ABSTRACT
X-linked adrenoleukodystrophy (X-ALD) is an inherited neurodegenerative disorder associated with mutations of the ABCD1 gene that encodes a peroxisomal transmembrane protein. It results in accumulation of very long chain fatty acids in tissues and body fluid. Along with other factors such as epigenetic and environmental involvement, ABCD1 mutation-provoked disorders can present different phenotypes including cerebral adrenoleukodystrophy (cALD), adrenomyeloneuropathy (AMN), and peripheral neuropathy. cALD is the most severe form that causes death in young childhood. Bone marrow transplantation and hematopoietic stem cell gene therapy are only effective when performed at an early stage of onsets in cALD. Nonetheless, current research and development of novel therapies are hampered by a lack of in-depth understanding disease pathophysiology and a lack of reliable cALD models. The Abcd1 and Abcd1/Abcd2 knock-out mouse models as well as the deficiency of Abcd1 rabbit models created in our lab, do not develop cALD phenotypes observed in human beings. In this review, we summarize the clinical and biochemical features of X-ALD, the progress of pre-clinical and clinical studies. Challenges and perspectives for future X-ALD studies are also discussed.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D, Member 1/genetics , Adrenoleukodystrophy/therapy , Mutation , ATP Binding Cassette Transporter, Subfamily D/genetics , ATP Binding Cassette Transporter, Subfamily D/metabolism , ATP Binding Cassette Transporter, Subfamily D, Member 1/metabolism , Adrenoleukodystrophy/genetics , Adrenoleukodystrophy/metabolism , Animals , Disease Models, Animal , Genetic Predisposition to Disease , Humans , Mice, Knockout , Phenotype , Prognosis , Rabbits , Species SpecificityABSTRACT
ATP-binding cassette (ABC) transporters constitute one of the largest superfamilies of conserved proteins from bacteria to mammals. In humans, three members of this family are expressed in the peroxisomal membrane and belong to the subfamily D: ABCD1 (ALDP), ABCD2 (ALDRP), and ABCD3 (PMP70). These half-transporters must dimerize to form a functional transporter, but they are thought to exist primarily as tetramers. They possess overlapping but specific substrate specificity, allowing the transport of various lipids into the peroxisomal matrix. The defects of ABCD1 and ABCD3 are responsible for two genetic disorders called X-linked adrenoleukodystrophy and congenital bile acid synthesis defect 5, respectively. In addition to their role in peroxisome metabolism, it has recently been proposed that peroxisomal ABC transporters participate in cell signaling and cell control, particularly in cancer. This review presents an overview of the knowledge on the structure, function, and mechanisms involving these proteins and their link to pathologies. We summarize the different in vitro and in vivo models existing across the species to study peroxisomal ABC transporters and the consequences of their defects. Finally, an overview of the known and possible interactome involving these proteins, which reveal putative and unexpected new functions, is shown and discussed.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily D/genetics , ATP-Binding Cassette Transporters/genetics , Adrenoleukodystrophy/genetics , Adrenoleukodystrophy/pathology , Cholestasis/genetics , Cholestasis/pathology , Fatty Acids/genetics , Humans , Peroxisomes/geneticsABSTRACT
Unlike nucleobase modifications in canonical restriction-modification systems, DNA phosphorothioate (PT) epigenetic modification occurs in the DNA sugar-phosphate backbone when the nonbridging oxygen is replaced by sulfur in a double-stranded (ds) or single-stranded (ss) manner governed by DndABCDE or SspABCD, respectively. SspABCD coupled with SspE constitutes a defense barrier in which SspE depends on sequence-specific PT modifications to exert its antiphage activity. Here, we identified a new type of ssDNA PT-based SspABCD-SspFGH defense system capable of providing protection against phages through a mode of action different from that of SspABCD-SspE. We provide further evidence that SspFGH damages non-PT-modified DNA and exerts antiphage activity by suppressing phage DNA replication. Despite their different defense mechanisms, SspFGH and SspE are compatible and pair simultaneously with one SspABCD module, greatly enhancing the protection against phages. Together with the observation that the sspBCD-sspFGH cassette is widely distributed in bacterial genomes, this study highlights the diversity of PT-based defense barriers and expands our knowledge of the arsenal of phage defense mechanisms.IMPORTANCE We recently found that SspABCD, catalyzing single-stranded (ss) DNA phosphorothioate (PT) modification, coupled with SspE provides protection against phage infection. SspE performs both PT-simulated NTPase and DNA-nicking nuclease activities to damage phage DNA, rendering SspA-E a PT-sensing defense system. To our surprise, ssDNA PT modification can also pair with a newly identified 3-gene sspFGH cassette to fend off phage infection with a different mode of action from that of SspE. Interestingly, both SspFGH and SspE can pair with the same SspABCD module for antiphage defense, and their combination provides Escherichia coli JM109 with additive phage resistance up to 105-fold compared to that for either barrier alone. This agrees with our observation that SspFGH and SspE coexist in 36 bacterial genomes, highlighting the diversity of the gene contents and molecular mechanisms of PT-based defense systems.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D/genetics , Bacterial Proteins/genetics , DNA, Bacterial/genetics , Phosphates , Vibrio/genetics , ATP Binding Cassette Transporter, Subfamily D/classification , ATP Binding Cassette Transporter, Subfamily D/metabolism , Bacterial Proteins/metabolism , Bacteriophages/genetics , Bacteriophages/pathogenicity , Escherichia coli/genetics , Genome, Bacterial , Vibrio/metabolismABSTRACT
OBJECTIVE: To identify a pharmacological compound targeting macrophages, the most affected immune cells in inflammatory X-linked adrenoleukodystrophy (cerebral X-ALD) caused by ABCD1 mutations and involved in the success of hematopoietic stem cell transplantation and gene therapy. METHODS: A comparative database analysis elucidated the epigenetic repressing mechanism of the related ABCD2 gene in macrophages and identified the histone deacetylase (HDAC) inhibitor Vorinostat as a compound to induce ABCD2 in these cells to compensate for ABCD1 deficiency. In these cells, we investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ß-oxidation activity, accumulation of very long-chain fatty acids (VLCFAs) and their differentiation status. We investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ß-oxidation activity, accumulation of very long-chain fatty acids (VLCFA) and differentiation status. Three advanced cerebral X-ALD patients received Vorinostat and CSF and MRI diagnostics was carried out in one patient after 80 days of treatment. RESULTS: Vorinostat improved the metabolic defects in X-ALD macrophages by stimulating ABCD2 expression, peroxisomal ß-oxidation, and ameliorating VLCFA accumulation. Vorinostat interfered with pro-inflammatory skewing of X-ALD macrophages by correcting IL12B expression and further reducing monocyte differentiation. Vorinostat normalized the albumin and immunoglobulin CSF-serum ratios, but not gadolinium enhancement upon 80 days of treatment. INTERPRETATION: The beneficial effects of HDAC inhibitors on macrophages in X-ALD and the improvement of the blood-CSF/blood-brain barrier are encouraging for future investigations. In contrast with Vorinostat, less toxic macrophage-specific HDAC inhibitors might improve also the clinical state of X-ALD patients with advanced inflammatory demyelination.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D, Member 1/deficiency , ATP Binding Cassette Transporter, Subfamily D/drug effects , Adrenoleukodystrophy/drug therapy , Histone Deacetylase Inhibitors/pharmacology , Inflammation/drug therapy , Macrophages/drug effects , Vorinostat/pharmacology , Acute Disease , Adrenoleukodystrophy/cerebrospinal fluid , Adrenoleukodystrophy/diagnostic imaging , Coenzyme A Ligases/drug effects , Humans , Magnetic Resonance Imaging , Outcome Assessment, Health Care , PeroxisomesABSTRACT
BACKGROUND: Enterococci intrinsically resistant to cephalosporins represent a major cause of healthcare-associated infections, and the emergence of MDR makes therapeutic approaches particularly challenging. OBJECTIVES: Teichoic acids are cell wall glycopolymers present in Gram-positive bacteria. Teichoic acids can be modified by d-alanylation, which requires four proteins encoded by the dltABCD operon. Our objective was to evaluate the Dlt system as a druggable target to treat enterococcal infections. METHODS: The susceptibility of a d-alanylation-deficient strain of Enterococcus faecalis to ß-lactam antibiotics individually and/or in combination was analysed. Moreover, a DltA inhibitor was synthesized to test pharmacological inhibition of d-alanylation in vivo and in host using the animal model Galleria mellonella with different clinical isolates of E. faecalis and Enterococcus faecium. RESULTS: Most cephalosporins used as mono treatment had no impact on survival of the parental strain, but were slightly lethal for the dltA mutant of E. faecalis. Addition of a very low concentration of amoxicillin significantly increased killing of the dltA mutant under these conditions. The most spectacular effect was obtained with a combination of cefotaxime (1 mg/L) and amoxicillin (0.03 mg/L). In the presence of the inhibitor, the WT strain was as susceptible to this combination treatment as the dltA mutant. This molecule associated with the antibiotics was also effective in killing other E. faecalis clinical isolates and successfully prevented death of Galleria infected with either E. faecalis or E. faecium. CONCLUSIONS: The combined results support the potential usefulness of the Dlt system as a target to potentiate antibiotic combination therapies for the treatment of drug-resistant enterococci.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Enterococcus/drug effects , Enterococcus/growth & development , Teichoic Acids/genetics , beta-Lactams/pharmacology , ATP Binding Cassette Transporter, Subfamily D/genetics , Animals , Bacterial Proteins/antagonists & inhibitors , Enterococcus/genetics , Enterococcus faecalis/drug effects , Enterococcus faecalis/genetics , Enterococcus faecium/drug effects , Enterococcus faecium/genetics , Gram-Positive Bacterial Infections/microbiology , Larva/microbiology , Microbial Sensitivity Tests , Moths/microbiology , Teichoic Acids/chemistryABSTRACT
X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder, is associated with mutation in the ABCD1 gene which encodes a peroxisomal ATP-binding cassette transporter for very long-chain fatty acids (VLCFA). The biochemical hallmark of the disease is the accumulation of VLCFA. Peroxisomal defect in microglia being now considered a priming event in the pathology, we have therefore generated murine microglial cells mutated in the Abcd1 gene and its closest homolog, the Abcd2 gene. Using CRISPR/Cas9 gene editing strategy, we obtained 3 cell clones with a single or double deficiency. As expected, only the combined absence of ABCD1 and ABCD2 proteins resulted in the accumulation of VLCFA. Ultrastructural analysis by electron microscopy revealed in the double mutant cells the presence of lipid inclusions similar to those observed in brain macrophages of patients. These observations are likely related to the increased level of cholesterol and the accumulation of neutral lipids that we noticed in mutant cells. A preliminary characterization of the impact of peroxisomal defects on the expression of key microglial genes such as Trem2 suggests profound changes in microglial functions related to inflammation and phagocytosis. The expression levels of presumed modifier genes have also been found modified in mutant cells, making these novel cell lines relevant for use as in vitro models to better understand the physiopathogenesis of X-ALD and to discover new therapeutic targets.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily D/genetics , Adrenoleukodystrophy/genetics , ATP Binding Cassette Transporter, Subfamily D/metabolism , ATP Binding Cassette Transporter, Subfamily D, Member 1/metabolism , Adrenoleukodystrophy/metabolism , Adrenoleukodystrophy/pathology , Animals , CRISPR-Cas Systems , Cell Line , Fatty Acids/metabolism , Female , Gene Deletion , Mice, Inbred C57BL , Microglia/metabolism , Microglia/pathologyABSTRACT
BACKGROUND: ATP-binding cassette (ABC) transporters are involved in a huge range of physiological processes. Mutations in the ABCC6 gene cause pseudoxanthoma elasticum, a metabolic disease with progressive soft tissue calcification. METHODS: The aim of the present study was to analyze gene expression levels of selected ABC transporters associated with cholesterol homeostasis in metabolic active tissues, such as the liver, kidney and white adipose tissue (WAT) of Abcc6-/- mice from an early and late disease stage (six-month-old and 12-month-old mice). RESULTS: The strongest regulation of ABC transporter genes was observed in the liver tissue of six-month-old Abcc6-/- mice. Here, we found a significant increase of mRNA expression levels of phospholipid, bile salt and cholesterol/sterol transporters Abcb1b, Abcb11, Abcg1, Abcg5 and Abcg8. Abcd2 mRNA expression was increased by 3.2-fold in the liver tissue. We observed strong upregulation of Abca3 and Abca1 mRNA expression up to 3.3-fold in kidney and WAT, and a 2-fold increase of Abca9 mRNA in the WAT of six-month-old Abcc6 knockout mice. Gene expression levels of Abcb1b and Abcg1 remained increased in the liver tissue after an age-related disease progression, while we observed lower mRNA expression of Abca3 and Abca9 in the kidney and WAT of 12-month-old Abcc6-/- mice. CONCLUSIONS: These data support previous findings that Abcc6 deficiency leads to an altered gene expression of other ABC transporters depending on the status of disease progression. The increased expression of fatty acid, bile salt and cholesterol/sterol transporters may be linked to an altered cholesterol and lipoprotein metabolism due to a loss of Abcc6 function.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Adipose Tissue, White/metabolism , Kidney/metabolism , Liver/metabolism , Pseudoxanthoma Elasticum/genetics , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics , ATP Binding Cassette Transporter, Subfamily B, Member 11/metabolism , ATP Binding Cassette Transporter, Subfamily D/genetics , ATP Binding Cassette Transporter, Subfamily D/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 1/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 5/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 5/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 8/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 8/metabolism , ATP-Binding Cassette Transporters/deficiency , ATP-Binding Cassette Transporters/metabolism , Adipose Tissue, White/pathology , Animals , Bile Acids and Salts/metabolism , Biological Transport , Cholesterol/metabolism , Disease Models, Animal , Disease Progression , Female , Gene Expression Regulation , Kidney/pathology , Lipoproteins/genetics , Lipoproteins/metabolism , Liver/pathology , Male , Mice , Mice, Knockout , Multidrug Resistance-Associated Proteins , Pseudoxanthoma Elasticum/metabolism , Pseudoxanthoma Elasticum/pathologyABSTRACT
PURPOSE: Burn patients are particularly susceptible to microbial infection. Staphylococcus aureus causes burn wound, impetigo and cellulitis. Although sub-lethal antimicrobial photodynamic therapy (aPDT) would not result in microorganism killing, it can considerably influence microbial virulence factor. METHODS: Twelve methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) isolated from burns patients. To determine the sub-lethal dose of aPDT, 12 clinical isolates of S. aureus photosensitized with 100 µg ml -1 toluidine blue O (TBO) and irradiated by light emitting diode (LED) with a wavelength of 630 ± 10 nm and energy densities of 52.0, 104.1, and 156.2 J/cm2, then bacterial viability was measured. The effects of sub-lethal aPDT on the expression levels of ica ABCD and ica R genes were assessed by quantitative Real-time PCR (qRT-PCR) method. RESULT: Fifty and 100 µg ml-1 of TBO significantly reduced the mean cell survival in the MRSA (2.5 - 3 log10) and MSSA (2.75-3.1 log10) isolates. The average expression levels of icaA, ica B, ica C, and ica D in the MRSA and MSSA isolates were decreased by (12, 14, 11, and 9) and (13, 14.5, 12, and 9.5) fold change, respectively (P < 0.05). However, the expression of ica R gene was decreased by 6 and 8 folds change in MRSA and MSSA, respectively. CONCLUSION: The potential of TBO-mediated aPDT could reduce the expression of ica ABCD as important genes involved in biofilm formation and ica R gene as a repressor of the ica operon. Therefore, the use of aPDT agents as a complementary therapy in wound infections of burn patients is recommended.
Subject(s)
Biofilms/drug effects , Burns/microbiology , Photochemotherapy/methods , Photosensitizing Agents/pharmacology , Staphylococcus aureus/drug effects , Wound Infection/microbiology , ATP Binding Cassette Transporter, Subfamily D/drug effects , Dose-Response Relationship, Drug , Humans , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/genetics , Microbial Sensitivity Tests , Real-Time Polymerase Chain Reaction , Staphylococcus aureus/genetics , Tolonium Chloride/pharmacologyABSTRACT
Even though increasing evidence indicates the importance of peroxisomal lipid metabolism in regulating biological and pathological events, its involvement in cartilage development has not been well studied. Here, we identified the importance of peroxisomal function, particularly the functional integrity of ABCD2, in the pathogenesis of osteoarthritis (OA). Knockdown of ABCD2 in OA chondrocytes induced the accumulation of very long chain fatty acids (VLCFAs) and apoptotic cell death. Moreover, knockdown of ABCD2 altered profiles of miRNAs that affect the expression level of ACSL4, a known direct regulator of lipid metabolism. Suppression of ACSL4 in human chondrocytes-induced VLCFA accumulation, MMP-13 expression, and apoptotic cell death. In vivo morph-down of the ACSL4 homologue in zebrafish resulted in significant defects in cartilage development and in vivo knockdown of ACSL4 in cartilage tissue of an OA model mice promoted severe cartilage degradation. In summary, to the best of our knowledge, this is the first report suggesting that the regulatory network among peroxisomal ABCD2:ACSL4:VLCFA serves as a novel regulator of cartilage homeostasis, and these data may provide novel insights into the role of peroxisomal fatty acid metabolism in pathogenesis of human OA. SIGNIFICANCE OF THE STUDY: Our study indicates that peroxisomal dysfunction is closely related to OA pathogenesis. Particularly, the functional integrity of ABCD2 may play an important role in OA pathogenesis via the accumulation of VLCFAs and stimulation of apoptotic death through altering profiles of miRNAs that target ACSL4. Our findings suggest that targeting the regulatory network among the peroxisomal ABCD2:ACSL4:VLCFA axis may provide a new potential therapeutic strategy for OA pathogenesis.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D/metabolism , Coenzyme A Ligases/metabolism , Lipid Metabolism , MicroRNAs/metabolism , Osteoarthritis/metabolism , ATP Binding Cassette Transporter, Subfamily D/genetics , Adult , Animals , Apoptosis , Chondrocytes/metabolism , Chondrocytes/pathology , Coenzyme A Ligases/genetics , Disease Models, Animal , Fatty Acids/metabolism , Female , Gene Expression Profiling , Humans , Male , Mice , MicroRNAs/genetics , Middle Aged , Osteoarthritis/genetics , Osteoarthritis/pathology , Peroxisomes/metabolism , ZebrafishABSTRACT
Periplasmic thiol/disulfide oxidoreductases participate in the formation and isomerization of disulfide bonds and contribute to the virulence of pathogenic microorganisms. Among the systems encoded in the Salmonella genome, the system encoded by the scsABCD locus was shown to be required to cope with Cu and H2O2 stress. Here we report that this locus forms an operon whose transcription is driven by a promoter upstream of scsA and depends on CpxR/CpxA and on Cu. Furthermore, genes homologous to scsB, scsC, and scsD are always detected immediately downstream of scsA and in the same genetic arrangement in all scsA-harboring enterobacterial species. Also, a CpxR-binding site is detected upstream of scsA in most of those species, providing evidence of evolutionarily conserved function and regulation. Each individual scs gene shows a different role in copper and/or H2O2 resistance, indicating hierarchical contributions of these factors in the defense against these intoxicants. A protective effect of Cu preincubation against H2O2 toxicity and the increased Cu-mediated activation of cpxP in the ΔscsABCD mutant suggest that the CpxR/CpxA-controlled transcription of the ScsABCD system contributes to prevent Cu toxicity and to restore the redox balance at the Salmonella envelope.IMPORTANCE Copper intoxication triggers both specific and nonspecific responses in Salmonella The scs locus, which codes for periplasmic thiol/disulfide-oxidoreductase/isomerase-like proteins, has been the focus of attention because it is necessary for copper resistance, oxidative stress responses, and virulence and because it is not present in nonpathogenic Escherichia coli Still, the conditions under which the scs locus is expressed and the roles of its individual components remain unknown. In this report, we examine the contribution of each Scs factor to survival under H2O2 and copper stress. We establish that the scs genes form a copper-activated operon controlled by the CpxR/CpxA signal transduction system, and we provide evidence of its conserved gene arrangement and regulation in other bacterial pathogens.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D/genetics , Bacterial Proteins/genetics , Copper/pharmacology , Oxidative Stress , Protein Kinases/genetics , Salmonella typhi/genetics , Gene Expression Regulation, Bacterial , Hydrogen Peroxide/pharmacology , Operon , Periplasm/metabolism , Salmonella typhi/drug effects , Salmonella typhi/pathogenicity , Signal Transduction , Virulence Factors/geneticsABSTRACT
In mammals, four ATP-binding cassette (ABC) proteins belonging to subfamily D have been identified. ABCD1â3 are located on peroxisomal membrane and play an important role in the transportation of various fatty acid-CoA derivatives, including very long chain fatty acid-CoA, into peroxisomes. ABCD4 is located on lysosomal membrane and is suggested to be involved in the transport of vitamin B12 from lysosomes to the cytosol. However, the precise transport mechanism by which these ABC transporters facilitate the import or export of substrate has yet to be well elucidated. In this study, the overexpression of human ABCD1â4 in the methylotrophic yeast Pichia pastoris and a purification procedure were developed. The detergent-solubilized proteins were reconstituted into liposomes. ABCD1â4 displayed stable ATPase activity, which was inhibited by AlF3. Furthermore, ABCD1â4 were found to possess an equal levels of acyl-CoA thioesterase activity. Proteoliposomes is expected to be an aid in the further biochemical characterization of ABCD transporters.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D/chemistry , Liposomes/chemistry , Proteolipids/chemistry , Binding Sites , Enzyme Activation , Enzyme Stability , Kinetics , Protein BindingABSTRACT
ABCD1 and its homolog ABCD2 are peroxisomal ATP-binding cassette (ABC) half-transporters of fatty acyl-CoAs with both distinct and overlapping substrate specificities. Although it is established that ABC half-transporters have at least to dimerize to generate a functional unit, functional equivalents of tetramers (i.e. dimers of full-length transporters) have also been reported. However, oligomerization of peroxisomal ABCD transporters is incompletely understood but is of potential significance because more complex oligomerization might lead to differences in substrate specificity. In this work, we have characterized the quaternary structure of the ABCD1 and ABCD2 proteins in the peroxisomal membrane. Using various biochemical approaches, we clearly demonstrate that both transporters exist as both homo- and heterotetramers, with a predominance of homotetramers. In addition to tetramers, some larger molecular ABCD assemblies were also found but represented only a minor fraction. By using quantitative co-immunoprecipitation assays coupled with tandem mass spectrometry, we identified potential binding partners of ABCD2 involved in polyunsaturated fatty-acid metabolism. Interestingly, we identified calcium ATPases as ABCD2-binding partners, suggesting a role of ABCD2 in calcium signaling. In conclusion, we have shown here that ABCD1 and its homolog ABCD2 exist mainly as homotetramers in the peroxisomal membrane.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Peroxisomes/metabolism , ATP Binding Cassette Transporter, Subfamily D , ATP Binding Cassette Transporter, Subfamily D, Member 1 , Adenosine Triphosphate/metabolism , Animals , COS Cells , Calcium Signaling , Calcium-Transporting ATPases/metabolism , Carcinoma, Hepatocellular/metabolism , Cell Line , Chlorocebus aethiops , Green Fluorescent Proteins/metabolism , Liver Neoplasms/metabolism , Mass Spectrometry , Mice , Protein Binding , Protein Interaction Mapping , Protein Structure, Quaternary , Protein Transport , Rats , Tandem Mass SpectrometryABSTRACT
X-linked adrenoleukodystrophy (X-ALD) is a rare, genetic disorder characterized by adrenal insufficiency and central nervous system (CNS) demyelination. All patients with X-ALD have the biochemical abnormality of elevated blood and tissue levels of very long chain fatty acids (VLCFAs), saturated fatty acids with 24 to 26 carbons. X-ALD results from loss of function mutations in the gene encoding the peroxisomal transporter ABCD1, which is responsible for uptake of VLCFAs into peroxisomes for degradation by oxidation. One proposed therapeutic strategy for genetic complementation of ABCD1 is pharmacologic upregulation of ABCD2, a gene encoding a homologous peroxisomal transporter. Here, we show that thyroid hormone or sobetirome, a clinical-stage selective thyroid hormone receptor agonist, increases cerebral Abcd2 and lowers VLCFAs in blood, peripheral organs, and brains of mice with defective Abcd1. These results support an approach to treating X-ALD that involves a thyromimetic agent that reactivates VLCFA disposal both in the periphery and the CNS.
Subject(s)
ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Acetates/therapeutic use , Adrenoleukodystrophy/drug therapy , Adrenoleukodystrophy/metabolism , Brain/metabolism , Phenols/therapeutic use , Thyroid Hormones/therapeutic use , ATP Binding Cassette Transporter, Subfamily D , ATP Binding Cassette Transporter, Subfamily D, Member 1 , ATP-Binding Cassette Transporters/deficiency , Acetates/administration & dosage , Adrenoleukodystrophy/blood , Adrenoleukodystrophy/genetics , Animals , Biological Transport , Cell Line , Fatty Acids/blood , Fatty Acids/metabolism , Fibroblasts , Humans , Male , Mice , Peroxisomes/metabolism , Phenols/administration & dosage , Receptors, Thyroid Hormone/agonists , Thyroid Hormones/administration & dosage , Thyroid Hormones/metabolismABSTRACT
3-Methyldiphenylether (MDE) is an important alkyl-substituted diphenyl ether compound that is widely used as an intermediate in the synthesis of pyrethroid insecticides. An efficient MDE-degrading strain QY7-2, identified as Hydrogenophaga atypical, was isolated from activated sludge for the first time. Strain QY7-2 can utilize MDE as the sole carbon and energy source and completely mineralize MDE. The degradation pathway of MDE was proposed in the strain through metabolites identification. A gene cluster involving in methy-oxidation of MDE was cloned from QY7-2 and expressed in Escherichia coli BL21 (DE3), and the products were purified by SDS-PAGE. The specific activities of the recombinant enzymes MdeAB, MdeC and MdeD were 113.8 ± 3.5, 274.5 ± 6.2 and 673.4 ± 8.7 nmol min-1 mg-1, respectively. These results provide the biochemical and genetic foundation of microbial degradation pathway of MDE and benefit the bioremediation of MDE-contaminated environments.
Subject(s)
ATP Binding Cassette Transporter, Subfamily D/metabolism , Cloning, Molecular , Comamonadaceae/metabolism , Ether/metabolism , ATP Binding Cassette Transporter, Subfamily D/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Biodegradation, Environmental , Chromatography, High Pressure Liquid , Escherichia coli/metabolism , Ether/analysis , Ether/chemistry , Kinetics , Mutagenesis , Tandem Mass SpectrometryABSTRACT
ATP-binding cassette (ABC) transporters are one of the largest families of membrane-bound proteins and transport a wide variety of substrates across both extra- and intracellular membranes. They play a critical role in maintaining cellular homeostasis. To date, four ABC transporters belonging to subfamily D have been identified. ABCD1-3 and ABCD4 are localized to peroxisomes and lysosomes, respectively. ABCD1 and ABCD2 are involved in the transport of long and very long chain fatty acids (VLCFA) or their CoA-derivatives into peroxisomes with different substrate specificities, while ABCD3 is involved in the transport of branched chain acyl-CoA into peroxisomes. On the other hand, ABCD4 is deduced to take part in the transport of vitamin B12 from lysosomes into the cytosol. It is well known that the dysfunction of ABCD1 results in X-linked adrenoleukodystrophy, a severe neurodegenerative disease. Recently, it is reported that ABCD3 and ABCD4 are responsible for hepatosplenomegaly and vitamin B12 deficiency, respectively. In this review, the targeting mechanism and physiological functions of the ABCD transporters are summarized along with the related disease.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , ATP Binding Cassette Transporter, Subfamily D , ATP Binding Cassette Transporter, Subfamily D, Member 1 , ATP-Binding Cassette Transporters/genetics , Adrenoleukodystrophy/genetics , Adrenoleukodystrophy/metabolism , Adrenoleukodystrophy/pathology , Fatty Acids/metabolism , Humans , Lysosomes/genetics , Lysosomes/metabolism , Peroxisomes/genetics , Peroxisomes/metabolism , Substrate Specificity , Vitamin B 12 Deficiency/genetics , Vitamin B 12 Deficiency/metabolism , Vitamin B 12 Deficiency/pathologyABSTRACT
X-linked adrenoleukodystrophy (X-ALD) is a progressive neurometabolic disease caused by mutations/deletions in the Abcd1 gene. Similar mutations/deletions in the Abcd1 gene often result in diagonally opposing phenotypes of mild adrenomyeloneuropathy and severe neuroinflammatory cerebral adrenoleukodystrophy (ALD), which suggests involvement of downstream modifier genes. We recently documented the first evidence of loss of AMP-activated protein kinase α1 (AMPKα1) in ALD patient-derived cells. Here, we report the novel loss of AMPKα1 in postmortem brain white matter of patients with ALD phenotype. Pharmacological activation of AMPK can rescue the mitochondrial dysfunction and inhibit the pro-inflammatory response. The FDA approved anti-diabetic drug Metformin, a well-known AMPK activator, induces mitochondrial biogenesis and is documented for its anti-inflammatory role. We observed a dose-dependent activation of AMPKα1 in metformin-treated X-ALD patient-derived fibroblasts. Metformin also induced mitochondrial oxidative phosphorylation and ATP levels in X-ALD patient-derived fibroblasts. Metformin treatment decreased very long chain fatty acid levels and pro-inflammatory cytokine gene expressions in X-ALD patient-derived cells. Abcd2 [adrenoleukodystrophy protein-related protein] levels were increased in metformin-treated X-ALD patient-derived fibroblasts and Abcd1-KO mice primary mixed glial cells. Abcd2 induction was AMPKα1-dependent since metformin failed to induce Abcd2 levels in AMPKα1-KO mice-derived primary mixed glial cells. In vivo metformin (100 mg/Kg) in drinking water for 60 days induced Abcd2 levels and mitochondrial oxidative phosphorylation protein levels in the brain and spinal cord of Abcd1-KO mice. Taken together, these results provide proof-of-principle for therapeutic potential of metformin as a useful strategy for correcting the metabolic and inflammatory derangements in X-ALD by targeting AMPK. There is no effective therapy for inherited peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD). We document the therapeutic potential of FDA approved drug, Metformin, for X-ALD by targeting AMPK. Metformin induced peroxisomal Abcd2 levels in vitro and in vivo. Metformin lowered VLCFA levels, improved mitochondrial function and ameliorated inflammatory gene expression in X-ALD patient-derived cells. Metformin-induced Abcd2 levels were dependent on AMPKα1, a metabolic and anti-inflammatory gene, recently documented by our laboratory to play a putative role in X-ALD pathology. Read the Editorial Highlight for this article on page 10.
Subject(s)
AMP-Activated Protein Kinases/metabolism , ATP-Binding Cassette Transporters/metabolism , Adrenoleukodystrophy/enzymology , Adrenoleukodystrophy/pathology , Hypoglycemic Agents/pharmacology , Metformin/pharmacology , Up-Regulation/drug effects , AMP-Activated Protein Kinases/genetics , ATP Binding Cassette Transporter, Subfamily D , ATP Binding Cassette Transporter, Subfamily D, Member 1 , ATP-Binding Cassette Transporters/deficiency , ATP-Binding Cassette Transporters/genetics , Adrenoleukodystrophy/genetics , Animals , Animals, Newborn , Brain/metabolism , Brain/pathology , Disease Models, Animal , Humans , Mice , Mice, Knockout , Mitochondria/drug effects , Mitochondria/genetics , Nitric Oxide Synthase Type II/metabolism , Oxidation-Reduction/drug effects , Oxygen Consumption/drug effects , Oxygen Consumption/genetics , Phosphorylation/drug effects , Phosphorylation/genetics , Time Factors , Up-Regulation/geneticsABSTRACT
The aim of the present study was to introduce methods for exome sequencing of two ATP-binding cassette (ABC) transporters ABCC8 and ABCD2 recently suggested to play a putative role in breast cancer progression and prognosis of patients. We performed next generation sequencing targeted at analysis of all exons in ABCC8 and ABCD2 genes and surrounding noncoding sequences in blood DNA samples from 24 patients with breast cancer. The revealed alterations were characterized by in silico tools. We then compared the most frequent functionally relevant polymorphism rs757110 in ABCC8 with clinical data of patients. In total, the study identified 113 genetic alterations (>70 % novel ones) in both genes. Of these alterations, 83 were noncoding, 13 synonymous, 10 frameshifts and 7 were missense alterations. Four in silico programs predicted pathogenicity of two polymorphisms and four newly identified alterations. Rs757110 polymorphism in ABCC8 did not significantly associate with clinical data of the patients. In conclusion, exome sequencing identified several functionally relevant alterations in ABCC8 and ABCD2 genes that may further be used for a larger follow-up study aiming to assess their clinical significance.
Subject(s)
ATP-Binding Cassette Transporters/genetics , Breast Neoplasms/genetics , Exome/genetics , Genetic Association Studies , Sequence Analysis, DNA , Sulfonylurea Receptors/genetics , ATP Binding Cassette Transporter, Subfamily D , Adult , Breast Neoplasms/diagnosis , Female , Genetic Association Studies/methods , Humans , Middle Aged , Polymorphism, Single Nucleotide/genetics , Sequence Analysis, DNA/methodsABSTRACT
BACKGROUND: The aim of this study was to refine clinical risk factor stratification and make an optimal intervention plan to prevent ischemic stroke. MATERIAL/METHODS: Clinical data, including diffusion-weighted imaging (DWI) findings, were collected in a cohort of hospitalized transient ischemic attack (TIA) patients from January 2010 to December 2011. Recurrent cerebrovascular events after TIA, including recurrent TIA, minor stroke, and major stroke, were identified by face-to-face follow-up. A multivariate, ordinal, logistic regression model was used to determine significant predictors of recurrent events. RESULTS: Of 106 TIA patients, 24 (22.6%) had recurrent TIA and 20 (18.9%) had a stroke within 7 days. Hypertension, dyslipidemia, a history of ischemic stroke or TIA, and ABCD2 score were significantly associated with the recurrent events after TIA (P<0.001, P=0.02, P<0.001, P=0.02). Hypertension (RR=9.21; 95% CI, 3.07-27.61, P<0.001) and duration of symptom (RR=1.10; 95% CI, 1.02-1.17, P=0.01) as an item of ABCD2 score were highly predictive of the severity of recurrent events, whereas ABCD2 score as a whole (P=0.18) proved to be less strongly predictive. CONCLUSIONS: A history of hypertension and long duration of symptom independently and significantly predict severe recurrent events after TIA within 7 days, but a high ABCD2 score was less strongly predictive of severe recurrent events.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Ischemic Attack, Transient/diagnosis , Ischemic Attack, Transient/metabolism , ATP Binding Cassette Transporter, Subfamily D , Aged , Cohort Studies , Diffusion Magnetic Resonance Imaging , Female , Hospitalization , Hospitals , Humans , Hypertension/complications , Hypertension/pathology , Ischemic Attack, Transient/therapy , Magnetic Resonance Imaging , Male , Middle Aged , Multivariate Analysis , Predictive Value of Tests , Recurrence , Risk Assessment , Risk FactorsABSTRACT
ATP-binding cassette transporter D2 (D2) is an ABC half transporter that is thought to promote the transport of very long-chain fatty acyl-CoAs into peroxisomes. Both D2 and peroxisomes increase during adipogenesis. Although peroxisomes are essential to both catabolic and anabolic lipid metabolism, their function, and that of D2, in adipose tissues remain largely unknown. Here, we investigated the D2 localization and the proteome of D2-containing organelles, in adipose tissue. Centrifugation of mouse adipose homogenates generated a fraction enriched with D2, but deficient in peroxisome markers including catalase, PEX19, and ABCD3 (D3). Electron microscopic imaging of this fraction confirmed the presence of D2 protein on an organelle with a dense matrix and a diameter of â¼ 200 nm, the typical structure and size of a microperoxisome. D2 and PEX19 antibodies recognized distinct structures in mouse adipose. Immunoisolation of the D2-containing compartment confirmed the scarcity of PEX19 and proteomic profiling revealed the presence of proteins associated with peroxisome, endoplasmic reticulum (ER), and mitochondria. D2 is localized to a distinct class of peroxisomes that lack many peroxisome proteins, and may associate physically with mitochondria and the ER.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adipose Tissue/metabolism , Peroxisomes/metabolism , ATP Binding Cassette Transporter, Subfamily D , Animals , Endoplasmic Reticulum/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron , Microscopy, Fluorescence , Mitochondria/metabolism , Proteome/metabolism , Proteomics , Signal TransductionABSTRACT
The inherited peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD), associated with neurodegeneration and inflammatory cerebral demyelination, is caused by mutations in the ABCD1 gene encoding the peroxisomal ATP-binding cassette (ABC) transporter ABCD1 (ALDP). ABCD1 transports CoA-esters of very long-chain fatty acids (VLCFA) into peroxisomes for degradation by ß-oxidation; thus, ABCD1 deficiency results in VLCFA accumulation. The closest homologue, ABCD2 (ALDRP), when overexpressed, compensates for ABCD1 deficiency in X-ALD fibroblasts and in Abcd1-deficient mice. Microglia/macrophages have emerged as important players in the progression of neuroinflammation. Human monocytes, lacking significant expression of ABCD2, display severely impaired VLCFA metabolism in X-ALD. Here, we used thioglycollate-elicited primary mouse peritoneal macrophages (MPMΦ) from Abcd1 and Abcd2 single- and double-deficient mice to establish how these mutations affect VLCFA metabolism. By quantitative RT-PCR, Abcd2 mRNA was about half as abundant as Abcd1 mRNA in wild-type and similarly abundant in Abcd1-deficient MPMΦ. VLCFA (C26â¶0) accumulated about twofold in Abcd1-deficient MPMΦ compared with wild-type controls, as measured by gas chromatography-mass spectrometry. In Abcd2-deficient macrophages VLCFA levels were normal. However, upon Abcd1/Abcd2 double-deficiency, VLCFA accumulation was markedly increased (sixfold) compared with Abcd1-deficient MPMΦ. Elovl1 mRNA, encoding the rate-limiting enzyme for elongation of VLCFA, was equally abundant across all genotypes. Peroxisomal ß-oxidation of C26â¶0 amounted to 62% of wild-type activity in Abcd1-deficient MPMΦ and was significantly more impaired (29% residual activity) upon Abcd1/Abcd2 double-deficiency. Single Abcd2 deficiency did not significantly compromise ß-oxidation of C26â¶0. Thus, the striking accumulation of VLCFA in double-deficient MPMΦ compared with single Abcd1 deficiency was due to the loss of ABCD2-mediated, compensatory transport of VLCFA into peroxisomes. We propose that moderate endogenous expression of Abcd2 in Abcd1-deficient murine macrophages prevents the severe metabolic phenotype observed in human X-ALD monocytes, which lack appreciable expression of ABCD2. This supports upregulation of ABCD2 as a therapeutic concept in X-ALD.