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1.
J Clin Immunol ; 43(1): 46-56, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36121535

RESUMEN

Almost 2 years into the pandemic and with vaccination of children significantly lagging behind adults, long-term pediatric humoral immune responses to SARS-CoV-2 are understudied. The C19.CHILD Hamburg (COVID-19 Child Health Investigation of Latent Disease) Study is a prospective cohort study designed to identify and follow up children and their household contacts infected in the early 2020 first wave of SARS-CoV-2. We screened 6113 children < 18 years by nasopharyngeal swab-PCR in a low-incidence setting after general lockdown, from May 11 to June 30, 2020. A total of 4657 participants underwent antibody testing. Positive tests were followed up by repeated PCR and serological testing of all household contacts over 6 months. In total, the study identified 67 seropositive children (1.44%); the median time after infection at first presentation was 83 days post-symptom onset (PSO). Follow-up of household contacts showed less than 100% seroprevalence in most families, with higher seroprevalence in families with adult index cases compared to pediatric index cases (OR 1.79, P = 0.047). Most importantly, children showed sustained seroconversion up to 9 months PSO, and serum antibody concentrations persistently surpassed adult levels (ratio serum IgG spike children vs. adults 90 days PSO 1.75, P < 0.001; 180 days 1.38, P = 0.01; 270 days 1.54, P = 0.001). In a low-incidence setting, SARS-CoV-2 infection and humoral immune response present distinct patterns in children including higher antibody levels, and lower seroprevalence in families with pediatric index cases. Children show long-term SARS-CoV-2 antibody responses. These findings are relevant to novel variants with increased disease burden in children, as well as for the planning of age-appropriate vaccination strategies.


Asunto(s)
Formación de Anticuerpos , COVID-19 , Adulto , Humanos , Niño , SARS-CoV-2 , COVID-19/diagnóstico , COVID-19/epidemiología , Estudios Prospectivos , Estudios Seroepidemiológicos , Control de Enfermedades Transmisibles , Anticuerpos Antivirales
2.
Int J Mol Sci ; 24(17)2023 Aug 24.
Artículo en Inglés | MEDLINE | ID: mdl-37685964

RESUMEN

Glutaric acidemia type 1 (GA1) is a neurotoxic metabolic disorder due to glutaryl-CoA dehydrogenase (GCDH) deficiency. The high number of missense variants associated with the disease and their impact on GCDH activity suggest that disturbed protein conformation can affect the biochemical phenotype. We aimed to elucidate the molecular basis of protein loss of function in GA1 by performing a parallel analysis in a large panel of GCDH missense variants using different biochemical and biophysical methodologies. Thirteen GCDH variants were investigated in regard to protein stability, hydrophobicity, oligomerization, aggregation, and activity. An altered oligomerization, loss of protein stability and solubility, as well as an augmented susceptibility to aggregation were observed. GA1 variants led to a loss of enzymatic activity, particularly when present at the N-terminal domain. The reduced cellular activity was associated with loss of tetramerization. Our results also suggest a correlation between variant sequence location and cellular protein stability (p < 0.05), with a more pronounced loss of protein observed with variant proximity to the N-terminus. The broad panel of variant-mediated conformational changes of the GCDH protein supports the classification of GA1 as a protein-misfolding disorder. This work supports research toward new therapeutic strategies that target this molecular disease phenotype.


Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos , Encefalopatías Metabólicas , Glutaril-CoA Deshidrogenasa , Glutaril-CoA Deshidrogenasa/química , Glutaril-CoA Deshidrogenasa/genética , Errores Innatos del Metabolismo de los Aminoácidos/enzimología , Errores Innatos del Metabolismo de los Aminoácidos/genética , Encefalopatías Metabólicas/enzimología , Encefalopatías Metabólicas/genética , Pliegue de Proteína , Mutación Missense , Dominios Proteicos , Humanos , Estabilidad de Enzimas , Solubilidad
3.
Hum Mol Genet ; 27(10): 1732-1742, 2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29514280

RESUMEN

Metabolic control of phenylalanine concentrations in body fluids is essential for cognitive development and executive function. The hepatic phenylalanine hydroxylating system is regulated by the ratio of l-phenylalanine, which is substrate of phenylalanine hydroxylase (PAH), to the PAH cofactor tetrahydrobiopterin (BH4). Physiologically, phenylalanine availability is governed by nutrient intake, whereas liver BH4 is kept at constant level. In phenylketonuria, PAH deficiency leads to elevated blood phenylalanine and is often caused by PAH protein misfolding with loss of function. Here, we report secondary hepatic BH4 deficiency in Pah-deficient mice. Alterations in de novo synthesis and turnover of BH4 were ruled out as molecular causes. We demonstrate that kinetically instable and aggregation-prone variant Pah proteins trap BH4, shifting the pool of free BH4 towards bound BH4. Interference of PAH protein misfolding with metabolite-based control of l-phenylalanine turnover suggests a mechanistic link between perturbation of protein homeostasis and disturbed regulation of metabolic pathways.


Asunto(s)
Biopterinas/análogos & derivados , Fenilalanina Hidroxilasa/genética , Fenilalanina/metabolismo , Fenilcetonurias/genética , Animales , Biopterinas/química , Biopterinas/genética , Biopterinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Inactivación Metabólica/genética , Cinética , Hígado/enzimología , Ratones , Fenilalanina/química , Fenilalanina/genética , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/metabolismo , Fenilcetonurias/metabolismo , Fenilcetonurias/patología , Pliegue de Proteína , Proteostasis/genética
4.
J Med Genet ; 52(3): 175-85, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25596310

RESUMEN

BACKGROUND: In phenylketonuria, genetic heterogeneity, frequent compound heterozygosity, and the lack of functional data for phenylalanine hydroxylase genotypes hamper reliable phenotype prediction and individualised treatment. METHODS: A literature search revealed 690 different phenylalanine hydroxylase genotypes in 3066 phenylketonuria patients from Europe and the Middle East. We determined phenylalanine hydroxylase function of 30 frequent homozygous and compound heterozygous genotypes covering 55% of the study population, generated activity landscapes, and assessed the phenylalanine hydroxylase working range in the metabolic (phenylalanine) and therapeutic (tetrahydrobiopterin) space. RESULTS: Shared patterns in genotype-specific functional landscapes were linked to biochemical and pharmacological phenotypes, where (1) residual activity below 3.5% was associated with classical phenylketonuria unresponsive to pharmacological treatment; (2) lack of defined peak activity induced loss of response to tetrahydrobiopterin; (3) a higher cofactor need was linked to inconsistent clinical phenotypes and low rates of tetrahydrobiopterin response; and (4) residual activity above 5%, a defined peak of activity, and a normal cofactor need were associated with pharmacologically treatable mild phenotypes. In addition, we provide a web application for retrieving country-specific information on genotypes and genotype-specific phenylalanine hydroxylase function that warrants continuous extension, updates, and research on demand. CONCLUSIONS: The combination of genotype-specific functional analyses with biochemical, clinical, and therapeutic data of individual patients may serve as a powerful tool to enable phenotype prediction and to establish personalised medicine strategies for dietary regimens and pharmacological treatment in phenylketonuria.


Asunto(s)
Estudios de Asociación Genética , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética , Medicina de Precisión , Europa (Continente) , Genotipo , Humanos , Medio Oriente , Mutación , Fenilcetonurias/fisiopatología
5.
Hum Mol Genet ; 20(13): 2628-41, 2011 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-21527427

RESUMEN

The discovery of a pharmacological treatment for phenylketonuria (PKU) raised new questions about function and dysfunction of phenylalanine hydroxylase (PAH), the enzyme deficient in this disease. To investigate the interdependence of the genotype, the metabolic state (phenylalanine substrate) and treatment (BH(4) cofactor) in the context of enzyme function in vitro and in vivo, we (i) used a fluorescence-based method for fast enzyme kinetic analyses at an expanded range of phenylalanine and BH(4) concentrations, (ii) depicted PAH function as activity landscapes, (iii) retraced the analyses in eukaryotic cells, and (iv) translated this into the human system by analyzing the outcome of oral BH(4) loading tests. PAH activity landscapes uncovered the optimal working range of recombinant wild-type PAH and provided new insights into PAH kinetics. They demonstrated how mutations might alter enzyme function in the space of varying substrate and cofactor concentrations. Experiments in eukaryotic cells revealed that the availability of the active PAH enzyme depends on the phenylalanine-to-BH(4) ratio. Finally, evaluation of data from BH(4) loading tests indicated that the patient's genotype influences the impact of the metabolic state on drug response. The results allowed for visualization and a better understanding of PAH function in the physiological and pathological state as well as in the therapeutic context of cofactor treatment. Moreover, our data underscore the need for more personalized procedures to safely identify and treat patients with BH(4)-responsive PAH deficiency.


Asunto(s)
Biopterinas/análogos & derivados , Coenzimas/uso terapéutico , Genotipo , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Fenilalanina/metabolismo , Fenilcetonurias , Biopterinas/farmacología , Biopterinas/uso terapéutico , Coenzimas/farmacología , Activación Enzimática/efectos de los fármacos , Células HEK293 , Humanos , Cinética , Chaperonas Moleculares/metabolismo , Mutación/genética , Fenilalanina Hidroxilasa/deficiencia , Fenilcetonurias/tratamiento farmacológico , Fenilcetonurias/enzimología , Fenilcetonurias/genética
6.
Hum Mol Genet ; 19(10): 2039-49, 2010 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-20179079

RESUMEN

The recent approval of sapropterin dihydrochloride, the synthetic form of 6[R]-l-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)), for the treatment of phenylketonuria (PKU) as the first pharmacological chaperone drug initiated a paradigm change in the treatment of monogenetic diseases. Symptomatic treatment is now replaced by a causal pharmacological therapy correcting misfolding of the defective phenylalanine hydroxylase (PAH) in numerous patients. Here, we disclose BH(4) responsiveness in Pah(enu1), a mouse model for PAH deficiency. Loss of function resulted from loss of PAH, a consequence of misfolding, aggregation, and accelerated degradation of the enzyme. BH(4) attenuated this triad by conformational stabilization augmenting the effective PAH concentration. This led to the rescue of the biochemical phenotype and enzyme function in vivo. Combined in vitro and in vivo analyses revealed a selective pharmaceutical action of BH(4) confined to the pathological metabolic state. Our data provide new molecular-level insights into the mechanisms underlying protein misfolding with loss of function and support a general model of pharmacological chaperone-induced stabilization of protein conformation to correct this intracellular phenotype. Pah(enu1) will be essential for pharmaceutical drug optimization and to design individually tailored therapies.


Asunto(s)
Biopterinas/análogos & derivados , Modelos Animales de Enfermedad , Chaperonas Moleculares/metabolismo , Fenilalanina Hidroxilasa/deficiencia , Sustitución de Aminoácidos/genética , Animales , Biopterinas/farmacología , Células COS , Chlorocebus aethiops , Humanos , Hidroxilación/efectos de los fármacos , Cinética , Ratones , Mutación/genética , Fenilalanina/metabolismo , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/metabolismo , Pliegue de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estructura Cuaternaria de Proteína
7.
Front Immunol ; 13: 867577, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35911689

RESUMEN

SARS-CoV-2 is still a major burden for global health despite effective vaccines. With the reduction of social distancing measures, infection rates are increasing in children, while data on the pediatric immune response to SARS-CoV-2 infection is still lacking. Although the typical disease course in children has been mild, emerging variants may present new challenges in this age group. Peripheral blood mononuclear cells (PBMC) from 51 convalescent children, 24 seronegative siblings from early 2020, and 51 unexposed controls were stimulated with SARS-CoV-2-derived peptide MegaPools from the ancestral and beta variants. Flow cytometric determination of activation-induced markers and secreted cytokines were used to quantify the CD4+ T cell response. The average time after infection was over 80 days. CD4+ T cell responses were detected in 61% of convalescent children and were markedly reduced in preschool children. Cross-reactive T cells for the SARS-CoV-2 beta variant were identified in 45% of cases after infection with an ancestral SARS-CoV-2 variant. The CD4+ T cell response was accompanied most predominantly by IFN-γ and Granzyme B secretion. An antiviral CD4+ T cell response was present in children after ancestral SARS-CoV-2 infection, which was reduced in the youngest age group. We detected significant cross-reactivity of CD4+ T cell responses to the more recently evolved immune-escaping beta variant. Our findings have epidemiologic relevance for children regarding novel viral variants of concern and vaccination efforts.


Asunto(s)
COVID-19 , SARS-CoV-2 , Linfocitos T CD4-Positivos , Niño , Preescolar , Humanos , Leucocitos Mononucleares
8.
J Biol Chem ; 285(40): 30686-97, 2010 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-20667834

RESUMEN

Protein misfolding with loss-of-function of the enzyme phenylalanine hydroxylase (PAH) is the molecular basis of phenylketonuria in many individuals carrying missense mutations in the PAH gene. PAH is complexly regulated by its substrate L-Phenylalanine and its natural cofactor 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)). Sapropterin dihydrochloride, the synthetic form of BH(4), was recently approved as the first pharmacological chaperone to correct the loss-of-function phenotype. However, current knowledge about enzyme function and regulation in the therapeutic setting is scarce. This illustrates the need for comprehensive analyses of steady state kinetics and allostery beyond single residual enzyme activity determinations to retrace the structural impact of missense mutations on the phenylalanine hydroxylating system. Current standard PAH activity assays are either indirect (NADH) or discontinuous due to substrate and product separation before detection. We developed an automated fluorescence-based continuous real-time PAH activity assay that proved to be faster and more efficient but as precise and accurate as standard methods. Wild-type PAH kinetic analyses using the new assay revealed cooperativity of activated PAH toward BH(4), a previously unknown finding. Analyses of structurally preactivated variants substantiated BH(4)-dependent cooperativity of the activated enzyme that does not rely on the presence of l-Phenylalanine but is determined by activating conformational rearrangements. These findings may have implications for an individualized therapy, as they support the hypothesis that the patient's metabolic state has a more significant effect on the interplay of the drug and the conformation and function of the target protein than currently appreciated.


Asunto(s)
Biopterinas/análogos & derivados , Coenzimas/química , Fenilalanina Hidroxilasa/química , Fenilalanina/química , Regulación Alostérica/genética , Biopterinas/química , Biopterinas/metabolismo , Biopterinas/uso terapéutico , Coenzimas/metabolismo , Coenzimas/uso terapéutico , Activación Enzimática/genética , Fluorescencia , Humanos , Cinética , Mutación Missense , Fenilalanina/genética , Fenilalanina/metabolismo , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Fenilcetonurias/tratamiento farmacológico , Fenilcetonurias/enzimología , Fenilcetonurias/genética
9.
Am J Hum Genet ; 83(1): 5-17, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18538294

RESUMEN

A significant share of patients with phenylalanine hydroxylase (PAH) deficiency benefits from pharmacological doses of tetrahydrobiopterin (BH(4)), the natural PAH cofactor. Phenylketonuria (PKU) is hypothesized to be a conformational disease, with loss of function due to protein destabilization, and the restoration of enzyme function that is observed in BH(4) treatment might be transmitted by correction of protein misfolding. To elucidate the molecular basis of functional impairment in PAH deficiency, we investigated the impact of ten PAH gene mutations identified in patients with BH(4)-responsiveness on enzyme kinetics, stability, and conformation of the protein (F55L, I65S, H170Q, P275L, A300S, S310Y, P314S, R408W, Y414C, Y417H). Residual enzyme activity was generally high, but allostery was disturbed in almost all cases and pointed to altered protein conformation. This was confirmed by reduced proteolytic stability, impaired tetramer assembly or aggregation, increased hydrophobicity, and accelerated thermal unfolding--with particular impact on the regulatory domain--observed in most variants. Three-dimensional modeling revealed the involvement of functionally relevant amino acid networks that may communicate misfolding throughout the protein. Our results substantiate the view that PAH deficiency is a protein-misfolding disease in which global conformational changes hinder molecular motions essential for physiological enzyme function. Thus, PKU has evolved from a model of a genetic disease that leads to severe neurological impairment to a model of a treatable protein-folding disease with loss of function.


Asunto(s)
Movimiento (Física) , Fenilalanina Hidroxilasa/deficiencia , Fenilalanina Hidroxilasa/metabolismo , Fenilcetonurias/enzimología , Fenilcetonurias/genética , Administración Oral , Regulación Alostérica , Errores Innatos del Metabolismo de los Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Biopterinas/administración & dosificación , Biopterinas/análogos & derivados , Biopterinas/uso terapéutico , Dominio Catalítico , Simulación por Computador , Dimerización , Endopeptidasa K/farmacología , Estabilidad de Enzimas , Femenino , Calor , Humanos , Enlace de Hidrógeno , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Recién Nacido , Cinética , Luminiscencia , Masculino , Modelos Moleculares , Mutación Missense , Fenilalanina/sangre , Fenilalanina/metabolismo , Fenilalanina Hidroxilasa/análisis , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/genética , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína/genética , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Electricidad Estática
10.
Biochim Biophys Acta Mol Cell Res ; 1866(3): 518-531, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30366024

RESUMEN

Peroxisomal biogenesis factor PEX26 is a membrane anchor for the multi-subunit PEX1-PEX6 protein complex that controls ubiquitination and dislocation of PEX5 cargo receptors for peroxisomal matrix protein import. PEX26 associates with the peroxisomal translocation pore via PEX14 and a splice variant (PEX26Δex5) of unknown function has been reported. Here, we demonstrate PEX26 homooligomerization mediated by two heptad repeat domains adjacent to the transmembrane domain. We show that isoform-specific domain organization determines PEX26 oligomerization and impacts peroxisomal ß-oxidation and proliferation. PEX26 and PEX26Δex5 displayed different patterns of interaction with PEX2-PEX10 or PEX13-PEX14 complexes, which relate to distinct pre-peroxisomes in the de novo synthesis pathway. Our data support an alternative PEX14-dependent mechanism of peroxisomal membrane association for the splice variant, which lacks a transmembrane domain. Structure-function relationships of PEX26 isoforms explain an extended function in peroxisomal homeostasis and these findings may improve our understanding of the broad phenotype of PEX26-associated human disorders.


Asunto(s)
Proteínas de la Membrana/metabolismo , Peroxisomas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Animales , Transferencia de Energía por Resonancia de Bioluminiscencia/métodos , Células COS , Chlorocebus aethiops , Fibroblastos/metabolismo , Células HEK293 , Humanos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/biosíntesis , Oxidación-Reducción , Receptor de la Señal 1 de Direccionamiento al Peroxisoma/metabolismo , Isoformas de Proteínas , Transporte de Proteínas
11.
Biochem Pharmacol ; 80(10): 1563-71, 2010 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-20705059

RESUMEN

Phenylketonuria (PKU), an autosomal recessive disease with phenylalanine hydroxylase (PAH) deficiency, was recently shown to be a protein misfolding disease with loss-of-function. It can be treated by oral application of the natural PAH cofactor tetrahydrobiopterin (BH(4)) that acts as a pharmacological chaperone and rescues enzyme function in vivo. Here we identified Pah(enu1/2) bearing a mild and a severe mutation (V106A/F363S) as a new mouse model for compound heterozygous mild PKU. Although BH(4) treatment has become established in clinical routine, there is substantial lack of knowledge with regard to BH(4) pharmacodynamics and the effect of the genotype on the response to treatment with the natural cofactor. To address these questions we applied an elaborate methodological setup analyzing: (i) blood phenylalanine elimination, (ii) blood phenylalanine/tyrosine ratios, and (iii) kinetics of in vivo phenylalanine oxidation using (13)C-phenylalanine breath tests. We compared pharmacodynamics in wild-type, Pah(enu1/1), and Pah(enu1/2) mice and observed crucial differences in terms of effect size as well as effect kinetics and dose response. Results from in vivo experiments were substantiated in vitro after overexpression of wild-type, V106A, and F263S in COS-7 cells. Pharmacokinetics did not differ between Pah(enu1/1) and Pah(enu1/2) indicating that the differences in pharmacodynamics were not induced by divergent pharmacokinetic behavior of BH(4). In conclusion, our findings show a significant impact of the genotype on the response to BH(4) in PAH deficient mice. This may lead to important consequences concerning the diagnostic and therapeutic management of patients with PAH deficiency underscoring the need for individualized procedures addressing pharmacodynamic aspects.


Asunto(s)
Biopterinas/análogos & derivados , Heterocigoto , Fenilalanina Hidroxilasa/deficiencia , Fenilcetonurias/tratamiento farmacológico , Animales , Biopterinas/farmacología , Biopterinas/uso terapéutico , Pruebas Respiratorias , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ratones , Ratones Mutantes , Mutación , Fenilalanina/sangre , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/sangre , Fenilcetonurias/enzimología , Fenilcetonurias/genética , Resultado del Tratamiento , Tirosina/sangre
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