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

Emmanuelle C Genin,Flore Geillon,Catherine Gondcaille,Anne Athias,Philippe Gambert,Doriane Trompier,Stéphane Savary

doi:10.1074/jbc.m110.211912

Emmanuelle C Genin, Flore Geillon + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.211912

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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 β-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 β-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 β-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.

Highlights

Mutations in the ABCD1 gene located in Xq28, which encodes a peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D called ALDP [2]
The exact nature of the substrates transported by ALDRP and PMP70, their relationship with ALDP, and their state of dimerization absolutely needs further investigations in situations of induced overexpression, a situation found in pharmacological induction based therapies
The mutant ALDRP-EGFP corresponds to a single change of the amino acid sequence (D207H) mimicking a natural mutation occurring in the ABCD1 gene (D196H ALDP), which results in a non-functional protein still able to dimerize and to be targeted at the peroxisomal membrane [25]

Summary

Introduction

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]. The mutant ALDRP-EGFP corresponds to a single change of the amino acid sequence (D207H) mimicking a natural mutation occurring in the ABCD1 gene (D196H ALDP), which results in a non-functional protein still able to dimerize and to be targeted at the peroxisomal membrane [25]. The basal level of the fusion protein is quasi null in clone 19 (not different from H4IIEC3 control cells), a leaky expression of ALDRP-EGFP was observed in clone 28.

Results

Conclusion