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Structure-function analysis of peroxisomal ATP-binding cassette transporters using chimeric dimers
- Source :
- Journal of Biological Chemistry, Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2014, 289 (35), pp.24511-24520. 〈10.1074/jbc.M114.575506〉, Journal of Biological Chemistry, 2014, 289 (35), pp.24511-24520. ⟨10.1074/jbc.M114.575506⟩, Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2014, 289 (35), pp.24511-24520. ⟨10.1074/jbc.M114.575506⟩, Journal of biological chemistry, 289(35), 24511-24520. American Society for Biochemistry and Molecular Biology Inc., Journal of Biological Chemistry 35 (289), 24511-24520. (2014)
- Publication Year :
- 2014
- Publisher :
- HAL CCSD, 2014.
-
Abstract
- Background: Peroxisomal ABC transporters are predicted to function as homodimers in mammals. [br/] Results: ABCD1 interacts with ABCD2. Chimeric proteins mimicking full-length dimers represent novel tools for functional study. Artificial homodimers and heterodimers are functional. [br/] Conclusion: Interchangeability between ABCD1 and ABCD2 is confirmed, but PUFA transport depends on ABCD2. [br/] Significance: For the first time, heterodimers in mammals are proven to be functional.[br/] ABCD1 and ABCD2 are two closely related ATP-binding cassette half-transporters predicted to homodimerize and form peroxisomal importers for fatty acyl-CoAs. Available evidence has shown that ABCD1 and ABCD2 display a distinct but overlapping substrate specificity, although much remains to be learned in this respect as well as in their capability to form functional heterodimers. Using a cell model expressing an ABCD2-EGFP fusion protein, we first demonstrated by proximity ligation assay and co-immunoprecipitation assay that ABCD1 interacts with ABCD2. Next, we tested in the pxa1/pxa2 yeast mutant the functionality of ABCD1/ABCD2 dimers by expressing chimeric proteins mimicking homo- or heterodimers. For further structure-function analysis of ABCD1/ABCD2 dimers, we expressed chimeric dimers fused to enhanced GFP in human skin fibroblasts of X-linked adrenoleukodystrophy patients. These cells are devoid of ABCD1 and accumulate very long-chain fatty acids (C26:0 and C26:1). We checked that the chimeric proteins were correctly expressed and targeted to the peroxisomes. Very long-chain fatty acid levels were partially restored in transfected X-linked adrenoleukodystrophy fibroblasts regardless of the chimeric construct used, thus demonstrating functionality of both homo- and heterodimers. Interestingly, the level of C24:6 n-3, the immediate precursor of docosahexaenoic acid, was decreased in cells expressing chimeric proteins containing at least one ABCD2 moiety. Our data demonstrate for the first time that both homo- and heterodimers of ABCD1 and ABCD2 are functionally active. Interestingly, the role of ABCD2 (in homo- and heterodimeric forms) in the metabolism of polyunsaturated fatty acids is clearly evidenced, and the chimeric dimers provide a novel tool to study substrate specificity of peroxisomal ATP-binding cassette transporters.
- Subjects :
- [SDV.BA] Life Sciences [q-bio]/Animal biology
protéine chimere
animal diseases
ATP-binding cassette transporter
Proximity ligation assay
Protein Chimera
biochimie structurale
[ SDV.BA ] Life Sciences [q-bio]/Animal biology
Polymerase Chain Reaction
Biochemistry
Green fluorescent protein
interaction moléculaire
Mice
[ CHIM.OTHE ] Chemical Sciences/Other
homodimère
reproductive and urinary physiology
Animal biology
hétérodimère
chemistry.chemical_classification
[SDV.BA]Life Sciences [q-bio]/Animal biology
mammifère
Transfection
Peroxisome
protéine de fusion
[CHIM.OTHE] Chemical Sciences/Other
Dimerization
Plasmids
ABC Transporter
Fatty Acid
Protein-Protein Interaction
transporteur abc
Biology
Cell Line
Protein–protein interaction
Structure-Activity Relationship
Membrane Biology
Biologie animale
parasitic diseases
Autre (Chimie)
Peroxisomes
Animals
Humans
Molecular Biology
DNA Primers
Base Sequence
ABCD2
fungi
ABCD1
Fatty acid
Cell Biology
Fusion protein
Rats
chemistry
ATP-Binding Cassette Transporters
Other
[CHIM.OTHE]Chemical Sciences/Other
Subjects
Details
- Language :
- English
- ISSN :
- 00219258 and 1083351X
- Database :
- OpenAIRE
- Journal :
- Journal of Biological Chemistry, Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2014, 289 (35), pp.24511-24520. 〈10.1074/jbc.M114.575506〉, Journal of Biological Chemistry, 2014, 289 (35), pp.24511-24520. ⟨10.1074/jbc.M114.575506⟩, Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2014, 289 (35), pp.24511-24520. ⟨10.1074/jbc.M114.575506⟩, Journal of biological chemistry, 289(35), 24511-24520. American Society for Biochemistry and Molecular Biology Inc., Journal of Biological Chemistry 35 (289), 24511-24520. (2014)
- Accession number :
- edsair.doi.dedup.....e621dc68fb33428162a27924e4e55e65