ABCMdb

ABCD2 p.Asp207His

None has been submitted yet.

Publications

PMID: 21209459 [PubMed] Genin EC et al: "Substrate specificity overlap and interaction between adrenoleukodystrophy protein (ALDP/ABCD1) and adrenoleukodystrophy-related protein (ALDRP/ABCD2)."

No. Sentence Comment

41 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
46 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
83 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
100 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
109 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
120 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
122 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 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 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 First, we used a proximity ligation assay (PLA duolink) to analyze in situ the proximity of D207H-ALDRP-EGFP with ALDP. Login to comment
132 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 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 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 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 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
138 Doxycycline-dependent expression of the WT or D207H Abcd2-EGFP gene in stable transfectant cell clones 28 and 19, respectively. Login to comment
143 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 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 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 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 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 Coimmunoprecipitation of ALDP and D207H-ALDRP-EGFP demonstrating interaction. Login to comment
165 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 Induction of D207H-ALDRP-EGFP resulted in a weak but significant decrease of the beta-oxidation level of C26:0. Login to comment
172 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 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 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 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 Duolink and coimmunoprecipitation experiments have evidenced interactions between D207H-ALDRP-EGFP and ALDP. Login to comment
204 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 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 Altogether, we can conclude that D207H-ALDRP-EGFP has a transdominant-negative effect on ALDP function. Login to comment
40 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
45 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
82 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
99 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 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 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 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
131 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
137 Doxycycline-dependent expression of the WT or D207H Abcd2-EGFP gene in stable transfectant cell clones 28 and 19, respectively. Login to comment
142 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

PMID: 16412981 [PubMed] Gueugnon F et al: "A novel cell model to study the function of the adrenoleukodystrophy-related protein."

No. Sentence Comment

42 Site directed mutagenesis was performed on the ALDRP-EGFP-pTRE2hyg to obtain the mutated construct D207H-ALDRP-EGFP-pTRE2hyg (GAC codon 619 switched to CAC). Login to comment
86 One microgram of the ALDRP, ALDRP-EGFP, or D207H- ALDRP-EGFP pcDNA3.1/Zeo(+) constructs was used as template in a final volume of 50 ll. Login to comment
149 or with D207H-ALDRP-EGFP-pTRE2hyg. Login to comment
162 Taken together, the results indicate that the clones 28 and 19 express ALDRP-EGFP and D207H-ALDRP-EGFP, respectively, at a high level in the peroxisome upon doxycycline treatment. Login to comment
165 Maximum level of induction (9-fold for the ALDRP-EGFP mRNA and 15-fold for D207H-ALDRP-EGFP mRNA) was reached with 2 lg/ml doxycycline. Login to comment
170 Analysis of the expression and of the subcellular localization of the normal and mutated (D207H) ALDRP-EGFP. Login to comment
171 (A) In vitro translation of the ALDRP, ALDRP-EGFP, or D207H-ALDRP-EGFP pcDNA3.1/Zeo(+) constructs using L[35 S]methionine. Login to comment
187 Characterization of the H4IIEC3-TetOn cell clones expressing a normal (clone 28) or mutated (D207H, clone 19) ALDRP-EGFP fusion protein upon doxycycline treatment. Login to comment
199 Quantification of the relative expression levels of mRNA of the fusion proteins by quantitative real-time RT-PCR with primers against Abcd2 in the H4IIEC3-TetOn cell clones expressing normal ALDRP-EGFP protein-clone 28 (open rectangles) and mutated (D207H) ALDRP-EGFP protein-clone 19 (filled rectangles). Login to comment
214 The D207H mutation in ALDRP is similar to the D194H mutation in ALDP that has been previously studied by stable transfection and has been shown to disrupt function without suppressing the capacity of dimerization and of import in the peroxisomal membrane [30]. Login to comment