Effect of dietary polyunsaturated fatty acids on the expression of peroxisomal ABC transporters

Peroxisomal ABC transporters encoded by the ABCD genes are thought to participate in the import of specific fatty acids in the peroxisomal matrix. ABCD1 deficiency is associated with X-linked adrenoleukodystrophy (X-ALD), the most frequent peroxisomal disorder which is characterized by the accumulation of saturated very-long-chain fatty acids (VLCFA). ABCD2 (the closest homolog of ABCD1) and ABCD3 have been shown to have partial functional redundancy with ABCD1; only when overexpressed, they can compensate for VLCFA accumulation. Other lipids, for instance polyunsaturated fatty acids (PUFA), should be possible candidate substrates for the ABCD2 and ABCD3 gene products, ALDRP and PMP70 respectively. Moreover, PUFA, which are known regulators of gene expression, could therefore represent potent inducers of the ABCD genes. To test this hypothesis, littermates of n-3-deficient rats were subjected to an n-3-deficient diet or equilibrated diets containing ALA (alpha-linolenic acid, 18:3n-3) as unique source of n-3 fatty acids or ALA plus DHA (docosahexaenoic acid, 22:6n-3) at two different doses. We analyzed the expression of peroxisomal ABC transporters and of the peroxisomal acyl-CoA oxidase gene 1 (Acox1) in adrenals, brain and liver. Whatever the diet, we did not observe any difference in gene expression in adrenals and brain. However, the hepatic expression level of Abcd2 and Abcd3 genes was found to be significantly higher in the n-3-deficient rats than in the rats fed the ALA diet or the DHA supplemented diets. This was accompanied by important changes in hepatic fatty acid composition. In summary, the hepatic expression of Abcd2 and Abcd3 but not of Abcd1 and Abcd4 appears to be highly sensitive towards dietary PUFA. This difference could be linked to the substrate specificity of the peroxisomal ABC transporters and a specific involvement of Abcd2 and Abcd3 in PUFA metabolism.     

ABC transporters and RNAi in Caenorhabditis elegans

RNAi is an evolutionarily conserved gene-silencing phenomenon that can be triggered by exogenous delivery of double stranded RNA to organisms. In Caenorhabditis elegans, the response to dsRNA is remarkably robust, and systemic RNAi responses are often observed. We have taken a genetic approach using this organism to better understand the mechanisms that facilitate RNAi. By analyzing strains of RNAi-defective mutants, we have uncovered an unexpected role for ABC transporters in RNAi and related silencing mechanisms. Ten of the sixty ABC transporter genes encoded in the C. elegans genome are required for robust RNAi. We will present data that highlights common features of these genes relative to their roles in RNAi, including genetic interactions with other components of the RNAi machinery. We will also describe unique roles for some transporter genes in endogenous RNAi-related processes.     

Targeted degradation of ABC transporters in health and disease

ATP binding cassette (ABC) transporters comprise an extended protein family involved in the transport of a broad spectrum of solutes across membranes. They consist of a common architecture including two ATP-binding domains converting chemical energy into conformational changes and two transmembrane domains facilitating transport via alternating access. This review focuses on the biogenesis, and more precisely, on the degradation of mammalian ABC transporters in the endoplasmic reticulum (ER). We enlighten the ER-associated degradation pathway in the context of misfolded, misassembled or tightly regulated ABC transporters with a closer view on the cystic fibrosis transmembrane conductance regulator (CFTR) and the transporter associated with antigen processing (TAP), which plays an essential role in the adaptive immunity. Three rather different scenarios affecting the stability and degradation of ABC transporters are discussed: (1) misfolded domains caused by a lack of proper intra- and intermolecular contacts within the ABC transporters, (2) deficient assembly with auxiliary factors, and (3) arrest and accumulation of an intermediate or ‘dead-end’ state in the transport cycle, which is prone to be recognized by the ER-associated degradation machinery.     

ABC transporters involved in the transport of plant secondary metabolites

Plants produce a large number of secondary metabolites, such as alkaloids, terpenoids, polyphenols, quinones and many further compounds having combined structures of those groups. Physiological roles of those metabolites for plants are still under investigation, but they play, at least in part, important functions as protectants for plant bodies against herbivores and pathogens, as well as from physical stresses like ultraviolet light and heat. In order to accomplish these functions, biosyntheses and accumulation of secondary metabolites are highly regulated in a temporal and spatial manner in plant organs, where they can appropriately accumulate. In this mini-review, I introduce the mechanism of accumulation and membrane transport of these metabolites, in particular, focusing on ATP-binding cassette transporters involved.     

Cholesterol fill-in model: mechanism for substrate recognition by ABC proteins

Many of the 48 or 49 human ABC proteins are involved in lipid homeostasis and in defence against hydrophobic substances in food and the environment. Defects in their functions cause various diseases, suggesting that they play very important roles in human health; however, the mechanism of how they handle enormous numbers of hydrophobic compounds with various structures and molecular weights, or phospholipids and cholesterol, major components of cellular membranes, is not known. We compared the functions of drug-transporting and lipid-transporting ABC proteins, and found that (1) ABC proteins, either lipid or drug transporters, have a similar substrate binding site which recognizes PL and cholesterol, or drugs and cholesterol; (2) Cholesterol in membranes binds to various ABC proteins together with PL or drugs, and plays an important role in substrate recognition, especially by ABCB1/MDR1, where cholesterol fills the empty space in the substrate binding site when small drugs bind to it. ABC proteins exert very flexible substrate recognition, i.e., one-to-many interaction rather than the conventional rigid one-to-one interaction. We propose calling the mechanism the “cholesterol fill-in model”.     

Multidrug resistance ABC transporters

Clinical multidrug resistance is caused by a group of integral membrane proteins that transport hydrophobic drugs and lipids across the cell membrane. One class of these permeases, known as multidrug resistance ATP binding cassette (ABC) transporters, translocate these molecules by coupling drug/lipid efflux with energy derived from the hydrolysis of ATP. In this review, we examine both the structures and conformational changes of multidrug resistance ABC transporters. Together with the available biochemical and structural evidence, we propose a general mechanism for hydrophobic substrate transport coupled to ATP hydrolysis.     

Peroxisomal membrane monocarboxylate transporters: evidence for a redox shuttle system?

One of the many functions of liver peroxisomes is the beta-oxidation of long-chain fatty acids. It is essential for the continuation of peroxisomal beta-oxidation that a redox shuttle system exist across the peroxisomal membrane to reoxidize NADH. We propose that this redox shuttle system consists of a substrate cycle between lactate and pyruvate. Here we present evidence that purified peroxisomal membranes contain both monocarboxylate transporter 1 (MCT 1) and MCT 2 and that along with peroxisomal lactate dehydrogenase (pLDH) form a Peroxisomal Lactate Shuttle. Peroxisomal beta-oxidation was greatly stimulated by the addition of pyruvate and this increase was partially inhibited by the addition of the MCT blocker alpha-cyano-4-hydroxycinnamate (CINN). We also found that peroxisomes generated lactate in the presence of pyruvate. Together these data provide compelling that the Peroxisome Lactate Shuttle helps maintain organelle redox and the proper functioning of peroxisomal beta-oxidation.     

Peroxisomal localization and function of NADP-specific isocitrate dehydrogenases in yeast

Yeast peroxisomal NADP⁺-specific isocitrate dehydrogenase (IDP3) contains a canonical type I peroxisomal targeting sequence (a carboxyl-terminal Cys-Lys-Leu tripeptide), and provides the NADPH required for β-oxidation of some fatty acids in that organelle. Cytosolic yeast IDP2 carrying a PTS1 (IDP2^+CKL) was only partially localized to peroxisomes, and the enzyme was able to function in lieu of either peroxisomal IDP3 or cytosolic IDP2. The analogous isocitrate dehydrogenase enzyme (IDPA) from Aspergillus nidulans, irrespective of the presence or absence of a putative PTS1, was found to exhibit patterns of dual compartmental distribution and of dual function in yeast similar to those observed for IDP2^+CKL. To test a potential cellular limit on peroxisomal levels, authentic yeast IDP3, which is normally strictly peroxisomal, was over-expressed. This also resulted in dual distribution and function of the enzyme in both the cytosol and in peroxisomes, supporting the possibility of a restriction on organellar amounts of IDP.     

The PDR-type ABC transporters AtrA and AtrG are involved in azole drug resistance in Aspergillus oryzae

For strain improvement of Aspergillus oryzae, development of the transformation system is essential, wherein dominant selectable markers, including drug-resistant genes, are available. However, A. oryzae generally has a relatively high resistance to many antifungal drugs effective against yeasts and other filamentous fungi. In the course of the study, while investigating azole drug resistance in A. oryzae, we isolated a spontaneous mutant that exhibited high resistance to azole fungicides and found that pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporter genes were upregulated in the mutant; their overexpression in the wild-type strain increased azole drug resistance. While deletion of the gene designated atrG resulted in increased azole susceptibility, double deletion of atrG and another gene (atrA) resulted in further azole hypersensitivity. Overall, these results indicate that the ABC transporters AtrA and AtrG are involved in azole drug resistance in A. oryzae.     

Analysis of the inhibition potential of zosuquidar derivatives on selected bacterial and fungal ABC transporters

Abstract

The increasing number of multidrug-resistant pathogenic microorganisms is a serious public health issue. Among the multitude of mechanisms that lead to multidrug resistance, the active extrusion of toxic compounds, mediated by MDR efflux pumps, plays an important role. In our study we analyzed the inhibitory capability of 26 synthesized zosuquidar derivatives on three ABC-type MDR efflux pumps, namely Saccharomyces cerevisiae Pdr5 as well as Lactococcus lactis LmrA and LmrCD. For Pdr5, five compounds could be identified that inhibited rhodamine 6G transport more efficiently than zosuquidar. One of these is a compound with a new catechol acetal structure that might represent a new lead compound. Furthermore, the determination of IC₅₀ values for rhodamine 6G transport of Pdr5 with representative compounds reveals values between 0.3 and 0.9 μM. Thus the identified compounds are among the most potent inhibitors known for Pdr5. For the ABC-type efflux pumps LmrA and LmrCD from L. lactis, seven and three compounds, which inhibit the transport activity more than the lead compound zosuquidar, were found. Interestingly, transport inhibition for LmrCD was very specific, with a drastic reduction by one compound while its diastereomers showed hardly an effect. Thus, the present study reveals new potent inhibitors for the ABC-type MDR efflux pumps studied with the inhibitors of Pdr5 and LmrCD being of particular interest as these proteins are well known model systems for their homologs in pathogenic fungi and Gram-positive bacteria.     

Complete Characterization of the Human ABC Gene Family

The human ATP-binding cassette (ABC) transporters comprise a large family of membrane transport proteins and play a vital role in many cellular processes. The genes provide functions as diverse as peptide transport, cholesterol and sterol transport, bile acid, retinoid, and iron transport. In addition some ABC genes play a role as regulatory elements. Many ABC genes play a role in human genetic diseases, and several are critical drug transport proteins overexpressed in drug resistant cells. Analysis of the gene products allows the genes to be grouped into seven different subfamilies.     

真菌的多向耐药性ABC转运蛋白

真菌的多向耐药性ABC转运蛋白(ATP-binding cassette transporters)是导致多药耐药性和抗真菌药物治疗效果明显下降的主要原因。文章对酿酒酵母(Saccharomyces cerevisiae)和主要致病真菌白色假丝酵母(Candida albicans)、新型隐球酵母(Cryptococcus neoformans)和烟曲霉(Aspergillus fumigatus)中的多向耐药性ABC转运蛋白的种类、药物外排机制以及基因表达调控网络的研究进展作一综述,为深入了解真菌的多向耐药性机制以及探讨克服多向耐药性的策略和提高药效提供参考。     

Peroxisomal ABC transporters: Structure, function and role in disease

ATP-binding cassette (ABC) transporters belong to one of the largest families of membrane proteins, and are present in almost all living organisms from eubacteria to mammals. They exist on plasma membranes and intracellular compartments such as the mitochondria, peroxisomes, endoplasmic reticulum, Golgi apparatus and lysosomes, and mediate the active transport of a wide variety of substrates in a variety of different cellular processes. These include the transport of amino acids, polysaccharides, peptides, lipids and xenobiotics, including drugs and toxins. Three ABC transporters belonging to subfamily D have been identified in mammalian peroxisomes. The ABC transporters are half-size and assemble mostly as a homodimer after posttranslational transport to peroxisomal membranes. ABCD1/ALDP and ABCD2/ALDRP are suggested to be involved in the transport of very long chain acyl-CoA with differences in substrate specificity, and ABCD3/PMP70 is involved in the transport of long and branched chain acyl-CoA. ABCD1 is known to be responsible for X-linked adrenoleukodystrophy (X-ALD), an inborn error of peroxisomal β-oxidation of very long chain fatty acids. Here, we summarize recent advances and important points in our advancing understanding of how these ABC transporters target and assemble to peroxisomal membranes and perform their functions in physiological and pathological processes, including the neurodegenerative disease, X-ALD. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Site-directed mutagenesis to enable and improve crystallizability of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase

The N-terminal part of Candida tropicalis MFE-2 (MFE-2(h2Delta)) having two (3R)-hydroxyacyl-CoA dehydrogenases with different substrate specificities has been purified and crystallized as a recombinant protein. The expressed construct was modified so that a stabile, homogeneous protein could be obtained instead of an unstabile wild-type form with a large amount of cleavage products. Cubic crystals with unit cell parameters a=74.895, b=78.340, c=95.445, and alpha=beta=gamma=90 degrees were obtained by using PEG 4000 as a precipitant. The crystals exhibit the space group P2(1)2(1)2(1) and contain one molecule, consisting of two different (3R)-hydroxyacyl-CoA dehydrogenases, in the asymmetric unit. The crystals diffract to a resolution of 2.2A at a conventional X-ray source.     

Lipid dependence of ABC transporter localization and function

Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. ATP-binding cassette (ABC) transporters have also been localized in these membrane domains. In this review the evidence for this specific localization will be evaluated and discussed in terms of relevance to ABC transporter function. We will focus on three ABC transporters of the A, B and C subfamily, respectively. Two of these transporters are relevant to multidrug resistance in tumor cells (Pgp/ABCB1 and MRP1/ABCC1), while the third (ABCA1) is extensively studied in relation to the reverse cholesterol pathway and cellular cholesterol homeostasis. We will attempt to derive a generalized model of lipid rafts to which they associate based on the use of various different lipid raft isolation procedures. In the context of lipid rafts, modulation of ABC transporter localization and function by two relevant lipid classes, i.e. sphingolipids and cholesterol, will be discussed.     

Nitrate transporters and peptide transporters

In higher plants, two types of nitrate transporters, NRT1 and NRT2, have been identified. In Arabidopsis, there are 53 NRT1 genes and 7 NRT2 genes. NRT2 are high-affinity nitrate transporters, while most members of the NRT1 family are low-affinity nitrate transporters. The exception is CHL1 (AtNRT1.1), which is a dual-affinity nitrate transporter, its mode of action being switched by phosphorylation and dephosphorylation of threonine 101. Two of the NRT1 genes, CHL1 and AtNRT1.2, and two of the NRT2 genes, AtNRT2.1 and AtNRT2.2, are known to be involved in nitrate uptake. In addition, AtNRT1.4 is required for petiole nitrate storage. On the other hand, some members of the NRT1 family are dipeptide transporters, called PTRs, which transport a broad spectrum of di/tripeptides. In barley, HvPTR1, expressed in the plasma membrane of scutellar epithelial cells, is involved in mobilizing peptides, produced by hydrolysis of endosperm storage protein, to the developing embryo. In higher plants, there is another family of peptide transporters, called oligopeptide transporters (OPTs), which transport tetra/pentapeptides. In addition, some OPTs transport GSH, GSSH, GSH conjugates, phytochelatins, and metals.     

Heterologous Expression of a Mammalian ABC Transporter in Plant and its Application to Phytoremediation

Mammalian ATP-binding cassette (ABC) transporters involved in the multidrug-resistance of cancer cells can efflux cytotoxic compounds that show a wide variety of chemical structures and biological activities. Human multidrug resistance-associated protein (hMRP1) is one of the most intensively studied ABC transporters and many substrates have been identified, including both organic and inorganic compounds. In an attempt at novel ‘transport engineering’ using hMRP1 as a molecular pump, we established transgenic tobacco plants that showed clear resistance to cadmium and daunorubicin, although they were not resistant to etoposide, another known substrate of hMRP1. When expressed in tobacco cells, hMRP1 protein was localized at vacuolar membrane, while members of the MRP family are localized at plasma membrane in mammalian cells to reduce the cellular accumulation of various drugs. Thus, the hMRP1-expressing tobacco cells were able to take up these substrates across the tonoplast and sequestrate them in the vacuolar matrix. These results suggest that it may be possible to use the transgenic tobacco in phytoremediation, where a single transformation with an ABC transporter with broad substrate specificity should be effective for extracting various environmental pollutants including both organic and inorganic compounds, and accumulate them in the plant body. This should be advantageous for the remediation of a complex polluted environment, which is commonly found in the real world. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. An erratum to this article is available at .