Substrate Specificity Overlap and Interaction between Adrenoleukodystrophy Protein (ALDP/ABCD1) and Adrenoleukodystrophy-related Protein (ALDRP/ABCD2)

Abstract

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes a peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D called ALDP. ALDP is supposed to function as a homodimer allowing the entry of CoA-esters of very-long chain fatty acids (VLCFA) into the peroxisome, the unique site of their ␤-oxidation. ALDP deficiency can be corrected by overexpression of ALDRP, its closest homolog. However, the exact nature of the substrates transported by ALDRP and its relationships with ALDP still remain unclear. To gain insight into the function of ALDRP, we used cell models allowing the induction in a dose-dependent manner of a wild type or a mutated non-functional ALDRP-EGFP fusion protein. We explored the consequences of the changes of ALDRP expression levels on the fatty acid content (saturated, monounsaturated, and polyunsaturated fatty acids) in phospholipids as well as on the levels of ␤-oxidation of 3 suspected substrates: C26:0, C24:0, and C22:6n-3 (DHA). We found an inverse correlation between the fatty acid content of saturated (C26:0, C24:0) and monounsaturated (C26:1, C24:1) VLCFA and the expression level of ALDRP. Interestingly, we obtained a transdominant-negative effect of the inactive ALDRP-EGFP on ALDP function. This effect is due to a physical interaction between ALDRP and ALDP that we evidenced by proximity ligation assays and coimmunoprecipitation. Finally, the ␤-oxidation assays demonstrate a role of ALDRP in the metabolism of saturated VLCFA (redundant with that of ALDP) but also a specific involvement of ALDRP in the metabolism of DHA. X-linked adrenoleukodystrophy (X-ALD)2 (OMIM 300100) is a very complex neurodegenerative disorder whose physiopathogenesis remains to be clarified (1). X-ALD is caused by mutations in the ABCD1 gene located in Xq28, which encodes a peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D called ALDP (adrenoleukodystrophy-protein) (2). This protein has the structure of a half ABC transporter, which is supposed to function as a homodimer (3, 4). However, heterodimerization with one of the other members of the ABCD subfamily (i.e. ALDRP (encoded by the ABCD2 gene (5)), PMP70 (ABCD3 (6)), and PMP69 (ABCD4 (7, 8)) cannot be excluded especially in the situation where these proteins are overexpressed. Although a mirror expression pattern is often observed between ALDP and ALDRP when specific cell types are analyzed (9), peroxisomal ABC transporters have overlapping expression patterns rendering possible such interactions (5, 10). Coimmunoprecipitation experiments or FRET analysis have demonstrated heterodimerization in cells overexpressing the peroxisomal ABC transporters (11,12). Although the peroxisomal localization of ALDP, ALDRP, and PMP70 is clearly demonstrated, PMP69 has recently been described to be localized in the endoplasmic reticulum and was found to be absent in the peroxisome. This excludes a possible interaction at the peroxisomal membrane with the other ABC...

Structure-Function Analysis of Peroxisomal ATP-binding Cassette Transporters Using Chimeric Dimers

“…The level of C24:6 was decreased only in the presence of ABCD2 and ABCD2-ABCD2, ABCD1-ABCD2, or ABCD2-ABCD1 chimeric proteins but not in the presence of ABCD1 or ABCD1-ABCD1 chimeric dimers. This result reinforces the supposed important role of ABCD2 in PUFA metabolism (8,10,49) and is in agreement with the fact that ABCD2 expression is regulated by PUFA (50). It also surprisingly shows that one TMD from ABCD2 is sufficient to ensure C24:6-CoA transport.…”

“…From the increased ␤-oxidation of C24:6, we could have expected an increase of DHA. However, we previously demonstrated that ABCD2 expression increased ␤-oxidation of DHA (10). It is therefore consistent to find that expression of ABCD2 or of chimeric proteins containing at least one ABCD2 moiety result in equilibrium.…”

“…Cell Culture-Rat H4IIEC3 cells stably expressing ABCD2-EGFP (clone 28.38) (10,26) were cultured in DMEM/Ham's F-12 (1:1) supplemented with 5% FCS at 37°C in a humidified atmosphere of 5% CO 2 in the presence of 200 g/ml G418 (InvivoGen) and 200 g/ml hygromycin B (InvivoGen). Culture of these cells under doxycycline treatment permits a dose-dependent induction of the expression of ABCD2-EGFP.…”

Transporteurs ABC peroxysomaux et adrénoleucodystrophie liée au chromosome X

“…En effet, à la suite d'étapes d'élon-gation et de désaturation de l'acide α-linolénique (C18:3 n-3) dans le réticulum endoplasmique, il y a formation de C24:6 n-3 qui subit ensuite un cycle de β-oxydation (perte de 2 atomes de carbone) dans le peroxysome pour former le DHA. ABCD2 pourrait ainsi participer à la fois au transport du DHA pour sa dégradation et au transport de son précurseur immédiat pour sa synthèse [12][13][14]. Concernant ABCD3, là encore, il apparaît que la surexpression d'ABCD3 peut compenser biochimiquement la déficience en ABCD1, signe qu'ABCD3 peut transporter des AGTLC saturés et monoinsaturés, mais avec une moins bonne efficacité [9].…”

“…Chez les mammifères, plusieurs études ont suggéré qu'ABCD1, ABCD2 et ABCD3 sont fonctionnels sous forme d'homodimères. En fait, diffé-rentes équipes, dont la nôtre, ont démontré l'existence d'interactions physiques entre les différents hémitrans-porteurs, indiquant qu'homodimères et hétérodimères coexistent [14,16]. Les niveaux d'expression relatifs de ces trois transporteurs varient fortement, que ce soit au cours du développement ou en fonction des tissus [17,18].…”