ABCMdb

PMID: 21209459

Genin EC, Geillon F, Gondcaille C, Athias A, Gambert P, Trompier D, Savary S
Substrate specificity overlap and interaction between adrenoleukodystrophy protein (ALDP/ABCD1) and adrenoleukodystrophy-related protein (ALDRP/ABCD2).
J Biol Chem. 2011 Mar 11;286(10):8075-84. Epub 2011 Jan 5., [PubMed]

Sentences

No. Mutations Sentence Comment

40 ABCD2 p.Asp207His WT (clone 28) and D207H (clone 19) ALDRP-EGFP Tet-on cell clones were established as described previously (20), and cultured in the presence of 200 òe;g/ml of G418 (InvivoGen) and 200 òe;g/ml of hygromycin B (InvivoGen). Login to comment 41 ABCD2 p.Asp207His WT (clone 28) and D207H (clone 19) ALDRP-EGFP Tet-on cell clones were established as described previously (20), and cultured in the presence of 200 ␮g/ml of G418 (InvivoGen) and 200 ␮g/ml of hygromycin B (InvivoGen). Login to comment 45 ABCD2 p.Asp207His To further select subclones expressing the highest expression levels of WT or D207H Abcd2-EGFP, cells were incubated or not for 24 h with 2 òe;g/ml of doxycycline (Clontech). Login to comment 46 ABCD2 p.Asp207His To further select subclones expressing the highest expression levels of WT or D207H Abcd2-EGFP, cells were incubated or not for 24 h with 2 ␮g/ml of doxycycline (Clontech). Login to comment 82 ABCD2 p.Asp207His Coimmunoprecipitation Assays and Western Blotting- H4IIEC3 cells stably expressing D207H-ALDRP-EGFP after overnight induction with 2 òe;g/ml of doxycycline or not were homogenized in solubilization buffer (100 mM Tris-HCl, pH 8, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1% PMSF and protease inhibitor mixtures (Roche Applied Science)). Login to comment 83 ABCD2 p.Asp207His Coimmunoprecipitation Assays and Western Blotting- H4IIEC3 cells stably expressing D207H-ALDRP-EGFP after overnight induction with 2 ␮g/ml of doxycycline or not were homogenized in solubilization buffer (100 mM Tris-HCl, pH 8, 100 mM NaCl, 10 mM EDTA, 1% Triton X-100, 1% PMSF and protease inhibitor mixtures (Roche Applied Science)). Login to comment 99 ABCD2 p.Asp207His 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). Login to comment 100 ABCD2 p.Asp207His 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). Login to comment 108 ABCD2 p.Asp207His Moreover, because the alteration of ALDRP (WT or D207H) expression was unlikely to result in strong modifications in normal conditions (ALDP and PMP70 are quite well expressed in H4IIEC3 cells), we challenged the cells with C26:0, the supposed preferential substrate of ALDP, and compared the different situations. Login to comment 109 ABCD2 p.Asp207His Moreover, because the alteration of ALDRP (WT or D207H) expression was unlikely to result in strong modifications in normal conditions (ALDP and PMP70 are quite well expressed in H4IIEC3 cells), we challenged the cells with C26:0, the supposed preferential substrate of ALDP, and compared the different situations. Login to comment 119 ABCD2 p.Asp207His ABCD2 p.Asp207His Transdominant Negative Effect of D207H-ALDRP-EGFP- Parallel experiments were conducted with cell clone 19 expressing the mutant non-functional D207H-ALDRP-EGFP protein. Login to comment 120 ABCD2 p.Asp207His ABCD2 p.Asp207His Transdominant Negative Effect of D207H-ALDRP-EGFP- Parallel experiments were conducted with cell clone 19 expressing the mutant non-functional D207H-ALDRP-EGFP protein. Login to comment 121 ABCD2 p.Asp207His However, whereas induction of ALDRP-EGFP in clone 28 resulted in a decreased content of C26:0 and C24:0, doxycycline-dependent induction of D207H-ALDRP-EGFP resulted in a dose-dependent increase of the level of C26:0 (Fig. 2). Login to comment 122 ABCD2 p.Asp207His ABCD2 p.Asp207His However, whereas induction of ALDRP-EGFP in clone 28 resulted in a decreased content of C26:0 and C24:0, doxycycline-dependent induction of D207H-ALDRP-EGFP resulted in a dose-dependent increase of the level of C26:0 (Fig. 2). Login to comment 123 ABCD2 p.Asp207His ABCD2 p.Asp207His ABCD2 p.Asp207His Because the basal level of expression of the endogenous ALDRP in cell clones is quasi null, this result may be the consequence of a transdominant-negative effect of D207H-ALDRP-EGFP on ALDP function. Login to comment 124 ABCD2 p.Asp207His ABCD2 p.Asp207His ABCD2 p.Asp207His D207H-ALDRP-EGFP Interacts with ALDP-To evidence the interaction between D207H-ALDRP-EGFP and ALDP, we used two complementary approaches. Login to comment 125 ABCD2 p.Asp207His First, we used a proximity ligation assay (PLA duolink) to analyze in situ the proximity of D207H-ALDRP-EGFP with ALDP. Login to comment 131 ABCD2 p.Asp207His PLA experiments resulted in a positive labeling only in the presence of doxycycline and with the couple of antibodies directed against ALDP and GFP (Fig. 3B) demonstrating the proximity between ALDP and D207H- ALDRP-EGFP. Login to comment 132 ABCD2 p.Asp207His ABCD2 p.Asp207His PLA experiments resulted in a positive labeling only in the presence of doxycycline and with the couple of antibodies directed against ALDP and GFP (Fig. 3B) demonstrating the proximity between ALDP and D207H- ALDRP-EGFP. Login to comment 133 ABCD2 p.Asp207His ABCD2 p.Asp207His This labeling does not result from the overabundance of proteins in a membrane context because no PLA labeling was obtained between D207H-ALDRP-EGFP and proteins PEX14 or PMP22, PMP22 being the most represented protein of the peroxisomal membrane (28). Login to comment 134 ABCD2 p.Asp207His ABCD2 p.Asp207His To confirm that proximity between D207H-ALDRP-EGFP and ALDP is due to a physical interaction, cross-coimmunoprecipitation experiments were carried out from cell lysates obtained from clone 19 cultivated in the presence of doxycycline. Login to comment 135 ABCD2 p.Asp207His ABCD2 p.Asp207His As shown in Fig. 4A, D207H-ALDRP-EGFP was coimmunoprecipitated with ALDP by anti-ALDP antibody only in the doxycycline-treated cell clone 19. Login to comment 136 ABCD2 p.Asp207His In the cross-experiment, ALDP was coimmunoprecipitated with D207H-ALDRP-EGFP by anti-GFP antibody only in doxycycline-treated cell clone 19 (Fig. 4B) demonstrating interaction. Login to comment 137 ABCD2 p.Asp207His Doxycycline-dependent expression of the WT or D207H Abcd2-EGFP gene in stable transfectant cell clones 28 and 19, respectively. Login to comment 138 ABCD2 p.Asp207His Doxycycline-dependent expression of the WT or D207H Abcd2-EGFP gene in stable transfectant cell clones 28 and 19, respectively. Login to comment 142 ABCD2 p.Asp207His Although we did not notice any change in the PUFA content nor in the n-7 monounsaturated fatty acid content whatever the conditions and the cell clones, the n-9 monounsaturated fatty acid content demonstrated alteration depending on the expression level of WT or D207H-ALDRP-EGFP protein (Fig. 5). Login to comment 143 ABCD2 p.Asp207His Although we did not notice any change in the PUFA content nor in the n-7 monounsaturated fatty acid content whatever the conditions and the cell clones, the n-9 monounsaturated fatty acid content demonstrated alteration depending on the expression level of WT or D207H-ALDRP-EGFP protein (Fig. 5).3 F. Geillon, D. Trompier, C. Gondcaille, and S. Savary, manuscript in preparation. Login to comment 145 ABCD2 p.Asp207His GC-MS analysis of the saturated fatty acid content in phospholipids of cell clones 28 (WT ALDRP-EGFP) and 19 (D207H-ALDRP-EGFP) cultivated in the presence of various doses of doxycycline and in the absence or presence of C26:0. Login to comment 150 ABCD2 p.Asp207His On the contrary, doxycycline-dependent induction of D207H-ALDRP-EGFP resulted in a dose-dependent increase of the level of C26:1n-9, although non-statistically significant. Login to comment 161 ABCD2 p.Asp207His However, the increased level of beta-oxidation of DHA was unexpected because the levels of PUFA in PL appeared to be insensitive to the level of expression of ALDRP- FIGURE3.InsituanalysisoftheproximityofALDRP-EGFPwithotherperoxisomalproteins.A,immunofluorescencedetection(IFsignal)ofD207H-ALDRP-EGFP, ALDP, PEX14, and PMP22 observed by confocal microscopy in clone 19 (D207H-ALDRP-EGFP) induced by doxycycline (DOX) (2 ␮g/ml for 48 h). Login to comment 162 ABCD2 p.Asp207His B, Duolink proximity ligation assay between D207H-ALDRP-EGFP and ALDP, PEX14, and PMP22 in clone 19 induced by doxycycline observed by confocal microscopy. Login to comment 164 ABCD2 p.Asp207His Coimmunoprecipitation of ALDP and D207H-ALDRP-EGFP demonstrating interaction. Login to comment 165 ABCD2 p.Asp207His Cell lysates from cell clone 19 (D207H-ALDRP-EGFP) treated or not with doxycycline (dox) (2 ␮g/ml) for 18 h were used for anti-ALDP(A)oranti-GFP(B)immunoprecipitation.Aliquotsofcelllysatesand eluted fractions were separated on 7.5% SDS gels, blotted, and probed with anti-ALDP or anti-GFP antibodies. Substrate Specificity of ALDRP 8080 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 10•MARCH 11, EGFP. Login to comment 166 ABCD2 p.Asp207His Induction of D207H-ALDRP-EGFP resulted in a weak but significant decrease of the beta-oxidation level of C26:0. Login to comment 172 ABCD2 p.Asp207His GC-MS analysis of the n-9 monounsaturated fatty acid content in phospholipids of cell clones 28 (WT ALDRP-EGFP) and 19 (D207H-ALDRP-EGFP) cultivated in the presence of various doses of doxycycline and in the absence or presence of C26:0. Login to comment 176 ABCD2 p.Asp207His On the contrary, induction of the D207H-ALDRP-EGFP protein was expected not to modify the fatty acid content of PL and serve as a control. Login to comment 201 ABCD2 p.Asp207His The direct correlation between the level of C26:0 and C26:1 in PL and the expression level of the D207H-ALDRP-EGFP fusion protein demonstrated a transdominant-negative effect of the mutant protein. Login to comment 202 ABCD2 p.Asp207His This effect was also found in beta-oxidation assays as a decreased beta-oxidation activity of C26:0 was observed upon induction of D207H-ALDRP-EGFP. Login to comment 203 ABCD2 p.Asp207His Duolink and coimmunoprecipitation experiments have evidenced interactions between D207H-ALDRP-EGFP and ALDP. Login to comment 204 ABCD2 p.Asp207His The formation of a non-functional heterodimer D207H-ALDRP-EGFP/ALDP would deprive the cell of functional ALDP/ALDP homodimers and then result in the accumulation of C26:0 and C26:1 mimicking a deficiency on ALDP. Login to comment 206 ABCD2 p.Asp207His beta-Oxidation levels of 14 C-C26:0 (A), C24:0 (B), and C22:6n-3 (C) in clones 28.38 (WT ALDRP-EGFP) and 19.55 (D207H-ALDRP-EGFP) cultivated in the absence or presence of doxycycline (2 ␮g/ml). Login to comment 211 ABCD2 p.Asp207His Altogether, we can conclude that D207H-ALDRP-EGFP has a transdominant-negative effect on ALDP function. Login to comment