RESUMEN
Inorganic pyrophosphate (PPi) is a crucial extracellular mineralization regulator. Low plasma PPi concentrations underlie the soft tissue calcification present in several rare hereditary mineralization disorders as well as in more common conditions like chronic kidney disease and diabetes. Even though deregulated plasma PPi homeostasis is known to be linked to multiple human diseases, there is currently no reliable assay for its quantification. We here describe a PPi assay that employs the enzyme ATP sulfurylase to convert PPi into ATP. Generated ATP is subsequently quantified by firefly luciferase-based bioluminescence. An internal ATP standard was used to correct for sample-specific interference by matrix compounds on firefly luciferase activity. The assay was validated and shows excellent precision (< 3.5%) and accuracy (93-106%) of PPi spiked into human plasma samples. We found that of several anticoagulants tested only EDTA effectively blocked conversion of ATP into PPi in plasma after blood collection. Moreover, filtration over a 300,000-Da molecular weight cut-off membrane reduced variability of plasma PPi and removed ATP present in a membrane-enclosed compartment, possibly platelets. Applied to plasma samples of wild-type and Abcc6-/- rats, an animal model with established low circulating levels of PPi, the new assay showed lower variability than the assay that was previously in routine use in our laboratory. In conclusion, we here report a new and robust assay to determine PPi concentrations in plasma, which outperforms currently available assays because of its high sensitivity, precision, and accuracy.
Asunto(s)
Calcinosis , Difosfatos , Humanos , Ratas , Animales , Luciferasas de Luciérnaga , Adenosina TrifosfatoRESUMEN
The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.
Asunto(s)
Huesos/metabolismo , Calcinosis/genética , Ácido Cítrico/metabolismo , Proteínas de Transporte de Fosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Desarrollo Óseo/genética , Calcinosis/metabolismo , Calcinosis/patología , Diferenciación Celular , Células Cultivadas , Difosfatos/metabolismo , Humanos , Fenómenos Mecánicos , Ratones , Mutación/genética , Proteínas de Transporte de Fosfato/metabolismoRESUMEN
ABCC6 promotes ATP efflux from hepatocytes to bloodstream. ATP is metabolized to pyrophosphate, an inhibitor of ectopic calcification. Pathogenic variants of ABCC6 cause pseudoxanthoma elasticum, a highly variable recessive ectopic calcification disorder. Incomplete penetrance may initiate disease heterogeneity, hence symptoms may not, or differently manifest in carriers. Here, we investigated whether incomplete penetrance is a source of heterogeneity in pseudoxanthoma elasticum. By integrating clinical and genetic data of 589 patients, we created the largest European cohort. Based on allele frequency alterations, we identified two incomplete penetrant pathogenic variants, c.2359G>A (p.Val787Ile) and c.1171A>G (p.Arg391Gly), with 6.5% and 2% penetrance, respectively. However, when penetrant, the c.1171A>G (p.Arg391Gly) manifested a clinically unaltered severity. After applying in silico and in vitro characterization, we suggest that incomplete penetrant variants are only deleterious if a yet unknown interacting partner of ABCC6 is mutated simultaneously. The low penetrance of these variants should be contemplated in genetic counseling.
Asunto(s)
Seudoxantoma Elástico , Humanos , Mutación , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/patología , Penetrancia , Adenosina Trifosfato , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genéticaRESUMEN
Inactivating mutations in ABCC6 underlie the rare hereditary mineralization disorder pseudoxanthoma elasticum. ABCC6 is an ATP-binding cassette (ABC) integral membrane protein that mediates the release of ATP from hepatocytes into the bloodstream. The released ATP is extracellularly converted into pyrophosphate, a key mineralization inhibitor. Although ABCC6 is firmly linked to cellular ATP release, the molecular details of ABCC6-mediated ATP release remain elusive. Most of the currently available data support the hypothesis that ABCC6 is an ATP-dependent ATP efflux pump, an un-precedented function for an ABC transporter. This hypothesis implies the presence of an ATP-binding site in the substrate-binding cavity of ABCC6. We performed an extensive mutagenesis study using a new homology model based on recently published structures of its close homolog, bovine Abcc1, to characterize the substrate-binding cavity of ABCC6. Leukotriene C4 (LTC4), is a high-affinity substrate of ABCC1. We mutagenized fourteen amino acid residues in the rat ortholog of ABCC6, rAbcc6, that corresponded to the residues in ABCC1 found in the LTC4 binding cavity. Our functional characterization revealed that most of the amino acids in rAbcc6 corresponding to those found in the LTC4 binding pocket in bovine Abcc1 are not critical for ATP efflux. We conclude that the putative ATP binding site in the substrate-binding cavity of ABCC6/rAbcc6 is distinct from the bovine Abcc1 LTC4-binding site.
Asunto(s)
Sitios de Unión , Modelos Moleculares , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Mutación , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Ligandos , Conformación Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutagénesis , Unión Proteica , Transporte de Proteínas , Ratas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Pseudoxanthoma elasticum (PXE, OMIM 264800) is a rare autosomal recessive disorder with ectopic mineralization and fragmentation of elastin fibers. It is caused by mutations of the ABCC6 gene that leads to decreased serum levels of inorganic pyrophosphate (PPi) anti-mineralization factor. The occurrence of severe complications among PXE patients highlights the importance of early diagnosis so that prompt multidisciplinary care can be provided to patients. We aimed to examine dermal connective tissue with nonlinear optical (NLO) techniques, as collagen emits second-harmonic generation (SHG) signal, while elastin can be excited by two-photon excitation fluorescence (TPF). We performed molecular genetic analysis, ophthalmological and cardiovascular assessment, plasma PPi measurement, conventional histopathological examination, and ex vivo SHG and TPF imaging in five patients with PXE and five age- and gender-matched healthy controls. Pathological mutations including one new variant were found in the ABCC6 gene in all PXE patients and their plasma PPi level was significantly lower compared with controls. Degradation and mineralization of elastin fibers and extensive calcium deposition in the mid-dermis was visualized and quantified together with the alterations of the collagen structure in PXE. Our data suggests that NLO provides high-resolution imaging of the specific histopathological features of PXE-affected skin. In vivo NLO may be a promising tool in the assessment of PXE, promoting early diagnosis and follow-up.
Asunto(s)
Microscopía Óptica no Lineal/métodos , Seudoxantoma Elástico/diagnóstico por imagen , Piel/diagnóstico por imagen , Estudios de Casos y Controles , Colágeno/metabolismo , Tejido Conectivo/patología , Elastina/metabolismo , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador , Masculino , Seudoxantoma Elástico/metabolismo , Seudoxantoma Elástico/patología , Piel/metabolismo , Piel/patologíaRESUMEN
ATP binding cassette type-C (ABCC) transporters move molecules across cell membranes upon hydrolysis of ATP; however, their coupling of ATP hydrolysis to substrate transport remains elusive. Drosophila multidrug resistance-associated protein (DMRP) is the functional ortholog of human long ABCC transporters, with similar substrate and inhibitor specificity, but higher activity. Exploiting its high activity, we kinetically dissected the catalytic mechanism of DMRP by using E2-d-glucuronide (E2G), the physiologic substrate of human ABCC. We examined the DMRP-mediated interdependence of ATP and E2G in biochemical assays. We observed E2G-dependent ATPase activity to be biphasic at subsaturating ATP concentrations, which implies at least 2 E2G binding sites on DMRP. Furthermore, transport measurements indicated strong nonreciprocal cooperativity between ATP and E2G. In addition to confirming these findings, our kinetic modeling with the Complex Pathway Simulator indicated a 10-fold decrease in the E2G-mediated activation of ATP hydrolysis upon saturation of the second E2G binding site. Surprisingly, the binding of the second E2G allowed for substrate transport with a constant rate, which tightly coupled ATP hydrolysis to transport. In summary, we show that the second E2G binding-similar to human ABCC2-allosterically stimulates transport activity of DMRP. Our data suggest that this is achieved by a significant increase in the coupling of ATP hydrolysis to transport.-Karasik, A., Ledwitch, K. V., Arányi, T., Váradi, A., Roberts, A., Szeri, F. Boosted coupling of ATP hydrolysis to substrate transport upon cooperative estradiol-17-ß-D-glucuronide binding in a Drosophila ATP binding cassette type-C transporter.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/química , Proteínas de Drosophila/química , Estradiol/análogos & derivados , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Estradiol/química , Estradiol/metabolismo , Humanos , Hidrólisis , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
Pseudoxanthoma elasticum (PXE) is an inherited metabolic disease with autosomal recessive inheritance caused by mutations in the ABCC6 gene. Since the first description of the disease in 1896, alleging a disease involving the elastic fibers, the concept evolved with the further discoveries of the pivotal role of ectopic mineralization that is preponderant in the elastin-rich tissues of the skin, eyes and blood vessel walls. After discovery of the causative gene of the disease in 2000, the function of the ABCC6 protein remains elusive. More than 300 mutations have been now reported and the concept of a dermal disease has progressively evolved toward a metabolic disorder resulting from the remote effects caused by lack of a circulating anti-mineralization factor. Very recently, evidence has accumulated that this anti-mineralizing factor is inorganic pyrophosphate (PPi). This leads to decreased PPi/Pi (inorganic phosphate) ratio that results from the lack of extracellular ATP release by hepatocytes and probably renal cells harboring the mutant ABCC6 protein. However, the mechanism by which ABCC6 dysfunction causes diminished ATP release remains an enigma. Studies of other ABC transporters, such as ABCC7 or ABCC1 could help our understanding of what ABCC6 exact function is. Data and a hypothesis on the possible roles of ABCC6 in acquired metabolic diseases are also discussed.
Asunto(s)
Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Seudoxantoma Elástico/etiología , Calcificación Vascular/etiología , Animales , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mutación , Fosfatos/metabolismo , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , Calcificación Vascular/genética , Calcificación Vascular/metabolismoRESUMEN
RATIONALE: ABCC6 plays a crucial role in ectopic calcification; mutations of the gene cause pseudoxanthoma elasticum and general arterial calcification of infancy. To elucidate the role of ABCC6 in cellular physiology and disease, it is crucial to establish the exact subcellular localization of the native ABCC6 protein. OBJECTIVE: In a recent article in Circulation Research, ABCC6 was reported to localize to the mitochondria-associated membrane and not the plasma membrane. As the suggested mitochondrial localization is inconsistent with published data and the presumed role of ABCC6, we performed experiments to determine the cellular localization of ABCC6 in its physiological environment. METHODS AND RESULTS: We performed immunofluorescent labeling of frozen mouse and human liver sections, as well as primary hepatocytes. We used several different antibodies recognizing human and mouse ABCC6. Our results unequivocally show that ABCC6 is in the basolateral membrane of hepatocytes and is not associated with the mitochondria, mitochondria-associated membrane, or the endoplasmic reticulum. CONCLUSIONS: Our findings support the model that ABCC6 is in the basolateral membrane, mediating the sinusoidal efflux of a metabolite from the hepatocytes to systemic circulation.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Membrana Celular/metabolismo , Hepatocitos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Biomarcadores/metabolismo , Polaridad Celular/fisiología , Retículo Endoplásmico/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
OBJECTIVE: Mutations in ABCC6 underlie the ectopic mineralization disorder pseudoxanthoma elasticum (PXE) and some forms of generalized arterial calcification of infancy, both of which affect the cardiovascular system. Using cultured cells, we recently showed that ATP-binding cassette subfamily C member 6 (ABCC6) mediates the cellular release of ATP, which is extracellularly rapidly converted into AMP and the mineralization inhibitor inorganic pyrophosphate (PPi). The current study was performed to determine which tissues release ATP in an ABCC6-dependent manner in vivo, where released ATP is converted into AMP and PPi, and whether human PXE ptients have low plasma PPi concentrations. APPROACH AND RESULTS: Using cultured primary hepatocytes and in vivo liver perfusion experiments, we found that ABCC6 mediates the direct, sinusoidal, release of ATP from the liver. Outside hepatocytes, but still within the liver vasculature, released ATP is converted into AMP and PPi. The absence of functional ABCC6 in patients with PXE leads to strongly reduced plasma PPi concentrations. CONCLUSIONS: Hepatic ABCC6-mediated ATP release is the main source of circulating PPi, revealing an unanticipated role of the liver in systemic PPi homeostasis. Patients with PXE have a strongly reduced plasma PPi level, explaining their mineralization disorder. Our results indicate that systemic PPi is relatively stable and that PXE, generalized arterial calcification of infancy, and other ectopic mineralization disorders could be treated with PPi supplementation therapy.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Adenosina Trifosfato/metabolismo , Difosfatos/sangre , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/fisiología , Adenosina Monofosfato/sangre , Anciano , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Femenino , Células HEK293 , Células HeLa , Hepatocitos/metabolismo , Homeostasis , Humanos , Hígado/irrigación sanguínea , Masculino , Ratones , Persona de Mediana Edad , Seudoxantoma Elástico/genética , Seudoxantoma Elástico/metabolismo , RatasRESUMEN
Calcification of the cerebral microvessels in the basal ganglia in the absence of systemic calcium and phosphate imbalance is a hallmark of primary familial brain calcification (PFBC), a rare neurodegenerative disorder. Mutation in genes encoding for sodium-dependent phosphate transporter 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), platelet-derived growth factor B (PDGFB), platelet-derived growth factor receptor beta (PDGFRB), myogenesis regulating glycosidase (MYORG), and junctional adhesion molecule 2 (JAM2) are known to cause PFBC. Loss-of-function mutations in XPR1, the only known inorganic phosphate exporter in metazoans, causing dominantly inherited PFBC was first reported in 2015 but until now no studies in the brain have addressed whether loss of one functional allele leads to pathological alterations in mice, a commonly used organism to model human diseases. Here we show that mice heterozygous for Xpr1 (Xpr1WT/lacZ ) present with reduced inorganic phosphate levels in the cerebrospinal fluid and age- and sex-dependent growth of vascular calcifications in the thalamus. Vascular calcifications are surrounded by vascular basement membrane and are located at arterioles in the smooth muscle layer. Similar to previously characterized PFBC mouse models, vascular calcifications in Xpr1WT/lacZ mice contain bone matrix proteins and are surrounded by reactive astrocytes and microglia. However, microglial activation is not confined to calcified vessels but shows a widespread presence. In addition to vascular calcifications, we observed vessel tortuosity and transmission electron microscopy analysis revealed microangiopathy-endothelial swelling, phenotypic alterations in vascular smooth muscle cells, and thickening of the basement membrane.
Asunto(s)
Encefalopatías , Enfermedades Neurodegenerativas , Calcificación Vascular , Humanos , Animales , Ratones , Encefalopatías/patología , Fosfatos/metabolismo , Encéfalo/patología , Receptor de Retrovirus Xenotrópico y Politrópico , Calcificación Vascular/metabolismo , Calcificación Vascular/patología , Enfermedades Neurodegenerativas/patología , Mutación , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo III/metabolismoRESUMEN
Vascular ageing, characterized by structural and functional changes in blood vessels of which arterial stiffness and endothelial dysfunction are key components, is associated with increased risk of cardiovascular and other age-related diseases. As the global population continues to age, understanding the underlying mechanisms and developing effective therapeutic interventions to mitigate vascular ageing becomes crucial for improving cardiovascular health outcomes. Therefore, this review provides an overview of the current knowledge on pharmacological modulation of vascular ageing, highlighting key strategies and promising therapeutic targets. Several molecular pathways have been identified as central players in vascular ageing, including oxidative stress and inflammation, the renin-angiotensin-aldosterone system, cellular senescence, macroautophagy, extracellular matrix remodelling, calcification, and gasotransmitter-related signalling. Pharmacological and dietary interventions targeting these pathways have shown potential in ameliorating age-related vascular changes. Nevertheless, the development and application of drugs targeting vascular ageing is complicated by various inherent challenges and limitations, such as certain preclinical methodological considerations, interactions with exercise training and sex/gender-related differences, which should be taken into account. Overall, pharmacological modulation of endothelial dysfunction and arterial stiffness as hallmarks of vascular ageing, holds great promise for improving cardiovascular health in the ageing population. Nonetheless, further research is needed to fully elucidate the underlying mechanisms and optimize the efficacy and safety of these interventions for clinical translation.
Asunto(s)
Envejecimiento , Rigidez Vascular , Humanos , Envejecimiento/metabolismo , Estrés Oxidativo , Senescencia Celular , Transducción de SeñalRESUMEN
The plasma membrane protein ankylosis homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question of whether PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi found in ENPP1-proficient cells. Ank activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1-/- mice contained <2.5% of the PPi found in bones of wild-type mice, showing that Enpp1 activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60% to 70% of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification therefore do so by extruding NTPs rather than PPi itself. © 2022 American Society for Bone and Mineral Research (ASBMR).
Asunto(s)
Adenosina Trifosfato , Calcinosis , Difosfatos , Proteínas de Transporte de Fosfato , Adenosina Trifosfato/metabolismo , Animales , Huesos/metabolismo , Huesos/patología , Calcificación Fisiológica , Calcinosis/metabolismo , Calcinosis/patología , Difosfatos/metabolismo , Células HEK293 , Humanos , Ratones , Proteínas de Transporte de Fosfato/metabolismoRESUMEN
Pseudoxanthoma elasticum (PXE, OMIM # 264800) is an autosomal recessive, multisystemic disorder, associated with mutations of the ABCC6 gene. Ectopic mineralization is in the background of the clinical manifestations of the disease. Calcium-salt crystals are deposited primarily in the skin, in the Bruch membrane of the eyes, and in the vascular endothelium. Thus, in addition to the skin lesions, visual impairment and cardiovascular involvement also occur. Clinical symptoms show varying severity and display heterogeneous appearance. The identification of the phenotype and care of the patients require a multidisciplinary perspective based on the collaboration of a dermatologist, ophthalmologist, cardiologist, and clinical geneticist. The aim of our work is to describe the development of symptoms of the disease, in order to facilitate the diagnosis. In addition, we aim to draw attention to the importance of early diagnosis of pseudoxanthoma elasticum, and to present modern diagnostic methods. Considering the development of severe systemic complications, the early diagnosis with the collaboration between related specialists is crucial to provide optimal clinical care and management of the patients.
Asunto(s)
Seudoxantoma Elástico , Lámina Basal de la Coroides , Humanos , Mutación , Fenotipo , Seudoxantoma Elástico/diagnóstico , Seudoxantoma Elástico/genética , Piel/patologíaRESUMEN
ABCC6 mediates release of ATP from hepatocytes into the blood. Extracellularly, ATP is converted into the mineralization inhibitor pyrophosphate. Consequently, inactivating mutations in ABCC6 give low plasma pyrophosphate and underlie the ectopic mineralization disorder pseudoxanthoma elasticum. How ABCC6 mediates cellular ATP release is still unknown. Fluorescent ABCC6 fusion proteins would allow mechanistic studies, but fluorophores attached to the ABCC6 N- or C-terminus result in intracellular retention and degradation. Here we describe that intramolecular introduction of fluorophores yields fully functional ABCC6 fusion proteins. A corresponding ABCC6 variant in which the catalytic glutamate of the second nucleotide binding domain was mutated, correctly routed to the plasma membrane but was inactive. Finally, N-terminal His10 or FLAG tags did not affect activity of the fusion proteins, allowing their purification for biochemical characterization.
Asunto(s)
Adenosina Trifosfato/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Recombinantes de Fusión/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Células HEK293 , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Dyskinesias are characterized by abnormal repetitive involuntary movements due to dysfunctional neuronal activity. Although levodopa-induced dyskinesia, characterized by tic-like abnormal involuntary movements, has no clinical treatment for Parkinson's disease patients, animal studies indicate that Riluzole, which interferes with glutamatergic neurotransmission, can improve the phenotype. The rat model of Levodopa-Induced Dyskinesia is a unilateral lesion with 6-hydroxydopamine in the medial forebrain bundle, followed by the repeated administration of levodopa. The molecular pathomechanism of Levodopa-Induced Dyskinesia is still not deciphered; however, the implication of epigenetic mechanisms was suggested. In this study, we investigated the striatum for DNA methylation alterations under chronic levodopa treatment with or without co-treatment with Riluzole. Our data show that the lesioned and contralateral striata have nearly identical DNA methylation profiles. Chronic levodopa and levodopa + Riluzole treatments led to DNA methylation loss, particularly outside of promoters, in gene bodies and CpG poor regions. We observed that several genes involved in the Levodopa-Induced Dyskinesia underwent methylation changes. Furthermore, the Riluzole co-treatment, which improved the phenotype, pinpointed specific methylation targets, with a more than 20% methylation difference relative to levodopa treatment alone. These findings indicate potential new druggable targets for Levodopa-Induced Dyskinesia.
Asunto(s)
Cuerpo Estriado , Metilación de ADN/efectos de los fármacos , Discinesia Inducida por Medicamentos/tratamiento farmacológico , Levodopa/toxicidad , Riluzol , Animales , Cuerpo Estriado/metabolismo , Cuerpo Estriado/patología , Ratas , Ratas Wistar , Riluzol/farmacología , Riluzol/uso terapéuticoRESUMEN
DMRP, an ABC transporter encoded by the dMRP/CG6214 gene, is the Drosophila melanogaster orthologue of the "long" human multidrug resistance-associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6, and MRP7/ABCC10). In order to provide a detailed biochemical characterisation we expressed DMRP in Sf9 insect cell membranes. We demonstrated DMRP as a functional orthologue of its human counterparts capable of transporting several human MRP substrates like beta-estradiol 17-beta-D-glucuronide, leukotriene C4, calcein, fluo3 and carboxydichlorofluorescein. Unexpectedly, we found DMRP to exhibit an extremely high turnover rate for the substrate transport as compared to its human orthologues. Furthermore, DMRP showed remarkably high basal ATPase activity (68-75 nmol Pi/mg membrane protein/min), which could be further stimulated by probenecid and the glutathione conjugate of N-ethylmaleimide. Surprisingly, this high level basal ATPase activity was inhibited by the transported substrates. We discussed this phenomenon in the light of a potential endogenous substrate (or activator) present in the Sf9 membrane.
Asunto(s)
Drosophila melanogaster/química , Drosophila melanogaster/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico , Línea Celular , Drosophila melanogaster/genética , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Spodoptera , Especificidad por SustratoRESUMEN
Intervertebral disc degeneration presents a wide spectrum of clinically degenerative disc phenotypes; however, the contribution of genetic background to the degenerative outcomes has not been established. We characterized the spinal phenotype of 3 mouse strains with varying cartilage-regenerative potential at 6 and 23 months: C57BL/6, LG/J and SM/J. All strains showed different aging phenotypes. Importantly, LG/J mice showed an increased prevalence of dystrophic disc calcification in caudal discs with aging. Quantitative-histological analyses of LG/J and SM/J caudal discs evidenced accelerated degeneration compared to BL6, with cellular disorganization and cell loss together with fibrosis of the NP, respectively. Along with the higher grades of disc degeneration, SM/J, at 6M, also differed the most in terms of NP gene expression compared to other strains. Moreover, although we found common DEGs between BL6 and LG/J aging, most of them were divergent between the strains. Noteworthy, the common DEGs altered in both LG/J and BL6 aging were associated with inflammatory processes, response to stress, cell differentiation, cell metabolism and cell division. Results suggested that disc calcification in LG/J resulted from a dystrophic calcification process likely aggravated by cell death, matrix remodelling, changes in calcium/phosphate homeostasis and cell transformation. Lastly, we report 7 distinct phenotypes of human disc degeneration based on transcriptomic profiles, that presented similar pathways and DEGs found in aging mouse strains. Together, our results suggest that disc aging and degeneration depends on the genetic background and involves changes in various molecular pathways, which might help to explain the diverse phenotypes seen during disc disease.
Asunto(s)
Senescencia Celular , Disco Intervertebral/patología , Animales , Disco Intervertebral/metabolismo , Ratones , Ratones Endogámicos , FenotipoRESUMEN
Epidemiological studies indicate that elevated alkaline phosphatase activity is associated with increased cardiovascular disease risk. Other epidemiological data demonstrate that mothers giving multiple childbirths (multipara) are also at increased risk of developing late-onset cardiovascular disease. We hypothesized that these two associations stem from a common cause, the insufficient plasma level of the ectopic mineralization inhibitor inorganic pyrophosphate, which is a substrate of alkaline phosphatase. As alkaline phosphatase activity is elevated in pregnancy, we hypothesized that pyrophosphate concentrations decrease gestationally, potentially leading to increased maternal vascular calcification and cardiovascular disease risk in multipara. We investigated plasma pyrophosphate kinetics pre- and postpartum in sheep and at term in humans and demonstrated its shortage in pregnancy, mirroring alkaline phosphatase activity. Next, we tested whether multiparity is associated with increased vascular calcification in pseudoxanthoma elasticum patients, characterized by low intrinsic plasma pyrophosphate levels. We demonstrated that these patients had increased vascular calcification when they give birth multiple times. We propose that transient shortages of pyrophosphate during repeated pregnancies might contribute to vascular calcification and multiparity-associated cardiovascular disease risk threatening hundreds of millions of healthy women worldwide. Future trials are needed to assess if gestational pyrophosphate supplementation might be a suitable prophylactic treatment to mitigate maternal cardiovascular disease risk in multiparous women.
RESUMEN
Methotrexate (MTX) is a widely used chemotherapeutic agent, immune suppressant and antimalarial drug. It is a substrate of several human ABC proteins that confer multidrug resistance to cancer cells and determine compartmentalization of a wide range of physiological metabolites and endo or xenobiotics, by their primary active transport across biological membranes. The substrate specificity and tissue distribution of these promiscuous human ABC transporters show a high degree of redundancy, providing robustness to these key physiological and pharmacological processes, such as the elimination of toxins, e.g. methotrexate from the body. A similar network of proteins capable of transporting methotrexate has been recently suggested to exist in Drosophila melanogaster. One of the key players of this putative network is Drosophila Multidrug-resistance Associated Protein (DMRP). DMRP has been shown to be a highly active and promiscuous ABC transporter, capable of transporting various organic anions. Here we provide the first direct evidence that DMRP, expressed alone in a heterologous system lacking other, potentially functionally overlapping D. melanogaster organic anion transporters, is indeed able to transport methotrexate. Our in vitro results support the hypothesized but debated role of DMRP in in vivo methotrexate excretion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Drosophila/metabolismo , Metotrexato/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Transporte Biológico Activo , Drosophila melanogaster/metabolismo , Técnicas In VitroRESUMEN
5-Hydroxymethylcytosine (5hmC) is produced from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) dioxygenases. The epigenetic modification 5hmC has crucial roles in both cellular development and differentiation. The 5hmC level is particularly high in the brain. While 5mC is generally associated with gene silencing/reduced expression, 5hmC is a more permissive epigenetic mark. To understand its physiological function, an easy and accurate quantification method is required. Here, we have developed a novel LC-MS/MS-based approach to quantify both genomic 5mC and 5hmC contents. The method is based on the liberation of nucleobases by formic acid. Applying this method, we characterized the levels of DNA methylation and hydroxymethylation in mouse brain and liver, primary hepatocytes, and various cell lines. Using this approach, we confirm that the treatment of different cell lines with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine leads to a decrease in 5mC content. This decrease was accompanied by an increase in 5hmC levels in cell lines of hematopoietic origin. Finally, we showed that ascorbate elevates the levels of 5hmC and augments the effect of 5-aza-2'-deoxycytidine without significantly influencing 5mC levels.