RESUMO
Mevalonate kinase deficiency (MKD) is an autoinflammatory metabolic disorder caused by bi-allelic loss-of-function variants in the MVK gene, resulting in decreased activity of the encoded mevalonate kinase (MK). Clinical presentation ranges from the severe early-lethal mevalonic aciduria to the milder hyper-IgD syndrome (MKD-HIDS), and is in the majority of patients associated with recurrent inflammatory episodes with often unclear cause. Previous studies with MKD-HIDS patient cells indicated that increased temperature, as caused by fever during an inflammatory episode, lowers the residual MK activity, which causes a temporary shortage of non-sterol isoprenoids that promotes the further development of inflammation. Because an increase of the residual MK activity is expected to make MKD-HIDS patients less sensitive to developing inflammatory episodes, we established a cell-based screen that can be used to identify compounds and/or therapeutic targets that promote this increase. Using a reporter HeLa cell line that stably expresses the most common MKD-HIDS variant, MK-V377I, C-terminally tagged with bioluminescent NanoLuc luciferase (nLuc), we screened the Prestwick Chemical Library®, which includes 1280 FDA-approved compounds. Multiple compounds increased MK-V377I-nLuc bioluminescence, including steroids (i.e., glucocorticoids, estrogens, and progestogens), statins and antineoplastic drugs. The glucocorticoids increased MK-V377I-nLuc bioluminescence through glucocorticoid receptor signaling. Subsequent studies in MKD-HIDS patient cells showed that the potent glucocorticoid clobetasol propionate increases gene transcription of MVK and other genes regulated by the transcription factor sterol regulatory element-binding protein 2 (SREBP-2). Our results suggest that increasing the flux through the isoprenoid biosynthesis pathway by targeting the glucocorticoid receptor or SREBP-2 could be a potential therapeutic strategy in MKD-HIDS.
Assuntos
Deficiência de Mevalonato Quinase , Humanos , Deficiência de Mevalonato Quinase/tratamento farmacológico , Deficiência de Mevalonato Quinase/genética , Células HeLa , Receptores de Glucocorticoides/uso terapêutico , Proteína de Ligação a Elemento Regulador de Esterol 1 , Fosfotransferases (Aceptor do Grupo Álcool)RESUMO
Peroxisomes are single-membrane bounded organelles that in humans play a dual role in lipid metabolism, including the degradation of very long-chain fatty acids and the synthesis of ether lipids/plasmalogens. The first step in de novo ether lipid synthesis is mediated by the peroxisomal enzyme glyceronephosphate O-acyltransferase, which has a strict substrate specificity reacting only with the long-chain acyl-CoAs. The aim of this study was to determine the origin of these long-chain acyl-CoAs. To this end, we developed a sensitive method for the measurement of de novo ether phospholipid synthesis in cells and, by CRISPR-Cas9 genome editing, generated a series of HeLa cell lines with deficiencies of proteins involved in peroxisomal biogenesis, beta-oxidation, ether lipid synthesis, or metabolite transport. Our results show that the long-chain acyl-CoAs required for the first step of ether lipid synthesis can be imported from the cytosol by the peroxisomal ABCD proteins, in particular ABCD3. Furthermore, we show that these acyl-CoAs can be produced intraperoxisomally by chain shortening of CoA esters of very long-chain fatty acids via beta-oxidation. Our results demonstrate that peroxisomal beta-oxidation and ether lipid synthesis are intimately connected and that the peroxisomal ABC transporters play a crucial role in de novo ether lipid synthesis.
Assuntos
Ácidos Graxos , Plasmalogênios , Humanos , Plasmalogênios/metabolismo , Células HeLa , Ácidos Graxos/metabolismo , Peroxissomos/metabolismo , Oxirredução , Acil Coenzima A/metabolismo , ÉteresRESUMO
Perrault syndrome (PS) is a rare recessive disorder characterized by ovarian dysgenesis and sensorineural deafness. It is clinically and genetically heterogeneous, and previously mutations have been described in different genes, mostly related to mitochondrial proteostasis. We diagnosed three unrelated females with PS and set out to identify the underlying genetic cause using exome sequencing. We excluded mutations in the known PS genes, but identified a single homozygous mutation in the ERAL1 gene (c.707A > T; p.Asn236Ile). Since ERAL1 protein binds to the mitochondrial 12S rRNA and is involved in the assembly of the small mitochondrial ribosomal subunit, the identified variant represented a likely candidate. In silico analysis of a 3D model for ERAL1 suggested that the mutated residue hinders protein-substrate interactions, potentially affecting its function. On a molecular basis, PS skin fibroblasts had reduced ERAL1 protein levels. Complexome profiling of the cells showed an overall decrease in the levels of assembled small ribosomal subunit, indicating that the ERAL1 variant affects mitochondrial ribosome assembly. Moreover, levels of the 12S rRNA were reduced in the patients, and were rescued by lentiviral expression of wild type ERAL1. At the physiological level, mitochondrial respiration was markedly decreased in PS fibroblasts, confirming disturbed mitochondrial function. Finally, knockdown of the C. elegans ERAL1 homologue E02H1.2 almost completely blocked egg production in worms, mimicking the compromised fertility in PS-affected women. Our cross-species data in patient cells and worms support the hypothesis that mutations in ERAL1 can cause PS and are associated with changes in mitochondrial metabolism.
Assuntos
Proteínas de Ligação ao GTP/genética , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/genética , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos/genética , Animais , Caenorhabditis elegans/genética , Exoma , Feminino , Proteínas de Ligação ao GTP/metabolismo , Disgenesia Gonadal 46 XX/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Homozigoto , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares/metabolismo , Mutação , Mutação de Sentido Incorreto/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequenciamento do ExomaRESUMO
X-linked adrenoleukodystrophy (ALD) is a severe neurodegenerative disorder caused by the accumulation of very long-chain fatty acids (VLCFA) due to mutations in the ABCD1 gene. The phenotypic spectrum ranges from a fatal cerebral demyelinating disease in childhood (cerebral ALD) to a progressive myelopathy without cerebral involvement in adulthood (adrenomyeloneuropathy). Because ABCD1 mutations have no predictive value with respect to clinical outcome a role for modifier genes was postulated. We report that the CYP4F2 polymorphism rs2108622 increases the risk of developing cerebral ALD in Caucasian patients. The rs2108622 polymorphism (c.1297G>A) results in an amino acid substitution valine for methionine at position 433 (p.V433M). Using cellular models of VLCFA accumulation, we show that p.V433M decreases the conversion of VLCFA into very long-chain dicarboxylic acids by ω-oxidation, a potential escape route for the deficient peroxisomal ß-oxidation of VLCFA in ALD. Although p.V433M does not affect the catalytic activity of CYP4F2 it reduces CYP4F2 protein levels markedly. These findings open perspectives for therapeutic interventions in a disease with currently limited treatment options.
Assuntos
Adrenoleucodistrofia , Família 4 do Citocromo P450 , Ácidos Graxos , Polimorfismo Genético , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/metabolismo , Linhagem Celular , Família 4 do Citocromo P450/genética , Família 4 do Citocromo P450/metabolismo , Ácidos Graxos/genética , Ácidos Graxos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease that is caused by mutations in the ABCD1 gene and characterized by elevated levels of very long-chain fatty acids (VLCFA) in plasma and tissues, with the most pronounced increase in the central nervous system. Virtually all male patients develop adrenal insufficiency and myelopathy (adrenomyeloneuropathy), but a subset develops a fatal cerebral demyelinating disease (known as cerebral ALD). Female patients may also develop myelopathy, but adrenal insufficiency or leukodystrophy are very rare. ALD has been associated with mitochondrial dysfunction, oxidative stress and bioenergetic failure, but the mechanism by which VLCFA accumulation triggers these effects has not been resolved thus far. In this study, we used primary human fibroblasts from normal subjects and ALD patients to investigate whether VLCFA can induce endoplasmic reticulum stress. We show that saturated VLCFA (C26:0) induce endoplasmic reticulum stress in fibroblasts from ALD patients, but not in controls. Furthermore, there is a clear correlation between the chain-length of the fatty acid and the induction of endoplasmic reticulum stress. Exposure of ALD fibroblasts to C26:0, resulted in increased expression of additional endoplasmic reticulum stress markers (EDEM1, GADD34 and CHOP) and in lipoapoptosis. This new insight into the underlying mechanism of VLCFA-induced toxicity is of great importance for the development of a disease modifying treatment for ALD aimed at the normalization of VLCFA levels in tissues.
Assuntos
Adrenoleucodistrofia/metabolismo , Ácidos Graxos/metabolismo , Fibroblastos/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/patologia , Estresse do Retículo Endoplasmático , Ácidos Graxos/genética , Fibroblastos/patologia , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/genética , Mitocôndrias/patologia , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismoRESUMO
X-linked adrenoleukodystrophy (X-ALD) is a neurometabolic disease that is caused by mutations in the ABCD1 gene. ABCD1 protein deficiency impairs peroxisomal very long-chain fatty acid (VLCFA) degradation resulting in increased cytosolic VLCFA-CoA levels, which are further elongated by the VLCFA-specific elongase, ELOVL1. In adulthood, X-ALD most commonly manifests as a gradually progressive myelopathy (adrenomyeloneuropathy; AMN) without any curative or disease modifying treatments. We recently showed that bezafibrate reduces VLCFA accumulation in X-ALD fibroblasts by inhibiting ELOVL1. Although, in a clinical trial, bezafibrate was unable to lower VLCFA levels in plasma or lymphocytes in X-ALD patients, inhibition of ELOVL1 remains an attractive therapeutic option. In this study, we investigated the kinetic characteristics of ELOVL1 using X-ALD fibroblasts and microsomal fractions from ELOVL1 over-expressing HEK293 cell lines and analyzed the inhibition kinetics of a series of fibrates. Our data show that the CoA esters of bezafibrate and gemfibrozil reduce chain elongation by specifically inhibiting ELOVL1. These fibrates can therefore serve as lead compounds for the development of more potent and more specific inhibitors for ELOVL1.
Assuntos
Acetiltransferases/metabolismo , Adrenoleucodistrofia/enzimologia , Ácidos Graxos/biossíntese , Fibroblastos/enzimologia , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Adrenoleucodistrofia/genética , Bezafibrato/farmacologia , Inibidores Enzimáticos/farmacologia , Elongases de Ácidos Graxos , Fibroblastos/efeitos dos fármacos , Genfibrozila/farmacologia , Células HEK293 , Humanos , Hipolipemiantes/farmacologia , Cinética , Microssomos/efeitos dos fármacos , Microssomos/enzimologia , TransfecçãoRESUMO
Mitochondria integrate metabolic networks for maintaining bioenergetic requirements. Deregulation of mitochondrial metabolic networks can lead to mitochondrial dysfunction, which is a common hallmark of many diseases. Reversible post-translational protein acetylation modifications are emerging as critical regulators of mitochondrial function and form a direct link between metabolism and protein function, via the metabolic intermediate acetyl-CoA. Sirtuins catalyze protein deacetylation, but how mitochondrial acetylation is determined is unclear. We report here a mechanism that explains mitochondrial protein acetylation dynamics in vivo. Food withdrawal in mice induces a rapid increase in hepatic protein acetylation. Furthermore, using a novel LC-MS/MS method, we were able to quantify protein acetylation in human fibroblasts. We demonstrate that inducing fatty acid oxidation in fibroblasts increases protein acetylation. Furthermore, we show by using radioactively labeled palmitate that fatty acids are a direct source for mitochondrial protein acetylation. Intriguingly, in a mouse model that resembles human very-long chain acyl-CoA dehydrogenase (VLCAD) deficiency, we demonstrate that upon food-withdrawal, hepatic protein hyperacetylation is absent. This indicates that functional fatty acid oxidation is necessary for protein acetylation to occur in the liver upon food withdrawal. Furthermore, we now demonstrate that protein acetylation is abundant in human liver peroxisomes, an organelle where acetyl-CoA is solely generated by fatty acid oxidation. Our findings provide a mechanism for metabolic control of protein acetylation, which provides insight into the pathophysiogical role of protein acetylation dynamics in fatty acid oxidation disorders and other metabolic diseases associated with mitochondrial dysfunction.
Assuntos
Acetilcoenzima A/metabolismo , Ácidos Graxos/metabolismo , Acetilação , Animais , Western Blotting , Cromatografia Líquida , Eletroforese em Gel de Poliacrilamida , Fibroblastos/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Peroxissomos/metabolismo , Espectrometria de Massas em TandemRESUMO
X-linked adrenoleukodystrophy (ALD), a progressive neurodegenerative disease, is caused by mutations in ABCD1 and characterized by very-long-chain fatty acids (VLCFA) accumulation. In male patients, an increased plasma VLCFA levels in combination with a pathogenic mutation in ABCD1 confirms the diagnosis. Recent studies have shown that many women with ALD also develop myelopathy. Correct diagnosis is important for management including genetic counseling. Diagnosis in women can only be confirmed when VLCFA levels are elevated or when a known pathogenic ABCD1 mutation is identified. However, in 15-20% of women with ALD VLCFA plasma levels are not elevated. Demonstration that a novel sequence variant is pathogenic can be a challenge when VLCFA levels are in the normal range. Here we report two women with a clinical presentation compatible with ALD, an ABCD1 variation (p.Arg17His and p.Ser358Pro) of unknown significance, but with normal VLCFA levels. We developed a diagnostic test that is based on generating clonal cell lines that express only one of the two alleles. Subsequent biochemical studies enabled us to show that the two sequence variants were not pathogenic, thereby excluding the diagnosis ALD in these women. We conclude that the clonal approach is an important addition to the existing diagnostic array.
Assuntos
Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/genética , Adrenoleucodistrofia/diagnóstico , Adrenoleucodistrofia/genética , Triagem de Portadores Genéticos/métodos , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP/metabolismo , Adrenoleucodistrofia/sangue , Adulto , Alelos , Linhagem Celular , Análise Mutacional de DNA , Ácidos Graxos/sangue , Feminino , Variação Genética , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Peroxissomos/metabolismo , Doenças da Medula Espinal/diagnóstico , Doenças da Medula Espinal/genéticaRESUMO
Oxidation of unsaturated fatty acids requires the action of auxiliary enzymes, such as Δ(3),Δ(2)-enoyl-CoA isomerases. Here we describe a detailed biochemical, molecular, histological, and evolutionary characterization of Eci3, the fourth member of the mammalian enoyl-CoA isomerase family. Eci3 specifically evolved in rodents after gene duplication of Eci2. Eci3 is with 79% identity homologous to Eci2 and contains a peroxisomal targeting signal type 1. Subcellular fractionation of mouse kidney and immunofluorescence studies revealed a specific peroxisomal localization for Eci3. Expression studies showed that mouse Eci3 is almost exclusively expressed in kidney. By using immunohistochemistry, we found that Eci3 is not only expressed in cells of the proximal tubule, but also in a subset of cells in the tubulointerstitium and the glomerulus. In vitro, Eci3 catalyzed the isomerization of trans-3-nonenoyl-CoA to trans-2-nonenoyl-CoA equally efficient as Eci2, suggesting a role in oxidation of unsaturated fatty acids. However, in contrast to Eci2, in silico gene coexpression and enrichment analysis for Eci3 in kidney did not yield carboxylic acid metabolism, but diverse biological functions, such as ion transport (P=7.1E-3) and tissue morphogenesis (P=1.0E-3). Thus, Eci3 picked up a novel and unexpected role in kidney function during rodent evolution.
Assuntos
Dodecenoil-CoA Isomerase/metabolismo , Rim/enzimologia , Animais , Sequência de Bases , Primers do DNA , Imunofluorescência , Humanos , CamundongosRESUMO
Mevalonate kinase deficiency (MKD) is an autosomal recessive metabolic disorder associated with recurrent autoinflammatory episodes. The disorder is caused by bi-allelic loss-of-function variants in the MVK gene, which encodes mevalonate kinase (MK), an early enzyme in the isoprenoid biosynthesis pathway. To identify molecular and cellular consequences of MKD, we studied primary fibroblasts from severely affected patients with mevalonic aciduria (MKD-MA) and more mildly affected patients with hyper IgD and periodic fever syndrome (MKD-HIDS). As previous findings indicated that the deficient MK activity in MKD impacts protein prenylation in a temperature-sensitive manner, we compared the subcellular localization and activation of the small Rho GTPases RhoA, Rac1 and Cdc42 in control, MKD-HIDS and MKD-MA fibroblasts cultured at physiological and elevated temperatures. This revealed a temperature-induced altered subcellular localization and activation in the MKD cells. To study if and how the temperature-induced ectopic activation of these signalling proteins affects cellular processes, we performed comparative transcriptome analysis of control and MKD-MA fibroblasts cultured at 37 °C or 40 °C. This identified cell cycle and actin cytoskeleton organization as respectively most down- and upregulated gene clusters. Further studies confirmed that these processes were affected in fibroblasts from both patients with MKD-MA and MKD-HIDS. Finally, we found that, similar to immune cells, the MK deficiency causes metabolic reprogramming in MKD fibroblasts resulting in increased expression of genes involved in glycolysis and the PI3K/Akt/mTOR pathway. We postulate that the ectopic activation of small GTPases causes inappropriate signalling contributing to the molecular and cellular aberrations observed in MKD.
Assuntos
Fibroblastos , Deficiência de Mevalonato Quinase , Deficiência de Mevalonato Quinase/genética , Deficiência de Mevalonato Quinase/metabolismo , Deficiência de Mevalonato Quinase/patologia , Humanos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Células Cultivadas , Transdução de SinaisRESUMO
Objective: Bi-allelic pathogenic variants in the MVK gene, which encodes mevalonate kinase (MK), an essential enzyme in isoprenoid biosynthesis, cause the autoinflammatory metabolic disorder mevalonate kinase deficiency (MKD). We generated and characterized MK-deficient monocytic THP-1 cells to identify molecular and cellular mechanisms that contribute to the pro-inflammatory phenotype of MKD. Methods: Using CRISPR/Cas9 genome editing, we generated THP-1 cells with different MK deficiencies mimicking the severe (MKD-MA) and mild end (MKD-HIDS) of the MKD disease spectrum. Following confirmation of previously established disease-specific biochemical hallmarks, we studied the consequences of the different MK deficiencies on LPS-stimulated cytokine release, glycolysis versus oxidative phosphorylation rates, cellular chemotaxis and protein kinase activity. Results: Similar to MKD patients' cells, MK deficiency in the THP-1 cells caused a pro-inflammatory phenotype with a severity correlating with the residual MK protein levels. In the MKD-MA THP-1 cells, MK protein levels were barely detectable, which affected protein prenylation and was accompanied by a profound pro-inflammatory phenotype. This included a markedly increased LPS-stimulated release of pro-inflammatory cytokines and a metabolic switch from oxidative phosphorylation towards glycolysis. We also observed increased activity of protein kinases that are involved in cell migration and proliferation, and in innate and adaptive immune responses. The MKD-HIDS THP-1 cells had approximately 20% residual MK activity and showed a milder phenotype, which manifested mainly upon LPS stimulation or exposure to elevated temperatures. Conclusion: MK-deficient THP-1 cells show the biochemical and pro-inflammatory phenotype of MKD and are a good model to study underlying disease mechanisms and therapeutic options of this autoinflammatory disorder.
Assuntos
Lipopolissacarídeos , Deficiência de Mevalonato Quinase , Fosfotransferases (Aceptor do Grupo Álcool) , Humanos , Lipopolissacarídeos/metabolismo , Células THP-1 , Fenótipo , Deficiência de Mevalonato Quinase/metabolismo , Fosforilação OxidativaRESUMO
We report on a 25-year-old female with intellectual disability, mildly unusual face, and a pervasive developmental disorder, in whom routine aCGH showed a 298 kb de novo deletion at chromosome 2q24.1(156869529-157167986 × 1). The region contained two genes (NR4A2; GPD2). Molecular studies in the proposita showed an additional variant in GPD2 (c.614C > T, p.Pro205Leu), which was predicted to be pathogenic. The variant was also present in the healthy mother and sister. Functional analysis showed absent GPD2 activity in the proposita and 50% activity in mother and sister. We conclude that we have been able to find circumstantial evidence for the causative effect of the hemizygous GPD2 mutation but full proof remained lacking. Total costs for the work-up in these patients were high (21,975 [$27,029]). Similar results will increasingly be found when Next Generation Techniques will be applied widely in patients with intellectual disability, and proving pathogenicity by functional studies or in animal models will be expensive. We advocate the use of freely accessible international databases combining phenotype and genotype data using standard nomenclatures to facilitate proving pathogenicity of research data and to decrease costs of health care.
Assuntos
Glicerolfosfato Desidrogenase/genética , Hemizigoto , Deficiência Intelectual/genética , Adulto , Hibridização Genômica Comparativa , Feminino , Humanos , Masculino , Mutação , Análise de Sequência de DNA/economiaRESUMO
X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene encoding ALDP, an ATP-binding-cassette (ABC) transporter located in the peroxisomal membrane. ALDP deficiency results in impaired peroxisomal ß-oxidation and the subsequent accumulation of very long-chain fatty acids (VLCFA; > C22:0) in plasma and tissues. VLCFA are primarily derived from endogenous synthesis by ELOVL1. Therefore inhibiting this enzyme might constitute a feasible therapeutic approach. In this paper we demonstrate that bezafibrate, a PPAR pan agonist used for the treatment of patients with hyperlipidaemia reduces VLCFA levels in X-ALD fibroblasts. Surprisingly, the VLCFA-lowering effect was independent of PPAR activation and not caused by the increase in either mitochondrial or peroxisomal fatty acid ß-oxidation capacity. In fact, our results show that bezafibrate reduces VLCFA synthesis by decreasing the synthesis of C26:0 through a direct inhibition of fatty acid elongation activity. Taken together, our data indicate bezafibrate as a potential pharmacotherapeutic treatment for X-ALD. A clinical trial is currently ongoing to evaluate the effect in patients with X-ALD.
Assuntos
Adrenoleucodistrofia/tratamento farmacológico , Adrenoleucodistrofia/metabolismo , Bezafibrato/farmacologia , Ácidos Graxos/metabolismo , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/genética , Acetiltransferases/antagonistas & inibidores , Acetiltransferases/genética , Adrenoleucodistrofia/genética , Animais , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Elongases de Ácidos Graxos , Ácidos Graxos/química , Humanos , Hipolipemiantes/farmacologia , Masculino , Camundongos , Camundongos Transgênicos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Oxirredução , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genéticaRESUMO
ABC (ATP-binding cassette) subfamily D transporters are found in all eukaryotic kingdoms and are known to play essential roles in mammals and plants; however, their number, organization and physiological contexts differ. Via cross-kingdom expression experiments, we have explored the conservation of targeting, protein stability and function between mammalian and plant ABCD transporters. When expressed in tobacco epidermal cells, the mammalian ABCD proteins ALDP (adrenoleukodystrophy protein), ALDR (adrenoleukodystrophy-related protein) and PMP70 (70 kDa peroxisomal membrane protein) targeted faithfully to peroxisomes and P70R (PMP70-related protein) targeted to the ER (endoplasmic reticulum), as in the native host. The Arabidopsis thaliana peroxin AtPex19_1 interacted with human peroxisomal ABC transporters both in vivo and in vitro, providing an explanation for the fidelity of targeting. The fate of X-linked adrenoleukodystrophy disease-related mutants differed between fibroblasts and plant cells. In fibroblasts, levels of ALDP in some 'protein-absent' mutants were increased by low-temperature culture, in some cases restoring function. In contrast, all mutant ALDP proteins examined were stable and correctly targeted in plant cells, regardless of their fate in fibroblasts. ALDR complemented the seed germination defect of the Arabidopsis cts-1 mutant which lacks the peroxisomal ABCD transporter CTS (Comatose), but neither ALDR nor ALDP was able to rescue the defect in fatty acid ß-oxidation in establishing seedlings. Taken together, our results indicate that the mechanism for trafficking of peroxisomal membrane proteins is shared between plants and mammals, but suggest differences in the sensing and turnover of mutant ABC transporter proteins and differences in substrate specificity and/or function.
Assuntos
Transportadores de Cassetes de Ligação de ATP/fisiologia , Arabidopsis/metabolismo , Peroxissomos/fisiologia , Subfamília D de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília D de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/fisiopatologia , Adulto , Animais , Arabidopsis/efeitos dos fármacos , Proteínas de Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Especificidade da Espécie , Nicotiana/metabolismoRESUMO
Peroxisome biogenesis disorders within the Zellweger spectrum (PBD-ZSDs) are most frequently associated with the c.2528G>A (p.G843D) mutation in the PEX1 gene (PEX1-G843D), which results in impaired import of peroxisomal matrix proteins and, consequently, defective peroxisomal functions. A recent study suggested that treatment with autophagy inhibitors, in particular hydroxychloroquine, would be a potential therapeutic option for PBD-ZSD patients carrying the PEX1-G843D mutation. Here, we studied whether autophagy inhibition by chloroquine, hydroxychloroquine and 3-methyladenine indeed can improve peroxisomal functions in four different cell types with the PEX1-G843D mutation, including primary patient cells. Furthermore, we studied whether autophagy inhibition may be the mechanism underlying the previously reported improvement of peroxisomal functions by L-arginine in PEX1-G843D cells. In contrast to L-arginine, we observed no improvement but a worsening of peroxisomal metabolic functions and peroxisomal matrix protein import by the autophagy inhibitors, while genetic knock-down of ATG5 and NBR1 in primary patient cells resulted in only a minimal improvement. Our results do not support the use of autophagy inhibitors as potential treatment for PBD-ZSD patients, whereas L-arginine remains a therapeutically promising compound.
RESUMO
Very-long-chain fatty acids (VLCFAs) have long been known to be degraded exclusively in peroxisomes via beta-oxidation. A defect in peroxisomal beta-oxidation results in elevated levels of VLCFAs and is associated with the most frequent inherited disorder of the central nervous system white matter, X-linked adrenoleukodystrophy. Recently, we demonstrated that VLCFAs can also undergo omega-oxidation, which may provide an alternative route for the breakdown of VLCFAs. The omega-oxidation of VLCFA is initiated by CYP4F2 and CYP4F3B, which produce omega-hydroxy-VLCFAs. In this article, we characterized the enzymes involved in the formation of very-long-chain dicarboxylic acids from omega-hydroxy-VLCFAs. We demonstrate that very-long-chain dicarboxylic acids are produced via two independent pathways. The first is mediated by an as yet unidentified, microsomal NAD(+)-dependent alcohol dehydrogenase and fatty aldehyde dehydrogenase, which is encoded by the ALDH3A2 gene and is deficient in patients with Sjögren-Larsson syndrome. The second pathway involves the NADPH-dependent hydroxylation of omega-hydroxy-VLCFAs by CYP4F2, CYP4F3B, or CYP4F3A. Enzyme kinetic studies show that oxidation of omega-hydroxy-VLCFAs occurs predominantly via the NAD(+)-dependent route. Overall, our data demonstrate that in humans all enzymes are present for the complete conversion of VLCFAs to their corresponding very-long-chain dicarboxylic acids.
Assuntos
Ácidos Graxos/metabolismo , Metabolismo dos Lipídeos , Adrenoleucodistrofia , Aldeído Oxirredutases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450 , Ácidos Dicarboxílicos/metabolismo , Humanos , Redes e Vias Metabólicas , NAD/metabolismo , OxirreduçãoRESUMO
BACKGROUND: Sjögren-Larsson syndrome is a metabolic disorder characterized by accumulation of long-chain fatty alcohols in plasma of patients due to mutations in the ALDH3A2 gene, that codes for a microsomal fatty aldehyde dehydrogenase (FALDH). Recent studies have demonstrated that FALDH is involved in the last step of the conversion of 22-hydroxy-C22:0 into the dicarboxylic acid of C22:0 (C22:0-DCA). METHODS: FALDH activity was determined by incubating fibroblast homogenates with omega-hydroxy-C22:0 in the presence of NAD(+). Electrospray ionization mass spectrometry (ESI-MS) was used to quantify the amounts of C22:0-DCA produced. RESULTS: All SLS patients were deficient in C22:0-DCA productions with activities ranging from 3.2-26.3% of mean control. CONCLUSIONS: The new assay described in this paper has substantial advantages over previous assays, and allows for the easy, reliable and rapid diagnosis of SLS.
Assuntos
Aldeído Oxirredutases/genética , Síndrome de Sjogren-Larsson/diagnóstico , Espectrometria de Massas por Ionização por Electrospray/métodos , Humanos , Síndrome de Sjogren-Larsson/enzimologia , Síndrome de Sjogren-Larsson/genéticaRESUMO
Zellweger spectrum disorders (ZSDs) are autosomal recessive diseases caused by defective peroxisome assembly. They constitute a clinical continuum from severe early lethal to relatively milder presentations in adulthood. Liver disease is a prevalent symptom in ZSD patients. The underlying pathogenesis for the liver disease, however, is not fully understood. We report a hypomorphic ZSD mouse model, which is homozygous for Pex1-c.2531G>A (p.G844D), the equivalent of the most common pathogenic variant found in ZSD, and which predominantly presents with liver disease. After introducing the Pex1-G844D allele by knock-in, we characterized homozygous Pex1-G844D mice for survival, biochemical parameters, including peroxisomal and mitochondrial functions, organ histology, and developmental parameters. The first 20 post-natal days (P20) were critical for survival of homozygous Pex1-G844D mice (~20% survival rate). Lethality was likely due to a combination of cholestatic liver problems, liver dysfunction and caloric deficit, probably as a consequence of defective bile acid biosynthesis. Survival beyond P20 was nearly 100%, but surviving mice showed a marked delay in growth. Surviving mice showed similar hepatic problems as described for mild ZSD patients, including hepatomegaly, bile duct proliferation, liver fibrosis and mitochondrial alterations. Biochemical analyses of various tissues showed the absence of functional peroxisomes accompanied with aberrant levels of peroxisomal metabolites predominantly in the liver, while other tissues were relatively spared. ur findings show that homozygous Pex1-G844D mice have a predominant liver disease phenotype, mimicking the hepatic pathology of ZSD patients, and thus constitute a good model to study pathogenesis and treatment of liver disease in ZSD patients.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Hepatopatias/etiologia , Hepatopatias/metabolismo , Fígado/metabolismo , Síndrome de Zellweger/complicações , ATPases Associadas a Diversas Atividades Celulares/genética , Alelos , Animais , Modelos Animais de Doenças , Feminino , Fibroblastos , Humanos , Fígado/patologia , Hepatopatias/patologia , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Peroxissomos , FenótipoAssuntos
Adrenoleucodistrofia/tratamento farmacológico , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Lovastatina/uso terapêutico , Adrenoleucodistrofia/sangue , LDL-Colesterol/sangue , Estudos Cross-Over , Método Duplo-Cego , Ácidos Graxos/análise , Ácidos Graxos/sangue , Humanos , Projetos PilotoRESUMO
Coenzyme A (CoASH) is an obligate cofactor for lipids undergoing beta-oxidation in peroxisomes. Although the peroxisomal membrane appears to be impermeable to CoASH, peroxisomes contain their own pool of CoASH. It is believed that CoASH enters peroxisomes as acyl-CoAs, but it is not known how this pool is regulated. The mouse nudix hydrolase 7 (NUDT7alpha) was previously identified in peroxisomes as a CoA-diphosphatase, and therefore suggested to be involved in regulation of peroxisomal CoASH levels. Here we show that mouse NUDT7alpha mainly acts as an acyl-CoA diphosphatase, with highest activity towards medium-chain acyl-CoAs, and much lower activity with CoASH. Nudt7alpha mRNA is highly expressed in liver, brown adipose tissue and heart, similar to enzymes involved in peroxisomal lipid degradation. Nudt7alpha mRNA is down-regulated by Wy-14,643, a peroxisome proliferator-activated receptor alpha (PPARalpha) ligand, in a PPARalpha-dependent manner in mouse liver. In highly purified peroxisomes, nudix hydrolase activity is highest with C(6)-CoA and is decreased by fibrate treatment. Under certain conditions, such as treatment with peroxisome proliferators or fasting, an increase in peroxisomal CoASH levels has been reported, which is in line with a decreased expression/activity of NUDT7alpha. Taken together these data suggest that NUDT7alpha function is tightly linked to peroxisomal CoASH/acyl-CoA homeostasis.