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1.
Eur J Immunol ; : e2350716, 2024 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-38837757

RESUMEN

Immune mediators affect multiple biological functions of intestinal epithelial cells (IECs) and, like Paneth and Paneth-like cells, play an important role in intestinal epithelial homeostasis. IFN-γ a prototypical proinflammatory cytokine disrupts intestinal epithelial homeostasis. However, the mechanism underlying the process remains unknown. In this study, using in vivo and in vitro models we demonstrate that IFN-γ is spontaneously secreted in the small intestine. Furthermore, we observed that this cytokine stimulates mitochondrial activity, ROS production, and Paneth and Paneth-like cell secretion. Paneth and Paneth-like secretion downstream of IFN-γ, as identified here, is mTORC1 and necroptosis-dependent. Thus, our findings revealed that the pleiotropic function of IFN-γ also includes the regulation of Paneth cell function in the homeostatic gut.

2.
Biochem Soc Trans ; 52(3): 1275-1291, 2024 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-38813865

RESUMEN

Parkinsonism is the primary type of movement disorder in adults, encompassing a set of clinical symptoms, including rigidity, tremors, dystonia, bradykinesia, and postural instability. These symptoms are primarily caused by a deficiency in dopamine (DA), an essential neurotransmitter in the brain. Currently, the DA precursor levodopa (synthetic L-DOPA) is the standard medication to treat DA deficiency, but it only addresses symptoms rather than provides a cure. In this review, we provide an overview of disorders associated with DA dysregulation and deficiency, particularly Parkinson's disease and rare inherited disorders leading predominantly to dystonia and/or parkinsonism, even in childhood. Although levodopa is relatively effective for the management of motor dysfunctions, it is less effective for severe forms of parkinsonism and is also associated with side effects and a loss of efficacy over time. We present ongoing efforts to reinforce the effect of levodopa and to develop innovative therapies that target the underlying pathogenic mechanisms affecting DA synthesis and transport, increasing neurotransmission through disease-modifying approaches, such as cell-based therapies, nucleic acid- and protein-based biologics, and small molecules.


Asunto(s)
Dopamina , Levodopa , Enfermedad de Parkinson , Humanos , Dopamina/metabolismo , Levodopa/uso terapéutico , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Trastornos Parkinsonianos/tratamiento farmacológico , Trastornos Parkinsonianos/metabolismo , Animales , Transporte Biológico
3.
Mol Genet Metab ; 142(3): 108514, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38905920

RESUMEN

Phenylketonuria (PKU) is a genetic disorder caused by variations in the phenylalanine hydroxylase (PAH) gene. Among the 3369 reported PAH variants, 33.7% are missense alterations. Unfortunately, 30% of these missense variants are classified as variants of unknown significance (VUS), posing challenges for genetic risk assessment. In our study, we focused on analyzing 836 missense PAH variants following the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines specified by ClinGen PAH Variant Curation Expert Panel (VCEP) criteria. We utilized and compared variant annotator tools like Franklin and Varsome, conducted 3D structural analysis of PAH, and examined active and regulatory site hotspots. In addition, we assessed potential splicing effect of apparent missense variants. By evaluating phenotype data from 22962 PKU patients, our aim was to reassess the pathogenicity of missense variants. Our comprehensive approach successfully reclassified 309 VUSs out of 836 missense variants as likely pathogenic or pathogenic (37%), upgraded 370 likely pathogenic variants to pathogenic, and reclassified one previously considered likely benign variant as likely pathogenic. Phenotypic information was available for 636 missense variants, with 441 undergoing 3D structural analysis and active site hotspot identification for 180 variants. After our analysis, only 6% of missense variants were classified as VUSs, and three of them (c.23A>C/p.Asn8Thr, c.59_60delinsCC/p.Gln20Pro, and c.278A >T/p.Asn93Ile) may be influenced by abnormal splicing. Moreover, a pathogenic variant (c.168G>T/p.Glu56Asp) was identified to have a risk exceeding 98% for modifications of the consensus splice site, with high scores indicating a donor loss of 0.94. The integration of ACMG/AMP guidelines with in silico structural analysis and phenotypic data significantly reduced the number of missense VUSs, providing a strong basis for genetic counseling and emphasizing the importance of metabolic phenotype information in variant curation. This study also sheds light on the current landscape of PAH variants.


Asunto(s)
Mutación Missense , Fenotipo , Fenilalanina Hidroxilasa , Fenilcetonurias , Humanos , Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/química , Fenilcetonurias/genética , Fenilcetonurias/patología , Simulación por Computador
4.
J Inherit Metab Dis ; 47(3): 533-550, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38168036

RESUMEN

Several mouse models have been developed to study human defects of primary and secondary inherited monoamine neurotransmitter disorders (iMND). As the field continues to expand, current defects in corresponding mouse models include enzymes and a molecular co-chaperone involved in monoamine synthesis and metabolism (PAH, TH, PITX3, AADC, DBH, MAOA, DNAJC6), tetrahydrobiopterin (BH4) cofactor synthesis and recycling (adGTPCH1/DRD, arGTPCH1, PTPS, SR, DHPR), and vitamin B6 cofactor deficiency (ALDH7A1), as well as defective monoamine neurotransmitter packaging (VMAT1, VMAT2) and reuptake (DAT). No mouse models are available for human DNAJC12 co-chaperone and PNPO-B6 deficiencies, disorders associated with recessive variants that result in decreased stability and function of the aromatic amino acid hydroxylases and decreased neurotransmitter synthesis, respectively. More than one mutant mouse is available for some of these defects, which is invaluable as different variant-specific (knock-in) models may provide more insights into underlying mechanisms of disorders, while complete gene inactivation (knock-out) models often have limitations in terms of recapitulating complex human diseases. While these mouse models have common phenotypic traits also observed in patients, reflecting the defective homeostasis of the monoamine neurotransmitter pathways, they also present with disease-specific manifestations with toxic accumulation or deficiency of specific metabolites related to the specific gene affected. This review provides an overview of the currently available models and may give directions toward selecting existing models or generating new ones to investigate novel pathogenic mechanisms and precision therapies.


Asunto(s)
Modelos Animales de Enfermedad , Neurotransmisores , Animales , Ratones , Humanos , Neurotransmisores/metabolismo , Monoaminas Biogénicas/metabolismo
5.
J Inherit Metab Dis ; 47(3): 494-508, 2024 05.
Artículo en Inglés | MEDLINE | ID: mdl-38196161

RESUMEN

Proteostatic regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, is crucial for maintaining proper brain neurotransmitter homeostasis. Variants of the TH gene are associated with tyrosine hydroxylase deficiency (THD), a rare disorder with a wide phenotypic spectrum and variable response to treatment, which affects protein stability and may lead to accelerated degradation, loss of TH function and catecholamine deficiency. In this study, we investigated the effects of the TH cofactor tetrahydrobiopterin (BH4) on the stability of TH in isolated protein and in DAn- differentiated from iPSCs from a human healthy subject, as well as from THD patients with the R233H variant in homozygosity (THDA) and R328W and T399M variants in heterozygosity (THDB). We report an increase in TH and dopamine levels, and an increase in the number of TH+ cells in control and THDA cells. To translate this in vitro effect, we treated with BH4 a knock-in THD mouse model with Th variant corresponding to R233H in patients. Importantly, treatment with BH4 significantly improved motor function in these mice, as demonstrated by increased latency on the rotarod test and improved horizontal activity (catalepsy). In conclusion, our study demonstrates the stabilizing effects of BH4 on TH protein levels and function in THD neurons and mice, rescuing disease phenotypes and improving motor outcomes. These findings highlight the therapeutic potential of BH4 as a treatment option for THDA patients with specific variants and provide insights into the modulation of TH stability and its implications for THD management.


Asunto(s)
Biopterinas , Modelos Animales de Enfermedad , Neuronas , Fenotipo , Tirosina 3-Monooxigenasa , Biopterinas/análogos & derivados , Animales , Humanos , Tirosina 3-Monooxigenasa/metabolismo , Ratones , Neuronas/metabolismo , Dopamina/metabolismo , Masculino , Fenilcetonurias/tratamiento farmacológico , Fenilcetonurias/genética , Fenilcetonurias/metabolismo , Femenino , Técnicas de Sustitución del Gen
6.
J Fish Biol ; 105(3): 1031-1035, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39392133

RESUMEN

We modeled feeding of the Atlantic salmon (Salmo salar) parr to understand the role of global (cross-continental) and regional (river) spatial scales for delineating feeding patterns. The diet composition differed between Eurasia and North America populations. Geographic location (latitude and elevation) had an influence for the most common prey (Ephemeroptera, Diptera and Plecoptera). The random factors (sampling location and river) had a strong explanatory power in our models, suggesting that local drivers may override the effects of large-scale drivers.


Asunto(s)
Conducta Alimentaria , Salmo salar , Animales , Salmo salar/fisiología , Ríos , Dieta/veterinaria , América del Norte , Geografía , Europa (Continente) , Modelos Biológicos
7.
Health Promot Pract ; : 15248399241275610, 2024 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-39230252

RESUMEN

Purpose. Caring Text Messages (CTM) is an evidence-based intervention, developed by the Northwest Portland Area Indian Health Board, modeled after the Caring Contacts (CC) intervention. CC has been shown to prevent suicide deaths, attempts, ideation, and hospitalizations in a variety of settings. Method. Three sets of CTM were developed by American Indian and Alaska Native (AI/AN) teens, college students, and veterans (tailored for each audience), which were reviewed by psychologists familiar with the intervention. To enroll in the service, participants texted a keyword to a text message short code and received two text messages per week with hopeful and encouraging messages. A robust multimedia social marketing campaign was designed to promote the service for each audience. Results. By September 2023, 387 participants enrolled in the Youth CTM intervention, 141 enrolled in the College CTM, and 31 enrolled in the Veterans CTM. Post surveys show elevated levels of user satisfaction. Conclusions. CTM can be tailored to reach populations at higher risk of suicide, including AI/AN youth, college students, and veterans, and connect them to culturally responsive peer and crisis support services. Continued monitoring and evaluation can guide next steps for marketing and outreach and will be useful to determine its impact on those who enroll.

8.
Am J Pathol ; 191(9): 1537-1549, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34139193

RESUMEN

Epithelial barrier impairment is a hallmark of several pathologic processes in the gut, including inflammatory bowel diseases. Several intracellular signals prevent apoptosis in intestinal epithelial cells. Herein, we show that in colonocytes, rictor/mammalian target of rapamycin complex 2 (mTORC2) signaling is a prosurvival stimulus. Mechanistically, mTORC2 activates Akt, which, in turn, inhibits apoptosis by phosphorylating B-cell lymphoma 2 (BCL2) associated agonist of cell death (Bad) and preventing caspase-3 activation. Nevertheless, during inflammation, rictor/mTORC2 signaling declines and Akt activity is reduced. Consequently, active caspase-3 increases in surface colonocytes undergoing apoptosis/anoikis and causes epithelial barrier breakdown. Likewise, Rictor ablation in intestinal epithelial cells interrupts mTORC2/Akt signaling and increases apoptosis/anoikis of surface colonocytes without affecting the crypt architecture. The increase in epithelial permeability induced by Rictor ablation produces a mild inflammatory response in the colonic mucosa, but minimally affects the development/establishment of colitis. The data identify a previously unknown mechanism by which rictor/mTORC2 signaling regulates apoptosis/anoikis in intestinal epithelial cells during colitis and clarify its role in the maintenance of the intestinal epithelial barrier.


Asunto(s)
Apoptosis/fisiología , Colitis/patología , Células Epiteliales/metabolismo , Mucosa Intestinal/patología , Proteína Asociada al mTOR Insensible a la Rapamicina/metabolismo , Animales , Colitis/metabolismo , Células Epiteliales/patología , Mucosa Intestinal/metabolismo , Ratones , Transducción de Señal/fisiología
9.
Rev Cardiovasc Med ; 23(12): 414, 2022 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-39076676

RESUMEN

Background: Several anthropometric measurements are used to assess cardiovascular risk and progress during clinical treatment. Most commonly used anthropometric measurements include total body weight and body mass index (BMI), with several other simple anthropometric measures typically underused in clinical practice. Herein, we review the evidence on the relationship between different anthropometric measurements and cardiovascular risk in patients with and without cardiovascular disease (CVD). Methods: Data for this review were identified by searches in PubMed, the Web of Science, Google Scholar, and references from relevant articles by using appropriate and related terms. The last search was performed on June 22, 2022. Articles published in English and Spanish were reviewed and included, if appropriate. We included studies detailing the relationship between skinfolds thickness, waist-to-hip ratio (WHR) and Conicity index with cardiovascular risk in adults with/without CVD. Results: In patients from the general population, elevated subscapular and triceps skinfolds showed a positive relationship with the development of hypertension, diabetes mellitus, hypercholesterolemia, cardiovascular mortality, and all-cause mortality. A higher subscapular skinfold was also associated with increased risk of coronary artery disease and stroke. A higher WHR, as well as other less common anthropometric measurements such as the Conicity index, was associated with an increased risk of myocardial infarction, incident CVD, major adverse cardiovascular events, and mortality in both patients with and without previous CVD. Conclusions: Non-traditional anthropometric measurements including skinfolds and WHR seem to improve the prediction of cardiovascular risk in the general population, and recurrent events in patients with previous CVD. Use of additional anthropometric techniques according to an objective and standardized method, may aid cardiovascular risk stratification in patients from the general population and the evaluation of therapeutic interventions for patients with CVD.

10.
Proc Natl Acad Sci U S A ; 116(23): 11229-11234, 2019 06 04.
Artículo en Inglés | MEDLINE | ID: mdl-31118288

RESUMEN

Phenylalanine hydroxylase (PAH) is a key enzyme in the catabolism of phenylalanine, and mutations in this enzyme cause phenylketonuria (PKU), a genetic disorder that leads to brain damage and mental retardation if untreated. Some patients benefit from supplementation with a synthetic formulation of the cofactor tetrahydrobiopterin (BH4) that partly acts as a pharmacological chaperone. Here we present structures of full-length human PAH (hPAH) both unbound and complexed with BH4 in the precatalytic state. Crystal structures, solved at 3.18-Å resolution, show the interactions between the cofactor and PAH, explaining the negative regulation exerted by BH4 BH4 forms several H-bonds with the N-terminal autoregulatory tail but is far from the catalytic FeII Upon BH4 binding a polar and salt-bridge interaction network links the three PAH domains, explaining the stability conferred by BH4 Importantly, BH4 binding modulates the interaction between subunits, providing information about PAH allostery. Moreover, we also show that the cryo-EM structure of hPAH in absence of BH4 reveals a highly dynamic conformation for the tetramers. Structural analyses of the hPAH:BH4 subunits revealed that the substrate-induced movement of Tyr138 into the active site could be coupled to the displacement of BH4 from the precatalytic toward the active conformation, a molecular mechanism that was supported by site-directed mutagenesis and targeted molecular dynamics simulations. Finally, comparison of the rat and human PAH structures show that hPAH is more dynamic, which is related to amino acid substitutions that enhance the flexibility of hPAH and may increase the susceptibility to PKU-associated mutations.


Asunto(s)
Biopterinas/análogos & derivados , Fenilalanina Hidroxilasa/química , Biopterinas/química , Biopterinas/genética , Humanos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida/métodos , Mutación/genética , Fenilalanina Hidroxilasa/genética , Fenilcetonurias/genética
11.
Mol Pharmacol ; 100(2): 155-169, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34031189

RESUMEN

The 14-3-3 proteins constitute a family of adaptor proteins with many binding partners and biological functions, and they are considered promising drug targets in cancer and neuropsychiatry. By screening 1280 small-molecule drugs using differential scanning fluorimetry (DSF), we found 15 compounds that decreased the thermal stability of 14-3-3ζ Among these compounds, ebselen was identified as a covalent, destabilizing ligand of 14-3-3 isoforms ζ, ε, γ, and η Ebselen bonding decreased 14-3-3ζ binding to its partner Ser19-phosphorylated tyrosine hydroxylase. Characterization of site-directed mutants at cysteine residues in 14-3-3ζ (C25, C94, and C189) by DSF and mass spectroscopy revealed covalent modification by ebselen of all cysteines through a selenylsulfide bond. C25 appeared to be the preferential site of ebselen interaction in vitro, whereas modification of C94 was the main determinant for protein destabilization. At therapeutically relevant concentrations, ebselen and ebselen oxide caused decreased 14-3-3 levels in SH-SY5Y cells, accompanied with an increased degradation, most probably by the ubiquitin-dependent proteasome pathway. Moreover, ebselen-treated zebrafish displayed decreased brain 14-3-3 content, a freezing phenotype, and reduced mobility, resembling the effects of lithium, consistent with its proposed action as a safer lithium-mimetic drug. Ebselen has recently emerged as a promising drug candidate in several medical areas, such as cancer, neuropsychiatric disorders, and infectious diseases, including coronavirus disease 2019. Its pleiotropic actions are attributed to antioxidant effects and formation of selenosulfides with critical cysteine residues in proteins. Our work indicates that a destabilization of 14-3-3 may affect the protein interaction networks of this protein family, contributing to the therapeutic potential of ebselen. SIGNIFICANCE STATEMENT: There is currently great interest in the repurposing of established drugs for new indications and therapeutic targets. This study shows that ebselen, which is a promising drug candidate against cancer, bipolar disorder, and the viral infection coronavirus disease 2019, covalently bonds to cysteine residues in 14-3-3 adaptor proteins, triggering destabilization and increased degradation in cells and intact brain tissue when used in therapeutic concentrations, potentially explaining the behavioral, anti-inflammatory, and antineoplastic effects of this drug.


Asunto(s)
Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Cisteína/genética , Isoindoles/farmacología , Compuestos de Organoselenio/farmacología , Proteínas 14-3-3/genética , Animales , Sitios de Unión/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Dicroismo Circular , Regulación hacia Abajo , Femenino , Humanos , Masculino , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica/efectos de los fármacos , Conformación Proteica , Estabilidad Proteica/efectos de los fármacos , Tirosina 3-Monooxigenasa/metabolismo , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo
12.
Mol Pharm ; 18(3): 976-985, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33417459

RESUMEN

Tyrosine hydroxylase (TH) is the enzyme catalyzing the rate-limiting step in the synthesis of dopamine in the brain. Developing enzyme replacement therapies using TH could therefore be beneficial to patient groups with dopamine deficiency, and the use of nanocarriers that cross the blood-brain barrier seems advantageous for this purpose. Nanocarriers may also help to maintain the structure and function of TH, which is complex and unstable. Understanding how TH may interact with a nanocarrier is therefore crucial for the investigation of such therapeutic applications. This work describes the interaction of TH with porous silicon nanoparticles (pSiNPs), chosen since they have been shown to deliver other macromolecular therapeutics successfully to the brain. Size distributions obtained by dynamic light scattering show a size increase of pSiNPs upon addition of TH and the changes observed at the surface of pSiNPs by transmission electron microscopy also indicated TH binding at pH 7. As pSiNPs are negatively charged, we also investigated the binding at pH 6, which makes TH less negatively charged than at pH 7. However, as seen by thioflavin-T fluorescence, TH aggregated at this more acidic pH. TH activity was unaffected by the binding to pSiNPs most probably because the active site stays available for catalysis, in agreement with calculations of the surface electrostatic potential pointing to the most positively charged regulatory domains in the tetramer as the interacting regions. These results reveal pSiNPs as a promising delivery device of enzymatically active TH to increase local dopamine synthesis.


Asunto(s)
Nanopartículas/metabolismo , Silicio/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Humanos , Porosidad , Electricidad Estática
13.
J Immunol ; 202(4): 1239-1249, 2019 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-30626693

RESUMEN

A single layer of polarized epithelial cells lining the colonic mucosa create a semipermeable barrier indispensable for gut homeostasis. The role of intestinal epithelial cell (IEC) polarization in the maintenance of the epithelial homeostasis and in the development of inflammatory bowel diseases is not fully understood. In this review, now we report that IEC polarization plays an essential role in the regulation of IL-6/STAT3 signaling in the colonic mucosa. Our results demonstrate that autocrine STAT3 activation in IECs is mediated by the apical secretion of IL-6 in response to the basolateral stimulation with IFN-γ. This process relies on the presence of functional, IFN-γ-producing CD4+ T cells. In the absence of basolateral IFN-γ, the compartmentalization of the IL-6/STAT3 signaling is disrupted, and STAT3 is activated mainly in macrophages. Thus, in this study, we show that during inflammation, IFN-γ regulates IL-6/STAT3 signaling in IEC in the colonic mucosa.


Asunto(s)
Colitis/metabolismo , Colon/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Células CACO-2 , Células Cultivadas , Células Epiteliales/metabolismo , Humanos , Inflamación/metabolismo , Interferón gamma/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL
14.
Mol Ther ; 28(2): 677-689, 2020 02 05.
Artículo en Inglés | MEDLINE | ID: mdl-31810863

RESUMEN

Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells overexpressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients' specific mutation.


Asunto(s)
Biomarcadores , Descubrimiento de Drogas , Porfiria Intermitente Aguda/metabolismo , Animales , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Humanos , Ratones , Ratones Noqueados , Terapia Molecular Dirigida , Porfiria Intermitente Aguda/etiología , Porfiria Intermitente Aguda/terapia , Pliegue de Proteína , Proteínas/antagonistas & inhibidores , Proteínas/química , Proteínas/metabolismo , Reproducibilidad de los Resultados , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad
15.
Int J Mol Sci ; 22(2)2021 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-33445488

RESUMEN

Acute intermittent porphyria (AIP) is an autosomal dominant inherited disease with low clinical penetrance, caused by mutations in the hydroxymethylbilane synthase (HMBS) gene, which encodes the third enzyme in the haem biosynthesis pathway. In susceptible HMBS mutation carriers, triggering factors such as hormonal changes and commonly used drugs induce an overproduction and accumulation of toxic haem precursors in the liver. Clinically, this presents as acute attacks characterised by severe abdominal pain and a wide array of neurological and psychiatric symptoms, and, in the long-term setting, the development of primary liver cancer, hypertension and kidney failure. Treatment options are few, and therapies preventing the development of symptomatic disease and long-term complications are non-existent. Here, we provide an overview of the disorder and treatments already in use in clinical practice, in addition to other therapies under development or in the pipeline. We also introduce the pathomechanistic effects of HMBS mutations, and present and discuss emerging therapeutic options based on HMBS stabilisation and the regulation of proteostasis. These are novel mechanistic therapeutic approaches with the potential of prophylactic correction of the disease by totally or partially recovering the enzyme functionality. The present scenario appears promising for upcoming patient-tailored interventions in AIP.


Asunto(s)
Porfiria Intermitente Aguda/terapia , Alelos , Animales , Terapia Combinada , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Predisposición Genética a la Enfermedad , Hemo/metabolismo , Humanos , Hidroximetilbilano Sintasa/química , Hidroximetilbilano Sintasa/genética , Redes y Vías Metabólicas , Mutación , Porfiria Intermitente Aguda/diagnóstico , Porfiria Intermitente Aguda/etiología , Relación Estructura-Actividad , Resultado del Tratamiento
16.
Hum Mutat ; 41(7): 1329-1338, 2020 07.
Artículo en Inglés | MEDLINE | ID: mdl-32333439

RESUMEN

Biallelic variants of the gene DNAJC12, which encodes a cochaperone, were recently described in patients with hyperphenylalaninemia (HPA). This paper reports the retrospective genetic analysis of a cohort of unsolved cases of HPA. Biallelic variants of DNAJC12 were identified in 20 patients (generally neurologically asymptomatic) previously diagnosed with phenylalanine hydroxylase (PAH) deficiency (phenylketonuria [PKU]). Further, mutations of DNAJC12 were identified in four carriers of a pathogenic variant of PAH. The genetic spectrum of DNAJC12 in the present patients included four new variants, two intronic changes c.298-2A>C and c.502+1G>C, presumably affecting the splicing process, and two exonic changes c.309G>T (p.Trp103Cys) and c.524G>A (p.Trp175Ter), classified as variants of unknown clinical significance (VUS). The variant p.Trp175Ter was detected in 83% of the mutant alleles, with 14 cases homozygous, and was present in 0.3% of a Spanish control population. Functional analysis indicated a significant reduction in PAH and its activity, reduced tyrosine hydroxylase stability, but no effect on tryptophan hydroxylase 2 stability, classifying the two VUS as pathogenic variants. Additionally, the effect of the overexpression of DNAJC12 on some destabilizing PAH mutations was examined and a mutation-specific effect on stabilization was detected suggesting that the proteostasis network could be a genetic modifier of PAH deficiency and a potential target for developing mutation-specific treatments for PKU.


Asunto(s)
Fenilcetonurias/genética , Proteínas Represoras/genética , Adolescente , Adulto , Alelos , Niño , Preescolar , Análisis Mutacional de ADN , Exones , Humanos , Lactante , Recién Nacido , Intrones , Empalme del ARN , Estudios Retrospectivos , España
17.
Am J Hum Genet ; 100(2): 257-266, 2017 02 02.
Artículo en Inglés | MEDLINE | ID: mdl-28132689

RESUMEN

Phenylketonuria (PKU, phenylalanine hydroxylase deficiency), an inborn error of metabolism, can be detected through newborn screening for hyperphenylalaninemia (HPA). Most individuals with HPA harbor mutations in the gene encoding phenylalanine hydroxylase (PAH), and a small proportion (2%) exhibit tetrahydrobiopterin (BH4) deficiency with additional neurotransmitter (dopamine and serotonin) deficiency. Here we report six individuals from four unrelated families with HPA who exhibited progressive neurodevelopmental delay, dystonia, and a unique profile of neurotransmitter deficiencies without mutations in PAH or BH4 metabolism disorder-related genes. In these six affected individuals, whole-exome sequencing (WES) identified biallelic mutations in DNAJC12, which encodes a heat shock co-chaperone family member that interacts with phenylalanine, tyrosine, and tryptophan hydroxylases catalyzing the BH4-activated conversion of phenylalanine into tyrosine, tyrosine into L-dopa (the precursor of dopamine), and tryptophan into 5-hydroxytryptophan (the precursor of serotonin), respectively. DNAJC12 was undetectable in fibroblasts from the individuals with null mutations. PAH enzyme activity was reduced in the presence of DNAJC12 mutations. Early treatment with BH4 and/or neurotransmitter precursors had dramatic beneficial effects and resulted in the prevention of neurodevelopmental delay in the one individual treated before symptom onset. Thus, DNAJC12 deficiency is a preventable and treatable cause of intellectual disability that should be considered in the early differential diagnosis when screening results are positive for HPA. Sequencing of DNAJC12 may resolve any uncertainty and should be considered in all children with unresolved HPA.


Asunto(s)
Distonía/genética , Discapacidad Intelectual/genética , Fenilcetonurias/genética , Proteínas Represoras/genética , Alelos , Secuencia de Aminoácidos , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Estudios de Casos y Controles , Dopamina/deficiencia , Dopamina/metabolismo , Exones , Femenino , Fibroblastos/metabolismo , Eliminación de Gen , Estudio de Asociación del Genoma Completo , Proteínas HSP70 de Choque Térmico/genética , Humanos , Masculino , Linaje , Fenilalanina/metabolismo , Fenilalanina Hidroxilasa/genética , Serotonina/deficiencia , Serotonina/metabolismo , Triptófano/metabolismo , Triptófano Hidroxilasa/genética , Triptófano Hidroxilasa/metabolismo , Tirosina/metabolismo , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismo
18.
Hum Mutat ; 40(4): 483-494, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30667134

RESUMEN

DNAJC12, a type III member of the HSP40/DNAJ family, has been identified as the specific co-chaperone of phenylalanine hydroxylase (PAH) and the other aromatic amino acid hydroxylases. DNAJ proteins work together with molecular chaperones of the HSP70 family to assist in proper folding and maintenance of intracellular stability of their clients. Autosomal recessive mutations in DNAJC12 were found to reduce PAH levels, leading to hyperphenylalaninemia (HPA) in patients without mutations in PAH. In this work, we investigated the interaction of normal wild-type DNAJC12 with mutant PAH in cells expressing several PAH variants associated with HPA in humans, as well as in the Enu1/1 mouse model, homozygous for the V106A-Pah variant, which leads to severe protein instability, accelerated PAH degradation and mild HPA. We found that mutant PAH exhibits increased ubiquitination, instability, and aggregation compared with normal PAH. In mouse liver lysates, we showed that DNAJC12 interacts with monoubiquitin-tagged PAH. This form represented a major fraction of PAH in the Enu1/1 but was also present in liver of wild-type PAH mice. Our results support a role of DNAJC12 in the processing of misfolded ubiquitinated PAH by the ubiquitin-dependent proteasome/autophagy systems and add to the evidence that the DNAJ proteins are important players both for proper folding and degradation of their clients.


Asunto(s)
Fenilalanina Hidroxilasa/genética , Fenilalanina Hidroxilasa/metabolismo , Proteínas Represoras/metabolismo , Alelos , Animales , Biomarcadores , Línea Celular Tumoral , Expresión Génica , Genotipo , Humanos , Inmunohistoquímica , Hígado/metabolismo , Ratones , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutación , Unión Proteica
19.
J Biol Chem ; 292(34): 14092-14107, 2017 08 25.
Artículo en Inglés | MEDLINE | ID: mdl-28637871

RESUMEN

Tyrosine hydroxylase (TH) catalyzes the conversion of l-tyrosine into l-DOPA, which is the rate-limiting step in the synthesis of catecholamines, such as dopamine, in dopaminergergic neurons. Low dopamine levels and death of the dopaminergic neurons are hallmarks of Parkinson's disease (PD), where α-synuclein is also a key player. TH is highly regulated, notably by phosphorylation of several Ser/Thr residues in the N-terminal tail. However, the functional role of TH phosphorylation at the Ser-31 site (THSer(P)-31) remains unclear. Here, we report that THSer(P)-31 co-distributes with the Golgi complex and synaptic-like vesicles in rat and human dopaminergic cells. We also found that the TH microsomal fraction content decreases after inhibition of cyclin-dependent kinase 5 (Cdk5) and ERK1/2. The cellular distribution of an overexpressed phospho-null mutant, TH1-S31A, was restricted to the soma of neuroblastoma cells, with decreased association with the microsomal fraction, whereas a phospho-mimic mutant, TH1-S31E, was distributed throughout the soma and neurites. TH1-S31E associated with vesicular monoamine transporter 2 (VMAT2) and α-synuclein in neuroblastoma cells, and endogenous THSer(P)-31 was detected in VMAT2- and α-synuclein-immunoprecipitated mouse brain samples. Microtubule disruption or co-transfection with α-synuclein A53T, a PD-associated mutation, caused TH1-S31E accumulation in the cell soma. Our results indicate that Ser-31 phosphorylation may regulate TH subcellular localization by enabling its transport along microtubules, notably toward the projection terminals. These findings disclose a new mechanism of TH regulation by phosphorylation and reveal its interaction with key players in PD, opening up new research avenues for better understanding dopamine synthesis in physiological and pathological states.


Asunto(s)
Neuronas Dopaminérgicas/enzimología , Aparato de Golgi/enzimología , Microtúbulos/enzimología , Procesamiento Proteico-Postraduccional , Serina/metabolismo , Vesículas Sinápticas/enzimología , Tirosina 3-Monooxigenasa/metabolismo , Sustitución de Aminoácidos , Animales , Línea Celular Tumoral , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Aparato de Golgi/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Microscopía Confocal , Microscopía Fluorescente , Microtúbulos/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Transporte de Proteínas , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Vesículas Sinápticas/metabolismo , Tirosina 3-Monooxigenasa/genética
20.
Mol Genet Metab ; 123(1): 1-5, 2018 01.
Artículo en Inglés | MEDLINE | ID: mdl-29174366

RESUMEN

Patients with hyperphenylalaninemia (HPA) are detected through newborn screening for phenylketonuria (PKU). HPA is known to be caused by deficiencies of the enzyme phenylalanine hydroxylase (PAH) or its cofactor tetrahydrobiopterin (BH4). Current guidelines for the differential diagnosis of HPA would, however, miss a recently described DNAJC12 deficiency. The co-chaperone DNAJC12 is, together with the 70kDa heat shock protein (HSP70), responsible for the proper folding of PAH. All DNAJC12-deficient patients investigated to date responded to a challenge with BH4 by lowering their blood phenylalanine levels. In addition, the patients presented with low levels of biogenic amine in CSF and responded to supplementation with BH4, L-dopa/carbidopa and 5-hydroxytryptophan. The phenotypic spectrum ranged from mild autistic features or hyperactivity to severe intellectual disability, dystonia and parkinsonism. Late diagnosis result in permanent neurological disability, while early diagnosed and treated patients develop normally. Molecular diagnostics for DNAJC12 variants are thus mandatory in all patients in which deficiencies of PAH and BH4 are genetically excluded.


Asunto(s)
Fenilalanina Hidroxilasa/genética , Fenilalanina/genética , Fenilcetonurias/genética , Proteínas Represoras/genética , Aminas Biogénicas/líquido cefalorraquídeo , Humanos , Recién Nacido , Levodopa/genética , Levodopa/metabolismo , Tamizaje Neonatal , Patología Molecular , Fenilalanina/metabolismo , Fenilalanina Hidroxilasa/metabolismo , Fenilcetonurias/líquido cefalorraquídeo , Fenilcetonurias/diagnóstico , Fenilcetonurias/patología , Pliegue de Proteína , Proteínas Represoras/deficiencia
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