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1.
Science ; 373(6555): 662-673, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34353949

RESUMO

The functional role of long noncoding RNAs (lncRNAs) in inherited metabolic disorders, including phenylketonuria (PKU), is unknown. Here, we demonstrate that the mouse lncRNA Pair and human HULC associate with phenylalanine hydroxylase (PAH). Pair-knockout mice exhibited excessive blood phenylalanine (Phe), musty odor, hypopigmentation, growth retardation, and progressive neurological symptoms including seizures, which faithfully models human PKU. HULC depletion led to reduced PAH enzymatic activities in human induced pluripotent stem cell-differentiated hepatocytes. Mechanistically, HULC modulated the enzymatic activities of PAH by facilitating PAH-substrate and PAH-cofactor interactions. To develop a therapeutic strategy for restoring liver lncRNAs, we designed GalNAc-tagged lncRNA mimics that exhibit liver enrichment. Treatment with GalNAc-HULC mimics reduced excessive Phe in Pair -/- and Pah R408W/R408W mice and improved the Phe tolerance of these mice.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Fenilalanina/metabolismo , Fenilcetonúrias/genética , RNA Longo não Codificante/genética , Acetilgalactosamina , Animais , Biopterina/análogos & derivados , Biopterina/metabolismo , Biopterina/uso terapêutico , Dieta , Modelos Animais de Doenças , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Knockout , Conformação de Ácido Nucleico , Fenilalanina/administração & dosagem , Fenilalanina Hidroxilase/deficiência , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/metabolismo , Ligação Proteica , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/uso terapêutico
2.
Orphanet J Rare Dis ; 16(1): 341, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344399

RESUMO

BACKGROUND: During the initial 26-week SPARK (Safety Paediatric efficAcy phaRmacokinetic with Kuvan®) study, addition of sapropterin dihydrochloride (Kuvan®; a synthetic formulation of the natural cofactor for phenylalanine hydroxylase, tetrahydrobiopterin; BH4), to a phenylalanine (Phe)-restricted diet, led to a significant improvement in Phe tolerance versus a Phe-restricted diet alone in patients aged 0-4 years with BH4-responsive phenylketonuria (PKU) or mild hyperphenylalaninaemia (HPA). Based on these results, the approved indication for sapropterin in Europe was expanded to include patients < 4 years of age. Herein, we present results of the SPARK extension study (NCT01376908), evaluating the long-term safety, dietary Phe tolerance, blood Phe concentrations and neurodevelopmental outcomes in patients < 4 years of age at randomisation, over an additional 36 months of treatment with sapropterin. RESULTS: All 51 patients who completed the 26-week SPARK study period entered the extension period. Patients who were previously treated with a Phe-restricted diet only ('sapropterin extension' group; n = 26), were initiated on sapropterin at 10 mg/kg/day, which could be increased up to 20 mg/kg/day. Patients previously treated with sapropterin plus Phe-restricted diet, remained on this regimen in the extension period ('sapropterin continuous' group; n = 25). Dietary Phe tolerance increased significantly at the end of the study versus baseline (week 0), by 38.7 mg/kg/day in the 'sapropterin continuous' group (95% CI 28.9, 48.6; p < 0.0001). In the 'sapropterin extension' group, a less pronounced effect was observed, with significant differences versus baseline (week 27) only observed between months 9 and 21; dietary Phe tolerance at the end of study increased by 5.5 mg/kg/day versus baseline (95% CI - 2.8, 13.8; p = 0.1929). Patients in both groups had normal neuromotor development and growth parameters. CONCLUSIONS: Long-term treatment with sapropterin plus a Phe-restricted diet in patients who initiated sapropterin at < 4 years of age with BH4-responsive PKU or mild HPA maintained improvements in dietary Phe tolerance over 3.5 years. These results continue to support the favourable risk/benefit profile for sapropterin in paediatric patients (< 4 years of age) with BH4-responsive PKU. Frequent monitoring of blood Phe levels and careful titration of dietary Phe intake to ensure adequate levels of protein intake is necessary to optimise the benefits of sapropterin treatment. Trial registration ClinicalTrials.gov, NCT01376908. Registered 17 June 2011, https://clinicaltrials.gov/ct2/show/NCT01376908 .


Assuntos
Fenilalanina Hidroxilase , Fenilcetonúrias , Biopterina/análogos & derivados , Biopterina/uso terapêutico , Criança , Pré-Escolar , Humanos , Fenilalanina , Fenilcetonúrias/tratamento farmacológico
3.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360752

RESUMO

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.


Assuntos
Quitosana , Enzimas Imobilizadas , Teste de Materiais , Nanopartículas/química , Fenilalanina Hidroxilase , Quitosana/química , Quitosana/farmacologia , Avaliação Pré-Clínica de Medicamentos , Estabilidade Enzimática , Enzimas Imobilizadas/química , Enzimas Imobilizadas/farmacologia , Células HEK293 , Células Hep G2 , Humanos , Fenilalanina Hidroxilase/química , Fenilalanina Hidroxilase/farmacologia
4.
Int J Mol Sci ; 22(12)2021 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207146

RESUMO

Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry.


Assuntos
Fenilalanina Hidroxilase/química , Desnaturação Proteica , Dobramento de Proteína , Sítios de Ligação , Varredura Diferencial de Calorimetria , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fenilalanina Hidroxilase/isolamento & purificação , Fenilcetonúrias , Conformação Proteica , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica , Temperatura , Termodinâmica , Ureia/química
5.
PLoS One ; 16(4): e0249608, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33822819

RESUMO

A timely detection of patients with tetrahydrobiopterin (BH4) -deficient types of hyperphenylalaninemia (HPABH4) is important for assignment of correct therapy, allowing to avoid complications. Often HPABH4 patients receive the same therapy as phenylalanine hydroxylase (PAH) -deficiency (phenylketonuria) patients-dietary treatment-and do not receive substitutive BH4 therapy until the diagnosis is confirmed by molecular genetic means. In this study, we present a cohort of 30 Russian patients with HPABH4 with detected variants in genes causing different types of HPA. Family diagnostics and biochemical urinary pterin spectrum analyses were carried out. HPABH4A is shown to be the prevalent type, 83.3% of all HPABH4 cases. The mutation spectrum for the PTS gene was defined, the most common variants in Russia were p.Thr106Met-32%, p.Asn72Lys-20%, p.Arg9His-8%, p.Ser32Gly-6%. We also detected 7 novel PTS variants and 3 novel QDPR variants. HPABH4 prevalence was estimated to be 0.5-0.9% of all HPA cases in Russia, which is significantly lower than in European countries on average, China, and Saudi Arabia. The results of this research show the necessity of introducing differential diagnostics for HPABH4 into neonatal screening practice.


Assuntos
Mutação , Fenilalanina Hidroxilase/deficiência , Fenilcetonúrias/epidemiologia , Fósforo-Oxigênio Liases/deficiência , Estudos de Casos e Controles , Humanos , Fenilcetonúrias/genética , Fenilcetonúrias/patologia , Fósforo-Oxigênio Liases/genética , Prognóstico , Estudos Retrospectivos , Federação Russa/epidemiologia
6.
Biomolecules ; 11(3)2021 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-33808760

RESUMO

Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.


Assuntos
Fenilalanina Hidroxilase/metabolismo , Quinolonas/química , Quinolonas/farmacologia , Domínio Catalítico , Espectroscopia de Ressonância de Spin Eletrônica , Fluorometria , Células HEK293 , Humanos , Doenças Metabólicas/metabolismo , Modelos Moleculares , Fenilalanina/metabolismo , Fenilcetonúrias/metabolismo , Ressonância de Plasmônio de Superfície , Tripsina
7.
Nat Commun ; 12(1): 2073, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824313

RESUMO

Phenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.


Assuntos
Amiloide/metabolismo , Fígado/enzimologia , Mutação/genética , Estresse Oxidativo , Fenilalanina Hidroxilase/genética , Agregados Proteicos , Animais , Autofagia , Biomarcadores/metabolismo , Peso Corporal , Cruzamento , Feminino , Regulação da Expressão Gênica , Genótipo , Metabolismo dos Lipídeos , Fígado/patologia , Masculino , Metaboloma , Camundongos , Proteínas Mutantes/metabolismo , Neurotransmissores/metabolismo , Estresse Oxidativo/genética , Fenilalanina/metabolismo , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/enzimologia , Pterinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Respiração , Ubiquitina/metabolismo , Ubiquitinação
8.
Mol Biol Rep ; 48(3): 2063-2070, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33677757

RESUMO

Phenylketonuria (PKU) is an autosomal recessive amino acid metabolism disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH; EC1.14.16.1). This study aimed to assess the specific heterogeneity of PAH variants found in Thai population as well as evaluate enzyme activity and expression of novel variants. PAH gene from 13 patients was analyzed by PCR amplification and direct Sanger-sequencing of 13 exons of the coding region. The novel variants were transiently transfected in COS-7 cells for functional verification. Eleven different PAH variants were identified: all pathogenic variants were missense variants, of which the most frequent variant was p.R169L, accounting for 24% (6/25) of all identified alleles. Two novel variants p.R169L and p.Y317N and previously reported variants with mutated residues at the same positions (p.R169H and p.Y317H) were expressed in COS-7 cells. These showed mildly impaired residual activity levels (42.3-63.1% of wild type), while the protein levels were well expressed (82.8-110%), except for p.R169L, which showed decreased protein expression of 55.7% compared to the wild type enzyme. All subjects with p.R169L identified in at least one of pathogenic alleles (one case is homozygous) had a metabolic phenotype of mild hyperphenylalaninemia (HPA). Our data has expanded the information on the genetic heterogeneity of Thai patients with PAH deficiency. This finding emphasizes the importance of genotyping in patients with HPA, and in vitro studies can provide additional information for prediction of phenotype.


Assuntos
Variação Genética , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/enzimologia , Fenilcetonúrias/genética , Animais , Células COS , Chlorocebus aethiops , Regulação Enzimológica da Expressão Gênica , Humanos , Mutação/genética , Fenótipo , Fenilalanina Hidroxilase/química , Tailândia
9.
Methods Mol Biol ; 2266: 239-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33759131

RESUMO

Molecular dynamics simulations can now routinely access the microsecond timescale, making feasible direct sampling of ligand association events. While Markov State Model (MSM) approaches offer a useful framework for analyzing such trajectory data to gain insight into binding mechanisms, accurate modeling of ligand association pathways and kinetics must be done carefully. We describe methods and good practices for constructing MSMs of ligand binding from unbiased trajectory data and discuss how to use time-lagged independent component analysis (tICA) to build informative models, using as an example recent simulation work to model the binding of phenylalanine to the regulatory ACT domain dimer of phenylalanine hydroxylase. We describe a variety of methods for estimating association rates from MSMs and discuss how to distinguish between conformational selection and induced-fit mechanisms using MSMs. In addition, we review some examples of MSMs constructed to elucidate the mechanisms by which p53 transactivation domain (TAD) and related peptides bind the oncoprotein MDM2.


Assuntos
Cadeias de Markov , Simulação de Dinâmica Molecular , Fenilalanina Hidroxilase/química , Fenilalanina/química , Proteínas Proto-Oncogênicas c-mdm2/química , Software , Proteína Supressora de Tumor p53/química , Cinética , Ligantes , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína
10.
Mol Genet Metab ; 133(1): 49-55, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33766497

RESUMO

BACKGROUND: In patients with phenylketonuria, stability of blood phenylalanine and tyrosine concentrations might influence brain chemistry and therefore patient outcome. This study prospectively investigated the effects of tetrahydrobiopterin (BH4), as a chaperone of phenylalanine hydroxylase on diurnal and day-to-day variations of blood phenylalanine and tyrosine concentrations. METHODS: Blood phenylalanine and tyrosine were measured in dried blood spots (DBS) four times daily for 2 days (fasting, before lunch, before dinner, evening) and once daily (fasting) for 6 days in a randomized cross-over design with a period with BH4 and a period without BH4. The sequence was randomized. Eleven proven BH4 responsive PKU patients participated, 5 of them used protein substitutes during BH4 treatment. Natural protein intake and protein substitute dosing was adjusted during the period without BH4 in order to keep DBS phenylalanine levels within target range. Patients filled out a 3-day food diary during both study periods. Variations of DBS phenylalanine and Tyr were expressed in standard deviations (SD) and coefficient of variation (CV). RESULTS: BH4 treatment did not significantly influence day-to-day phenylalanine and tyrosine variations nor diurnal phenylalanine variations, but decreased diurnal tyrosine variations (median SD 17.6 µmol/l, median CV 21.3%, p = 0.01) compared to diet only (median SD 34.2 µmol/l, median CV 43.2%). Consequently, during BH4 treatment diurnal phenylalanine/tyrosine ratio variation was smaller, while fasting tyrosine levels tended to be higher. CONCLUSION: BH4 did not impact phenylalanine variation but decreased diurnal tyrosine and phenylalanine/tyrosine ratio variations, possibly explained by less use of protein substitute and increased tyrosine synthesis.


Assuntos
Biopterina/análogos & derivados , Fenilalanina Hidroxilase/genética , Fenilalanina/sangue , Fenilcetonúrias/tratamento farmacológico , Tirosina/sangue , Adulto , Biopterina/efeitos adversos , Biopterina/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , Teste em Amostras de Sangue Seco , Feminino , Humanos , Masculino , Fenilalanina Hidroxilase/antagonistas & inibidores , Fenilcetonúrias/genética , Fenilcetonúrias/patologia
11.
Mol Genet Metab ; 132(4): 215-219, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33610470

RESUMO

BACKGROUND: A subset of patients with phenylketonuria benefit from treatment with tetrahydrobiopterin (BH4), although there is no consensus on the definition of BH4 responsiveness. The aim of this study therefore was to gain insight into the definitions of long-term BH4 responsiveness being used around the world. METHODS: We performed a web-based survey targeting healthcare professionals involved in the treatment of PKU patients. Data were analysed according to geographical region (Europe, USA/Canada, other). RESULTS: We analysed 166 responses. Long-term BH4 responsiveness was commonly defined using natural protein tolerance (95.6%), improvement of metabolic control (73.5%) and increase in quality of life (48.2%). When a specific value for a reduction in phenylalanine concentrations was reported (n = 89), 30% and 20% were most frequently used as cut-off values (76% and 19% of respondents, respectively). When a specific relative increase in natural protein tolerance was used to define long-term BH4 responsiveness (n = 71), respondents most commonly reported cut-off values of 30% and 100% (28% of respondents in both cases). Respondents from USA/Canada (n = 50) generally used less strict cut-off values compared to Europe (n = 96). Furthermore, respondents working within the same center answered differently. CONCLUSION: The results of this study suggest a very heterogeneous situation on the topic of defining long-term BH4 responsiveness, not only at a worldwide level but also within centers. Developing a strong evidence- and consensus-based definition would improve the quality of BH4 treatment.


Assuntos
Biopterina/análogos & derivados , Fenilalanina/genética , Fenilcetonúrias/tratamento farmacológico , Biopterina/efeitos adversos , Biopterina/uso terapêutico , Canadá/epidemiologia , Europa (Continente)/epidemiologia , Humanos , Fenilalanina/sangue , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/sangue , Fenilcetonúrias/epidemiologia , Fenilcetonúrias/patologia , Estados Unidos/epidemiologia
12.
Mol Genet Metab ; 132(3): 173-179, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33602601

RESUMO

Osteopenia occurs in a subset of phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) patients. While osteopenia is not fully penetrant in patients, the Pahenu2 classical PKU mouse is universally osteopenic, making it an ideal model of the phenotype. Pahenu2 Phe management, with a Phe-fee amino acid defined diet, does not improve bone density as histomorphometry metrics remain indistinguishable from untreated animals. Previously, we demonstrated Pahenu2 mesenchymal stem cells (MSCs) display impaired osteoblast differentiation. Oxidative stress is recognized in PKU patients and PKU animal models. Pahenu2 MSCs experience oxidative stress determined by intracellular superoxide over-representation. The deleterious impact of oxidative stress on mitochondria is recognized. Oximetry applied to Pahenu2 MSCs identified mitochondrial stress by increased basal respiration with concurrently reduced maximal respiration and respiratory reserve. Proton leak secondary to mitochondrial complex 1 dysfunction is a recognized superoxide source. Respirometry applied to Pahenu2 MSCs, in the course of osteoblast differentiation, identified a partial complex 1 deficit. Pahenu2 MSCs treated with the antioxidant resveratrol demonstrated increased mitochondrial mass by MitoTracker green labeling. In hyperphenylalaninemic conditions, resveratrol increased in situ alkaline phosphatase activity suggesting partial recovery of Pahenu2 MSCs osteoblast differentiation. Up-regulation of oxidative energy production is required for osteoblasts differentiation. Our data suggests impaired Pahenu2 MSC developmental competence involves an energy deficit. We posit energy support and oxidative stress reduction will enable Pahenu2 MSC differentiation in the osteoblast lineage to subsequently increase bone density.


Assuntos
Doenças Ósseas Metabólicas/genética , Estresse Oxidativo/genética , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/genética , Fosfatase Alcalina/genética , Animais , Densidade Óssea/genética , Doenças Ósseas Metabólicas/complicações , Doenças Ósseas Metabólicas/tratamento farmacológico , Doenças Ósseas Metabólicas/patologia , Diferenciação Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Fenilalanina/genética , Fenilcetonúrias/complicações , Fenilcetonúrias/tratamento farmacológico , Fenilcetonúrias/patologia , Resveratrol/farmacologia
13.
Pediatr Int ; 63(1): 8-12, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33423362

RESUMO

IMPORTANCE: Sapropterin hydrochloride, a natural coenzyme (6R-tetrahydrobiopterin) of phenylalanine hydroxylase, was first approved as a treatment for tetrahydrobiopterin deficiency in 1992 in Japan, and was then approved as a treatment for a tetrahydrobiopterin-responsive hyperphenylalaninemia in 2007 and 2008, in the USA and Japan, respectively. Guidelines are required on the proper use of sapropterin hydrochloride for tetrahydrobiopterin-responsive hyperphenylalaninemia. OBSERVATIONS: It is recommended that tetrahydrobiopterin-responsive hyperphenylalaninemia should be diagnosed in all cases of hyperphenylalaninemia, including phenylketonuria, by tetrahydrobiopterin administration tests rather than by phenotype or blood phenylalanine levels. CONCLUSIONS AND RELEVANCE: If tetrahydrobiopterin-responsive hyperphenylalaninemia is diagnosed, all ages can be treated with sapropterin hydrochloride. Although there are reports that sapropterin hydrochloride is effective and safe for the prevention of maternal phenylketonuria, further investigation is required.


Assuntos
Biopterina/análogos & derivados , Fenilcetonúrias , Biopterina/uso terapêutico , Feminino , Humanos , Japão , Fenótipo , Fenilalanina , Fenilalanina Hidroxilase , Fenilcetonúria Materna/prevenção & controle , Fenilcetonúrias/diagnóstico , Fenilcetonúrias/terapia , Gravidez
15.
Orphanet J Rare Dis ; 16(1): 35, 2021 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-33461585

RESUMO

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive inherited disorder characterised by a deficiency in phenylalanine hydroxylase. Untreated, PKU is associated with a wide range of cognitive and psychiatric sequelae. Contemporary management guidelines recommend lifetime dietary control of phenylalanine (Phe) levels, however many individuals who discontinue dietary control subsequently suffer symptoms of anxiety, depression and disturbances to cognition. We undertook a prospective cohort study of patients with early-treated phenylketonuria who had ceased dietary control to test the hypothesis that resumption of dietary control of PKU is associated with improvements in measures of psychiatric morbidity and cognitive functioning. METHODS: We re-initiated dietary control for early-treated patients with PKU and monitored cognitive and psychiatric outcomes over a twelve-month period. Assessments included objective cognitive function (measured by cognitive proficiency index (CPI)), anxiety and depression scales. General linear mixed model (GLMM) analyses were performed to assess change in psychometric variables from baseline over twelve months after resumption of dietary control. RESULTS: A total of nine patients were recruited. Mean age was 33 years (SD = 8.75), five were female. Mean time off dietary control was 19.1 years (SD = 11.3), and mean baseline phenylalanine (Phe) levels were 1108 µmol/L (SD = 293). GLMM analysis demonstrated a positive relationship between CPI and time on diet (b = 0.56 [95% CI = 0.17, 0.95]). Age, time off diet, Phe levels and depression scores were not associated with cognitive function. There was a negative relationship between time on diet and anxiety (b = - 0.88 95% CI = [- 1.26, - 0.50]) and depression ratings (b = - 0.61, 95% CI = [- 0.95, - 0.26]). CONCLUSIONS: This study demonstrated improvements in cognitive function, anxiety, and depression ratings associated with resumption of dietary control of PKU. Raw Phe levels were not strongly associated with psychiatric or cognitive scores in this cohort. These findings support the importance of lifelong treatment for PKU in improving the cognitive and psychiatric sequelae of the disease.


Assuntos
Fenilalanina Hidroxilase , Fenilcetonúrias , Adulto , Cognição , Feminino , Humanos , Masculino , Fenilalanina , Fenilcetonúrias/tratamento farmacológico , Estudos Prospectivos
16.
PLoS One ; 16(1): e0245831, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33493163

RESUMO

Phenylketonuria (PKU) is a metabolic disorder whereby phenylalanine metabolism is deficient due to allelic variations in the gene for phenylalanine hydroxylase (PAH). There is no cure for PKU other than orthotopic liver transplantation, and the standard of care for patients is limited to dietary restrictions and key amino acid supplementation. Therefore, Pah was edited in pig fibroblasts for the generation of PKU clone piglets that harbor a common and severe human mutation, R408W. Additionally, the proximal region to the mutation was further humanized by introducing 5 single nucleotide polymorphisms (SNPs) to allow for development of gene editing machinery that could be translated directly from the pig model to human PKU patients that harbor at least one classic R408W allele. Resulting piglets were hypopigmented (a single Ossabaw piglet) and had low birthweight (all piglets). The piglets had similar levels of PAH expression, but no detectable enzymatic activity, consistent with the human phenotype. The piglets were fragile and required extensive neonatal care to prevent failure to thrive and early demise. Phenylalanine levels rose sharply when dietary Phe was unrestricted but could be rapidly reduced with a low Phe diet. Fibroblasts isolated from R408W piglets show susceptibility to correction using CRISPR or TALEN, with subsequent homology-directed recombination to correct Pah. This pig model of PKU provides a powerful new tool for development of all classes of therapeutic candidates to treat or cure PKU, as well as unique value for proof-of-concept studies for in vivo human gene editing platforms in the context of this humanized PKU allele.


Assuntos
Edição de Genes/métodos , Mutação , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/genética , Animais , Sequência de Bases , Modelos Animais de Doenças , Humanos , Fenótipo , Segurança , Suínos
17.
Biochimie ; 183: 63-77, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33221376

RESUMO

Phenylalanine hydroxylase (PAH) is an allosteric enzyme that maintains phenylalanine (Phe) below neurotoxic levels; its failure results in phenylketonuria, an inborn error of amino acid metabolism. Wild type (WT) PAH equilibrates among resting-state (RS-PAH) and activated (A-PAH) conformations, whose equilibrium position depends upon allosteric Phe binding. The RS-PAH conformation of WT rat PAH (rPAH) contains a cation-π sandwich involving Phe80 that cannot exist in the A-PAH conformation. Phe80 variants F80A, F80D, F80L, and F80R were prepared and evaluated using native PAGE, size exclusion chromatography, ion exchange behavior, intrinsic protein fluorescence, enzyme kinetics, and limited proteolysis, each as a function of [Phe]. Like WT rPAH, F80A and F80D show allosteric activation by Phe while F80L and F80R are constitutively active. Maximal activity of all variants suggests relief of a rate-determining conformational change. Limited proteolysis of WT rPAH (minus Phe) reveals facile cleavage within a 4-helix bundle that is buried in the RS-PAH tetramer interface, reflecting dynamic dissociation of that tetramer. This cleavage is not seen for the Phe80 variants, which all show proteolytic hypersensitivity in a linker that repositions during the RS-PAH to A-PAH interchange. Hypersensitivity is corrected by addition of Phe such that all variants become like WT rPAH and achieve the A-PAH conformation. Thus, manipulation of Phe80 perturbs the conformational space sampled by PAH, increasing sampling of on-pathway intermediates in the RS-PAH and A-PAH interchange. The behavior of the Phe80 variants mimics that of disease-associated R68S and suggests a molecular basis for proteolytic susceptibility in PKU-associated human PAH variants.


Assuntos
Mutação de Sentido Incorreto , Fenilalanina Hidroxilase/química , Multimerização Proteica , Substituição de Aminoácidos , Animais , Estabilidade Enzimática , Humanos , Fenilalanina Hidroxilase/genética , Fenilalanina Hidroxilase/metabolismo , Fenilcetonúrias/enzimologia , Fenilcetonúrias/genética , Conformação Proteica em alfa-Hélice , Estrutura Quaternária de Proteína , Ratos
18.
Int J Mol Sci ; 21(23)2020 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-33260674

RESUMO

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by the dysfunction of the enzyme phenylalanine hydroxylase (PAH). Alterations in the level of PAH leads to the toxic accumulation of phenylalanine in the blood and brain. Protein degradation mediated by ubiquitination is a principal cellular process for maintaining protein homeostasis. Therefore, it is important to identify the E3 ligases responsible for PAH turnover and proteostasis. Here, we report that anaphase-promoting complex/cyclosome-Cdh1 (APC/C)Cdh1 is an E3 ubiquitin ligase complex that interacts and promotes the polyubiquitination of PAH through the 26S proteasomal pathway. Cdh1 destabilizes and declines the half-life of PAH. In contrast, the CRISPR/Cas9-mediated knockout of Cdh1 stabilizes PAH expression and enhances phenylalanine metabolism. Additionally, our current study demonstrates the clinical relevance of PAH and Cdh1 correlation in hepatocellular carcinoma (HCC). Overall, we show that PAH is a prognostic marker for HCC and Cdh1 could be a potential therapeutic target to regulate PAH-mediated physiological and metabolic disorders.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Fenilalanina Hidroxilase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Estabilidade Enzimática , Células HEK293 , Meia-Vida , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Fenilalanina/metabolismo , Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Ubiquitinação
19.
Genes (Basel) ; 12(1)2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-33375644

RESUMO

Phenylketonuria (PKU) is a common inborn error of amino acid metabolism in which the enzyme phenylalanine hydroxylase, which converts phenylalanine to tyrosine, is functionally impaired due to pathogenic variants in the PAH gene. Thirty-four Brazilian patients with a biochemical diagnosis of PKU, from 33 unrelated families, were analyzed through next-generation sequencing in the Ion Torrent PGM™ platform. Phenotype-genotype correlations were made based on the BioPKU database. Three patients required additional Sanger sequencing analyses. Twenty-six different pathogenic variants were identified. The most frequent variants were c.1315+1G>A (n = 8/66), c.473G>A (n = 6/66), and c.1162G>A (n = 6/66). One novel variant, c.524C>G (p.Pro175Arg), was found in one allele and was predicted as likely pathogenic by the American College of Medical Genetics and Genomics (ACMG) criteria. The molecular modeling of p.Pro175Arg indicated that this substitution can affect monomers binding in the PAH tetramer, which could lead to a change in the stability and activity of this enzyme. Next-generation sequencing was a fast and effective method for diagnosing PKU and is useful for patient phenotype prediction and genetic counseling.


Assuntos
Testes Genéticos/métodos , Fenilalanina Hidroxilase/genética , Fenilcetonúrias/diagnóstico , Brasil , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Aconselhamento Genético/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Masculino , Modelos Moleculares , Mutação , Fenilcetonúrias/genética , Estrutura Terciária de Proteína/genética
20.
JCI Insight ; 5(20)2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-33055427

RESUMO

Phenylalanine hydroxylase-deficient (PAH-deficient) phenylketonuria (PKU) results in systemic hyperphenylalaninemia, leading to neurotoxicity with severe developmental disabilities. Dietary phenylalanine (Phe) restriction prevents the most deleterious effects of hyperphenylalaninemia, but adherence to diet is poor in adult and adolescent patients, resulting in characteristic neurobehavioral phenotypes. Thus, an urgent need exists for new treatments. Additionally, rodent models of PKU do not adequately reflect neurocognitive phenotypes, and thus there is a need for improved animal models. To this end, we have developed PAH-null pigs. After selection of optimal CRISPR/Cas9 genome-editing reagents by using an in vitro cell model, zygote injection of 2 sgRNAs and Cas9 mRNA demonstrated deletions in preimplantation embryos, with embryo transfer to a surrogate leading to 2 founder animals. One pig was heterozygous for a PAH exon 6 deletion allele, while the other was compound heterozygous for deletions of exon 6 and of exons 6-7. The affected pig exhibited hyperphenylalaninemia (2000-5000 µM) that was treatable by dietary Phe restriction, consistent with classical PKU, along with juvenile growth retardation, hypopigmentation, ventriculomegaly, and decreased brain gray matter volume. In conclusion, we have established a large-animal preclinical model of PKU to investigate pathophysiology and to assess new therapeutic interventions.


Assuntos
Fígado/metabolismo , Fenilalanina Hidroxilase/genética , Fenilalanina/genética , Fenilcetonúrias/genética , Adolescente , Adulto , Animais , Sistemas CRISPR-Cas/genética , Dieta , Modelos Animais de Doenças , Edição de Genes , Humanos , Fígado/efeitos dos fármacos , Fenótipo , Fenilalanina/metabolismo , Fenilalanina/farmacologia , Fenilcetonúrias/dietoterapia , Fenilcetonúrias/metabolismo , Fenilcetonúrias/patologia , Suínos
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