collected from the Spanish national inflammatory bowel disease registry (ENEIDA), including information regarding demographics, clinical characteristics, disease complications, therapeutic interventions and smoking status. Patients were classified as nonsmokers, smokers and former smokers, according to their present and past smoking habits. RESULTS: In the univariate analysis, smokers had more strictures (22.6% vs. 19.3%, P < 0.05) and less colonic involvement (7.2% vs. 10.9%, P < 0.05), and were more frequently under treatment with steroids (91.6% vs. 85.8%, P < 0.05), immunosuppressants (73.5% vs. 63.6% P < 0.05) or anti-TNF drugs (31.4% vs. 25.1%, P < 0.05) than nonsmokers. In the time-dependent multivariate analysis, smokers were found to have a significantly decreased survival free of stricturing disease (HR: 1.5, CI 95% 1.18-1.90) or perianal complications (HR: 1.50, CI 95% 1.01-1.46), and had a higher risk for requiring thiopurine therapy (HR: 1.20, CI 95% 1.05-1.30). CONCLUSION: These results suggest that, despite the widespread use of immunosuppressants and anti-TNF drugs, smokers with Crohn's disease still have a more severe disease course, with increased therapeutic requirements when compared with nonsmokers.
Asunto(s)
Enfermedad de Crohn/fisiopatología , Factores Inmunológicos/uso terapéutico , Inmunosupresores/uso terapéutico , Fumar/efectos adversos , Adulto , Antiinflamatorios/uso terapéutico , Estudios de Cohortes , Enfermedad de Crohn/tratamiento farmacológico , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sistema de Registros , Estudios Retrospectivos , Riesgo , Índice de Severidad de la Enfermedad , España , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
BACKGROUND: Adalimumab is an effective treatment for Crohn's disease (CD), but may also be associated with loss of response. Few reports provide insight into the durability of treatment of CD with adalimumab for periods longer than 12 months in clinical practice. AIMS: To evaluate the long-term durability of adalimumab maintenance treatment and to identify predictive factors associated with loss of response. METHODS: CD patients who initially responded to adalimumab were evaluated in a historical cohort study. Maintenance of long-term response was estimated using Kaplan-Meier analysis. Cox regression analysis was performed to identify potential predictive factors for loss of efficacy. RESULTS: In all, 380 CD patients were included (mean age, 38 years; 52% female). Of these, 43% had ileocolic CD, 50% inflammatory CD, and 41% perianal CD. Median follow-up with adalimumab was 8 months (range, 4-75 months). The annual risk of loss of response to adalimumab was 18% per patient-year of follow-up. Twenty-eight percent of patients were anti-TNF-naïve and 72% anti-TNF-experienced. The loss of efficacy was 8% per patient-year of follow-up in the anti-TNF-naïve patients and 22% in the anti-TNF-experienced group (P < 0.01). In the multivariate analysis, the presence of extraintestinal manifestations (hazard ratio [HR] = 1.7; 95% confidence interval [CI] = 1.02-2.9) and previous experience with other anti-TNF agents (HR = 2.5,95% CI = 1.2-5.3) were associated with higher risk of loss of efficacy. CONCLUSIONS: A relevant proportion of CD patients on long-term adalimumab lost response. The risk of loss of response was higher (more than 2-fold) in anti-TNF-experienced than in anti-TNF-naïve patients (22% vs. 8% per patient-year of treatment). Having extraintestinal manifestations seems to increase the risk of loss of efficacy.
Asunto(s)
Antiinflamatorios/uso terapéutico , Anticuerpos Monoclonales Humanizados/uso terapéutico , Enfermedad de Crohn/tratamiento farmacológico , Adalimumab , Adolescente , Adulto , Anciano , Estudios de Cohortes , Femenino , Humanos , Masculino , Persona de Mediana Edad , Resultado del Tratamiento , Adulto JovenRESUMEN
BACKGROUND AND AIMS: Increased pancreatitis associated protein (PAP) mRNA has been reported in active inflammatory bowel disease (IBD). The aims of the current study were to characterise PAP production in IBD and the effects of PAP on inflammation. PATIENTS AND METHODS: Serum PAP levels were determined in healthy controls (n = 29), inflammatory controls (n = 14), and IBD patients (n = 171). Ex vivo PAP secretion in intestinal tissue was measured in 56 IBD patients and 13 healthy controls. Cellular origin of PAP was determined by immunohistochemistry. The effects of exogenous PAP on nuclear factor kappaB (NFkappaB) activation, proinflammatory cytokine production, and endothelial adhesion molecule expression were also analysed ex vivo. RESULTS: Patients with active IBD had increased serum PAP levels compared with controls, and these levels correlated with clinical and endoscopic disease severity. Ex vivo intestinal PAP synthesis was increased in active IBD and correlated with endoscopic and histological severity of inflammatory lesions. PAP localised to colonic Paneth cells. Incubation of mucosa from active Crohn's disease with PAP dose dependently reduced proinflammatory cytokines secretion. PAP prevented TNF-alpha induced NFkappaB activation in monocytic, epithelial, and endothelial cells and reduced proinflammatory cytokine mRNA levels and adhesion molecule expression. CONCLUSIONS: PAP is synthesised by Paneth cells and is overexpressed in colonic tissue of active IBD. PAP inhibits NFkappaB activation and downregulates cytokine production and adhesion molecule expression in inflamed tissue. It may represent an anti-inflammatory mechanism and new therapeutic strategy in IBD.
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Antígenos de Neoplasias/sangre , Biomarcadores de Tumor/sangre , Enfermedades Inflamatorias del Intestino/sangre , Lectinas Tipo C/sangre , Análisis de Varianza , Antígenos de Neoplasias/farmacología , Transporte Biológico , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/farmacología , Estudios de Casos y Controles , Moléculas de Adhesión Celular/metabolismo , Núcleo Celular/metabolismo , Células Cultivadas , Colitis/sangre , Colitis/inmunología , Colitis/patología , Colitis Ulcerosa/sangre , Colitis Ulcerosa/inmunología , Colitis Ulcerosa/patología , Enfermedad de Crohn/sangre , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/patología , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , FN-kappa B/metabolismo , Proteínas Asociadas a Pancreatitis , Células de Paneth/metabolismo , ARN Mensajero/análisis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Methylcrotonylglycinuria (MCG; MIM 210200) is an autosomal recessive inherited human disorder caused by the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC, E.C.6.4.1.4), involved in leucine catabolism. This mitochondrial enzyme is one of the four biotin-dependent carboxylases known in humans. MCC is composed of two different types of subunits, alpha and beta, encoded by the nuclear genes MCCA and MCCB, respectively, recently cloned and characterized. Several mutations have been identified, in both genes, the majority are missense mutations along with splicing mutations and small insertions/deletions. We have expressed four missense mutations, two MCCA and two MCCB mapping to highly evolutionarily conserved residues, by transient transfection of SV40-transformed deficient fibroblasts in order to confirm their pathogenic effect. All the missense mutations expressed resulted in null or severely diminished MCC activity providing direct evidence that they are disease-causing ones. The MCCA mutations have been analysed in the context of three-dimensional structural information modelling the changes in the crystallized biotin carboxylase subunit of the Escherichia coli acetyl-CoA carboxylase. The apparent severity of all the MCC mutations contrasts with the variety of the clinical phenotypes suggesting that there are other cellular and metabolic unknown factors that affect the resulting phenotype.
Asunto(s)
Ligasas de Carbono-Carbono/deficiencia , Ligasas de Carbono-Carbono/genética , Expresión Génica , Mutación Missense/genética , Secuencia de Aminoácidos , Secuencia Conservada/genética , Fibroblastos , Vectores Genéticos , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de ADN , Virus 40 de los Simios , TransfecciónRESUMEN
The single-copy pahA gene from Penicillium chrysogenum encodes a phenylacetate 2-hydroxylase that catalyzes the first step of phenylacetate catabolism, an oxidative route that decreases the precursor availability for penicillin G biosynthesis. PahA protein is homologous to cytochrome P450 monooxygenases involved in the detoxification of xenobiotic compounds, with 84% identity to the Aspergillus nidulans homologue PhacA. Expression level of pahA displays an inverse correlation with the penicillin productivity of the strain and is subject to induction by phenylacetic acid. Gene expression studies have revealed a reduced oxidative activity of the protein encoded by pahA genes from penicillin-overproducing strains of P. chrysogenum compared to the activity conferred by phacA of A. nidulans. Sequencing and expression of wild-type pahA from P. chrysogenum NRRL 1951 revealed that an L181F mutation was responsible for the reduced function in present industrial strains. The mutation has been tracked down to Wisconsin 49-133, a mutant obtained at the Department of Botany of the University of Wisconsin in 1949, at the beginning of the development of the Wisconsin family of strains.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Proteínas Fúngicas , Oxigenasas de Función Mixta/genética , Penicilinas/biosíntesis , Penicillium chrysogenum/enzimología , Fenilacetatos/metabolismo , Filogenia , Aspergillus nidulans/enzimología , Aspergillus nidulans/metabolismo , Farmacorresistencia Microbiana , Regulación Fúngica de la Expresión Génica , Oxigenasas de Función Mixta/metabolismo , Datos de Secuencia Molecular , Oxidación-Reducción , Oxigenasas/genética , Oxigenasas/metabolismo , Penicillium chrysogenum/efectos de los fármacos , Penicillium chrysogenum/genética , Penicillium chrysogenum/crecimiento & desarrollo , Fenilacetatos/farmacología , Transcripción GenéticaRESUMEN
Crucial for the establishment and development of biochemical genetics as a self-standing discipline was Beadle and Tatum's choice of Neurospora crassa as experimental organism some 60 years ago. Although Garrod's insights on biochemical genetics and his astonishingly modern concepts of biochemical individuality and susceptibility to disease had been ignored by their contemporaries, Beadle acknowledged on several occasions how close Garrod had come to the "one-gene-one-enzyme" hypothesis. In an unexpected turn of events, several genes involved in human inborn errors of metabolism, including the gene for Garrod's favorite disease, alkaptonuria, have been characterized by exploitation of the experimental advantages of another mold, Aspergillus nidulans, which shares with N. crassa the experimental advantages that prompted pioneers of biochemical genetics to use them: rapid growth, facile genetic manipulation, and an environment (the composition of the growth medium) that can be manipulated à la carte.
Asunto(s)
Alcaptonuria/genética , Aspergillus nidulans/genética , Errores Innatos del Metabolismo/genética , Alcaptonuria/metabolismo , Animales , Aspergillus nidulans/enzimología , Genes Fúngicos , Humanos , Errores Innatos del Metabolismo/enzimología , Modelos Químicos , Mutación , Fenilalanina/metabolismo , Tirosina/metabolismoRESUMEN
3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.
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Errores Innatos del Metabolismo de los Aminoácidos/genética , Ligasas de Carbono-Carbono/genética , Leucina/metabolismo , Adulto , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/patología , Secuencia de Aminoácidos , Aspergillus nidulans/efectos de los fármacos , Aspergillus nidulans/genética , Aspergillus nidulans/crecimiento & desarrollo , Secuencia de Bases , Northern Blotting , Ligasas de Carbono-Carbono/metabolismo , Preescolar , Mapeo Cromosómico , Cromosomas Humanos Par 3/genética , Cromosomas Humanos Par 5/genética , ADN/química , ADN/genética , Análisis Mutacional de ADN , ADN Complementario/química , ADN Complementario/genética , Exones , Femenino , Regulación Enzimológica de la Expresión Génica , Genes/genética , Humanos , Hibridación Fluorescente in Situ , Lactante , Intrones , Isoenzimas/genética , Isoenzimas/metabolismo , Leucina/farmacología , Datos de Secuencia Molecular , Mutación , Subunidades de Proteína , ARN/genética , ARN/metabolismo , Mapeo de Híbrido por Radiación , Análisis de Secuencia de ADN , Distribución Tisular , Transcripción GenéticaRESUMEN
Alkaptonuria (AKU), the prototypic inborn error of metabolism, was the first human disease to be interpreted as a Mendelian trait by Garrod and Bateson at the beginning of last century. AKU results from impaired function of homogentisate dioxygenase (HGO), an enzyme required for the catabolism of phenylalanine and tyrosine. With the novel 7 AKU and 22 fungal mutations reported here, a total of 84 mutations impairing this enzyme have been found in the HGO gene from humans and model organisms. Forty-three of these mutations result in single amino acid substitutions. This mutational information is analysed here in the context of the HGO structure and function using kinetic assays performed using purified AKU mutant enzymes and the crystal structure of human HGO. HGO is a topologically complex structure which assembles as a functional hexamer arranged as a dimer of trimers. We show how the intricate pattern of intra- and inter-subunit interactions and the extensive surfaces required for subunit folding and association of this oligomeric enzyme can be inactivated at multiple levels by single-residue substitutions. This explains, in part, the predominance of missense mutations (67%) in AKU.
Asunto(s)
Alcaptonuria/genética , Dioxigenasas , Oxigenasas/genética , Alcaptonuria/metabolismo , Alcaptonuria/patología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Dominio Catalítico , Homogentisato 1,2-Dioxigenasa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxigenasas/química , Oxigenasas/metabolismo , Conformación Proteica , Pliegue de Proteína , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Homogentisate dioxygenase (HGO) cleaves the aromatic ring during the metabolic degradation of Phe and Tyr. HGO deficiency causes alkaptonuria (AKU), the first human disease shown to be inherited as a recessive Mendelian trait. Crystal structures of apo-HGO and HGO containing an iron ion have been determined at 1.9 and 2.3 A resolution, respectively. The HGO protomer, which contains a 280-residue N-terminal domain and a 140-residue C-terminal domain, associates as a hexamer arranged as a dimer of trimers. The active site iron ion is coordinated near the interface between subunits in the HGO trimer by a Glu and two His side chains. HGO represents a new structural class of dioxygenases. The largest group of AKU associated missense mutations affect residues located in regions of contact between subunits.
Asunto(s)
Alcaptonuria/enzimología , Dioxigenasas , Oxigenasas/química , Alcaptonuria/genética , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Dimerización , Homogentisato 1,2-Dioxigenasa , Humanos , Hierro/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxigenasas/deficiencia , Oxigenasas/genética , Oxigenasas/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
In response to alkaline ambient pH, the Aspergillus nidulans PacC transcription factor mediating pH regulation of gene expression is activated by proteolytic removal of a negative-acting C-terminal domain. We demonstrate interactions involving the approximately 150 C-terminal PacC residues and two regions located immediately downstream of the DNA binding domain. Our data indicate two full-length PacC conformations whose relative amounts depend upon ambient pH: one 'open' and accessible for processing, the other 'closed' and inaccessible. The location of essential determinants for proteolytic processing within the two more upstream interacting regions probably explains why the interactions prevent processing, whereas the direct involvement of the C-terminal region in processing-preventing interactions explains why C-terminal truncating mutations result in alkalinity mimicry and pH-independent processing. A mutant PacC deficient in pH signal response and consequent processing behaves as though locked in the 'closed' form. Single-residue substitutions, obtained as mutations bypassing the need for pH signal transduction, identify crucial residues in each of the three interactive regions and overcome the processing deficiency in the 'permanently closed' mutant.
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Proteínas Fúngicas , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Sitios de Unión/genética , Endopeptidasas/metabolismo , Regulación de la Expresión Génica , Genes Fúngicos , Concentración de Iones de Hidrógeno , Modelos Biológicos , Datos de Secuencia Molecular , Mutación Puntual , Conformación Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Eliminación de Secuencia , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , Dedos de Zinc/genéticaRESUMEN
Completing the molecular analysis of the six pal genes of the ambient pH signal transduction pathway in Aspergillus nidulans, we report the characterization of palC and palH. The derived translation product of palH contains 760 amino acids with prediction of seven transmembrane domains in its N-terminal moiety. Remarkably, a palH frameshift mutant lacking just over half the PalH protein, including almost all of the long hydrophilic region C-terminal to the transmembrane domains, retains some PalH function. The palC-derived translation product contains 507 amino acids, and the null phenotype of a frameshift mutation indicates that at least one of the C-terminal 142 residues is essential for function. Uniquely among the A. nidulans pH-signalling pal genes, palC appears to have no Saccharomyces cerevisiae homologue, although it does have a Neurospora crassa expressed sequence tag homologue. In agreement with findings for the palA, palB and palI genes of this signalling pathway, levels of the palC and palH mRNAs do not appear to be pH regulated.
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Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Mutación del Sistema de Lectura , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
Aspergillus nidulans utilizes phenylacetate as a carbon source via homogentisate, which is degraded to fumarate and acetoacetate. Mutational evidence strongly suggested that phenylacetate is converted to homogentisate through two sequential hydroxylating reactions in positions 2 and 5 of the aromatic ring. Using cDNA substraction techniques, we have characterized a gene, denoted phacA, whose transcription is strongly induced by phenylacetate and which putatively encodes a cytochrome P450 protein. A disrupted phacA strain does not grow on phenylacetate but grows on 2-hydroxy- or 2, 5-dihydroxyphenylacetate. Microsomal extracts of the disrupted strain are deficient in the NADPH-dependent conversion of phenylacetate to 2-hydroxyphenylacetate. We conclude that PhacA catalyzes the ortho-hydroxylation of phenylacetate, the first step of A. nidulans phenylacetate catabolism. The involvement of a P450 enzyme in the ortho-hydroxylation of a monoaromatic compound has no precedent. In addition, PhacA shows substantial sequence divergence with known cytochromes P450 and defines a new family of these enzymes, suggesting that saprophytic fungi may represent a source of novel cytochromes P450. Phenylacetate is a precursor for benzylpenicillin production. phacA disruption increases penicillin production 3-5-fold, indicating that catabolism competes with antibiotic biosynthesis for phenylacetate and strongly suggesting strategies for Penicillium chrysogenum strain improvement by reverse genetics.
Asunto(s)
Aspergillus nidulans/enzimología , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/fisiología , Proteínas Fúngicas , Oxigenasas/genética , Penicilinas/biosíntesis , Fenilacetatos/metabolismo , Expresión Génica , Hidroxilación , Microsomas , Datos de Secuencia Molecular , FenotipoRESUMEN
The Aspergillus nidulans transcription factor PacC, which mediates pH regulation, is proteolytically processed to a functional form in response to ambient alkaline pH. The full-length PacC form is unstable in the presence of an operational pH signal transduction pathway, due to processing to the relatively stable short functional form. We have characterized and used an extensive collection of pacC mutations, including a novel class of "neutrality-mimicking" pacC mutations having aspects of both acidity- and alkalinity-mimicking phenotypes, to investigate a number of important features of PacC processing. Analysis of mutant proteins lacking the major translation initiation residue or truncated at various distances from the C terminus showed that PacC processing does not remove N-terminal residues, indicated that processing yields slightly heterogeneous products, and delimited the most upstream processing site to residues approximately 252 to 254. Faithful processing of three mutant proteins having deletions of a region including the predicted processing site(s) and of a fourth having 55 frameshifted residues following residue 238 indicated that specificity determinants reside at sequences or structural features located upstream of residue 235. Thus, the PacC protease cuts a peptide bond(s) remote from these determinants, possibly thereby resembling type I endonucleases. Downstream of the cleavage site, residues 407 to 678 are not essential for processing, but truncation at or before residue 333 largely prevents it. Ambient pH apparently regulates the accessibility of PacC to proteolytic processing. Alkalinity-mimicking mutations L259R, L266F, and L340S favor the protease-accessible conformation, whereas a protein with residues 465 to 540 deleted retains a protease-inaccessible conformation, leading to acidity mimicry. Finally, not only does processing constitute a crucial form of modulation for PacC, but there is evidence for its conservation during fungal evolution. Transgenic expression of a truncated PacC protein, which was processed in a pH-independent manner, showed that appropriate processing can occur in Saccharomyces cerevisiae.
Asunto(s)
Aspergillus nidulans/metabolismo , Proteínas Fúngicas/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Aspergillus nidulans/genética , Sitios de Unión , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Plásmidos/genética , Procesamiento Proteico-Postraduccional , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Factores de Transcripción/química , Factores de Transcripción/genética , Transformación GenéticaRESUMEN
Penicillin production by Penicillium chrysogenum is not only commercially important but arguably the most intensively investigated secondary-metabolic pathway in fungi. Isolation of the structural genes encoding the three main penicillin-biosynthetic enzymes has stimulated the use of molecular approaches to optimize yield and permitted genetic analysis of current production strains, which are themselves the products of 50 years of strain and process improvement. Parallel studies on the penicillin-producing genetic model organism Aspergillus nidulans are now addressing questions about the genetic regulation of primary and secondary metabolism, the compartmentalization of biosynthesis and the excretion of the end products.
Asunto(s)
Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Penicilinas/biosíntesis , Penicillium chrysogenum/genética , Penicillium chrysogenum/metabolismo , Regulación Fúngica de la Expresión Génica , Mutación , Oxidación-Reducción , Especificidad de la EspecieRESUMEN
The zinc finger regions of the Aspergillus nidulans PacC transcription factor, mediating regulation of gene expression by ambient pH, and the Saccharomyces cerevisiae Rim1p transcription factor, mediating control of meiosis and invasiveness, are homologous and both transcription factors undergo proteolytic processing of the C-terminus for conversion to the functional form. In both cases, functioning of a signal transduction pathway involving several gene products is a necessary prerequisite for processing. We now show that the Aspergillus PalI pH signal transduction component is homologous to the Saccharomyces Rim9p meiotic signal transduction component throughout a region containing four hydrophobic, putative membrane-spanning segments. This suggests that PalI might be a membrane sensor for ambient pH. Deletion of the palI gene established that the less extreme phenotype of palI mutations compared with mutations in the other five genes of the pH signalling pathway is a general feature of palI mutations.
Asunto(s)
Aspergillus nidulans/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Meiosis , Proteínas de la Membrana , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Transducción de Señal , Secuencia de Aminoácidos , Aspergillus nidulans/metabolismo , Secuencia de Bases , Northern Blotting , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Proteínas Represoras , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
pmaA, an Aspergillus nidulans gene encoding a P-ATPase, has been cloned by heterologous hybridization with the yeast PMA1 gene. The putative 990-residue PmaA polypeptide shows 50% identity to Saccharomyces cerevisiae and Neurospora crassa plasma membrane H(+)-ATPases and weak (19-26%) identity to other yeast P-type cation-translocating ATPases. PmaA contains all catalytic domains characterizing H(+)-ATPases. pmaA transcript levels are not regulated by PacC, the transcription factor mediating pH regulation, and were not significantly affected by an extreme creAd mutation resulting in carbon catabolite derepression. Deletion of pmaA causes lethality, but a single copy of the gene is sufficient to support normal growth rate in pmaA hemizygous diploids, even under acidic growth conditions. As compared to other fungal H(+)-ATPases, PmaA presents three insertions, 39, 7, and 16 residues long, in the conserved central region of the protein. Two of these insertions are predicted to be located in extracellular loops and might be of diagnostic value for the identification of Aspergillus species. Their absence from most mammalian P-type ATPases may have implications for antifungal therapy.