ABCD1 p.His667Asp
| Predicted by SNAP2: | A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), P: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] ABCD1 mutations and the X-linked adrenoleukodystro... Hum Mutat. 2001 Dec;18(6):499-515. Kemp S, Pujol A, Waterham HR, van Geel BM, Boehm CD, Raymond GV, Cutting GR, Wanders RJ, Moser HW
ABCD1 mutations and the X-linked adrenoleukodystrophy mutation database: role in diagnosis and clinical correlations.
Hum Mutat. 2001 Dec;18(6):499-515., [PMID:11748843]
Abstract [show]
X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene, which encodes a peroxisomal ABC half-transporter (ALDP) involved in the import of very long-chain fatty acids (VLCFA) into the peroxisome. The disease is characterized by a striking and unpredictable variation in phenotypic expression. Phenotypes include the rapidly progressive childhood cerebral form (CCALD), the milder adult form, adrenomyeloneuropathy (AMN), and variants without neurologic involvement. There is no apparent correlation between genotype and phenotype. In males, unambiguous diagnosis can be achieved by demonstration of elevated levels of VLCFA in plasma. In 15 to 20% of obligate heterozygotes, however, test results are false-negative. Therefore, mutation analysis is the only reliable method for the identification of heterozygotes. Since most X-ALD kindreds have a unique mutation, a great number of mutations have been identified in the ABCD1 gene in the last seven years. In order to catalog and facilitate the analysis of these mutations, we have established a mutation database for X-ALD ( http://www.x-ald.nl). In this review we report a detailed analysis of all 406 X-ALD mutations currently included in the database. Also, we present 47 novel mutations. In addition, we review the various X-ALD phenotypes, the different diagnostic tools, and the need for extended family screening for the identification of new patients.
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No. Sentence Comment
174 P560S 7 1678C>T n.d. # P560L 7 1679C>T Reduced P560L 7 1679C>T Reduced fs I588 7 1765delC n.d. # R591P 7 1772G>C Absent S606L 8 1817C>T Present E609K 8 1825G>A Absent E609K 8 1825G>A Absent R617C 8 1849C>T Absent R617H 8 1850G>A Absent R617H 8 1850G>A Absent A626T 9 1876G>A Absent A626T 9 1876G>A Absent A626D 9 1877C>A n.d. # E630G 9 1889A>G n.d. # C631Y 9 1892G>A n.d. # T632I 9 1895C>T n.d. # V635M 9 1903G>A n.d. # L654P 9 1961T>C Absent # R660W 9 1978C>T Absent fs L663 9 1988insT n.d. # fs L663 IVS 9 IVS9+1g>a n.d. # fs L663 IVS 9 IVS9-1g>a n.d. # H667D 10 1999C>G Absent # T668I 10 2003C>T Absent # T693M 10 2078C>T Present # exon1-5del 1-5 n.d. # The 47 mutations marked with a # are novel unique mutations reported for the first time in this paper.
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ABCD1 p.His667Asp 11748843:174:556
status: NEW[hide] Conservation of targeting but divergence in functi... Biochem J. 2011 Jun 15;436(3):547-57. Zhang X, De Marcos Lousa C, Schutte-Lensink N, Ofman R, Wanders RJ, Baldwin SA, Baker A, Kemp S, Theodoulou FL
Conservation of targeting but divergence in function and quality control of peroxisomal ABC transporters: an analysis using cross-kingdom expression.
Biochem J. 2011 Jun 15;436(3):547-57., [PMID:21476988]
Abstract [show]
ABC (ATP-binding cassette) subfamily D transporters are found in all eukaryotic kingdoms and are known to play essential roles in mammals and plants; however, their number, organization and physiological contexts differ. Via cross-kingdom expression experiments, we have explored the conservation of targeting, protein stability and function between mammalian and plant ABCD transporters. When expressed in tobacco epidermal cells, the mammalian ABCD proteins ALDP (adrenoleukodystrophy protein), ALDR (adrenoleukodystrophy-related protein) and PMP70 (70 kDa peroxisomal membrane protein) targeted faithfully to peroxisomes and P70R (PMP70-related protein) targeted to the ER (endoplasmic reticulum), as in the native host. The Arabidopsis thaliana peroxin AtPex19_1 interacted with human peroxisomal ABC transporters both in vivo and in vitro, providing an explanation for the fidelity of targeting. The fate of X-linked adrenoleukodystrophy disease-related mutants differed between fibroblasts and plant cells. In fibroblasts, levels of ALDP in some 'protein-absent' mutants were increased by low-temperature culture, in some cases restoring function. In contrast, all mutant ALDP proteins examined were stable and correctly targeted in plant cells, regardless of their fate in fibroblasts. ALDR complemented the seed germination defect of the Arabidopsis cts-1 mutant which lacks the peroxisomal ABCD transporter CTS (Comatose), but neither ALDR nor ALDP was able to rescue the defect in fatty acid beta-oxidation in establishing seedlings. Taken together, our results indicate that the mechanism for trafficking of peroxisomal membrane proteins is shared between plants and mammals, but suggest differences in the sensing and turnover of mutant ABC transporter proteins and differences in substrate specificity and/or function.
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153 Approximately 60% of X-ALD ABCD1 mutations are missense mutations, 65% of which result in no detectable ALDP, based on IF (immunofluorescence), indicating that they affect protein Table 1 Quantification of ALDP levels in X-ALD fibroblasts ALDP Mutation IF Immunoblot (% of control) p.Arg74Trp Absent 7.5 + - 0.6 p.Arg104Cys Reduced 35 + - 3.0 p.Ser149Asn Present 77 + - 3.0 p.Asp194His Present 60 + - 13.6 p.Leu220Pro Reduced 21.8 + - 5.4 p.Arg389His Present 40.6 + - 3.6 p.Arg554His Absent 1.0 + - 0.5 p.Ser606Leu Present 25 + - 1.5 p.Glu609Gly Absent 2.1 + - 1.3 p.Glu609Lys Absent 1.8 + - 0.9 p.Ala616Thr Absent 4.3 + - 1.7 p.Leu654Pro Absent 1.5 + - 1.3 p.Arg660Trp Absent 1.6 + - 0.8 p.His667Asp Absent 2.9 + - 1.0 p.Arg113fs Absent - Figure 3 Interaction of mammalian ABCD proteins with Arabidopsis Pex19 in vivo Tobacco plants stably expressing CFP-SKL were co-transfected with 35S::ABCD-YFP fusions andNLS-Pex19constructs.Leafepidermalcellswereimagedusingconfocalmicroscopy:(A-D) ALDP-YFP plus NLS-HsPex19; (E-H) ALDP-YFP plus NLS-AtPex19_1; (I-L) ALDR-YFP plus NLS-AtPex19_1.
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ABCD1 p.His667Asp 21476988:153:691
status: NEW181 These included relatively common mutants which are unstable in fibroblasts, but which can be rescued by the application of proteasome inhibitors (p.Ser606Leu, p.Arg617His and p.His667Asp), the p.Arg104Cys mutant in which degradation of ALDP cannot be prevented by proteasome inhibitors and the p.Tyr174Cys mutant which is Table 2 X-ALD mutants used for analysis of targeting in tobacco cells The occurrence is the number of documented patients bearing the mutation; source: X-ALD database (http://www.x-ald.nl).
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ABCD1 p.His667Asp 21476988:181:177
status: NEW154 Approximately 60% of X-ALD ABCD1 mutations are missense mutations, 65% of which result in no detectable ALDP, based on IF (immunofluorescence), indicating that they affect protein Table 1 Quantification of ALDP levels in X-ALD fibroblasts ALDP Mutation IF Immunoblot (% of control) p.Arg74Trp Absent 7.5 + - 0.6 p.Arg104Cys Reduced 35 + - 3.0 p.Ser149Asn Present 77 + - 3.0 p.Asp194His Present 60 + - 13.6 p.Leu220Pro Reduced 21.8 + - 5.4 p.Arg389His Present 40.6 + - 3.6 p.Arg554His Absent 1.0 + - 0.5 p.Ser606Leu Present 25 + - 1.5 p.Glu609Gly Absent 2.1 + - 1.3 p.Glu609Lys Absent 1.8 + - 0.9 p.Ala616Thr Absent 4.3 + - 1.7 p.Leu654Pro Absent 1.5 + - 1.3 p.Arg660Trp Absent 1.6 + - 0.8 p.His667Asp Absent 2.9 + - 1.0 p.Arg113fs Absent - Figure 3 Interaction of mammalian ABCD proteins with Arabidopsis Pex19 in vivo Tobacco plants stably expressing CFP-SKL were co-transfected with 35S::ABCD-YFP fusions andNLS-Pex19constructs.Leafepidermalcellswereimagedusingconfocalmicroscopy:(A-D) ALDP-YFP plus NLS-HsPex19; (E-H) ALDP-YFP plus NLS-AtPex19_1; (I-L) ALDR-YFP plus NLS-AtPex19_1.
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ABCD1 p.His667Asp 21476988:154:691
status: NEW182 These included relatively common mutants which are unstable in fibroblasts, but which can be rescued by the application of proteasome inhibitors (p.Ser606Leu, p.Arg617His and p.His667Asp), the p.Arg104Cys mutant in which degradation of ALDP cannot be prevented by proteasome inhibitors and the p.Tyr174Cys mutant which is c The Authors Journal compilation c Table 2 X-ALD mutants used for analysis of targeting in tobacco cells The occurrence is the number of documented patients bearing the mutation; source: X-ALD database (http://www.x-ald.nl).
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ABCD1 p.His667Asp 21476988:182:177
status: NEW[hide] Adrenoleukodystrophy: subcellular localization and... J Neurochem. 2007 Jun;101(6):1632-43. Takahashi N, Morita M, Maeda T, Harayama Y, Shimozawa N, Suzuki Y, Furuya H, Sato R, Kashiwayama Y, Imanaka T
Adrenoleukodystrophy: subcellular localization and degradation of adrenoleukodystrophy protein (ALDP/ABCD1) with naturally occurring missense mutations.
J Neurochem. 2007 Jun;101(6):1632-43., [PMID:17542813]
Abstract [show]
Mutation in the X-chromosomal adrenoleukodystrophy gene (ALD; ABCD1) leads to X-linked adrenoleukodystrophy (X-ALD), a severe neurodegenerative disorder. The encoded adrenoleukodystrophy protein (ALDP/ABCD1) is a half-size peroxisomal ATP-binding cassette protein of 745 amino acids in humans. In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior. When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs. Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation. Interestingly, the wild type ALDP co-expressed in these cells also disappeared. In the case of X-ALD fibroblasts from an ALD patient (R617H), the mutant ALDP was not detected in the cells, but appeared upon incubation with a proteasome inhibitor. When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared. In addition, mutant His-ALDP (Y174C), which has a mutation between transmembrane domain 2 and 3, did not exhibit peroxisomal localization by immunofluorescense study. These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization. Further, the region between transmembrane domain 2 and 3 is important for the targeting of ALDP to the peroxisome.
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No. Sentence Comment
2 In this study, we chose nine arbitrary mutant human ALDP forms (R104C, G116R, Y174C, S342P, Q544R, S606P, S606L, R617H, and H667D) with naturally occurring missense mutations and examined the intracellular behavior.
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ABCD1 p.His667Asp 17542813:2:124
status: NEW3 When expressed in X-ALD fibroblasts lacking ALDP, the expression level of mutant His-ALDPs (S606L, R617H, and H667D) was lower than that of wild type and other mutant ALDPs.
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ABCD1 p.His667Asp 17542813:3:110
status: NEW4 Furthermore, mutant ALDP-green fluorescence proteins (S606L and H667D) stably expressed in CHO cells were not detected due to rapid degradation.
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ABCD1 p.His667Asp 17542813:4:64
status: NEW7 When CHO cells expressing mutant ALDP-green fluorescence protein (H667D) were cultured in the presence of a proteasome inhibitor, both the mutant and wild type ALDP reappeared.
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ABCD1 p.His667Asp 17542813:7:66
status: NEW9 These results suggest that mutant ALDPs, which have a mutation in the COOH-terminal half of ALDP, including S606L, R617H, and H667D, were degraded by proteasomes after dimerization.
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ABCD1 p.His667Asp 17542813:9:126
status: NEW35 We found that mutant ALDPs with the missense mutations in the G116R S342P Q544R R617H H667D Y174C NH2 COOH C sequence Cytosol Membrane Matrix Walker A Walker B S606P, S606L R104C Fig. 1 A putative secondary structure of adrenoleukodystrophy protein.
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ABCD1 p.His667Asp 17542813:35:86
status: NEW71 CHO-K1 cells (5 · 105 cells) were cultured in Ham`s F-12 medium with 10% FBS, 70 lg/mL of penicillin, and 140 lg/mL of streptomycin and transfected with 5 lg of pMAM2/ Table 1 Oligonucleotide primer sequences used for the generation of mutant ALDP constructs Construct name Forward primer (5' to 3') (top) R104C GCCTTGGTGAGCTGCACCTTCCTGTCG G116R GCCCGCCTGGACAGAAGGCTGGCC Y174C GCCTACCGCCTCTGCTCCTCCCAG S342P TGGAGCGCCCCGGGCCTGCTCATG Q544R GCATGTTCTACATCCCGCGGAGGCCCTACATGTC S606P AAGGACGTCCTGCCGGGTGGCGAGAAG S606L AAGGACGTCCTGTTGGGTGGCGAGAAG R617H GCAGAGAATCGGCATGGCCCACATGTTCTACCACAGGC H667D TCCCTGTGGAAATACGACACACACTTGCTA The underlined letters indicate the single base mutation leading to an amino acid replacement.
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ABCD1 p.His667Asp 17542813:71:592
status: NEW120 However, the ratios of certain His-ALDPs (S606L, R617H, and H667D) were significantly lower than those of wild type His-ALDP and other mutant His-ALDPs.
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ABCD1 p.His667Asp 17542813:120:60
status: NEW123 Therefore, we focused our attention on mutant ALDP (S606H, R617H, and H667D) and examined the expression level with co-expression of GFP.
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ABCD1 p.His667Asp 17542813:123:70
status: NEW125 Taken together, it appears His-ALDP (S606L, R617H, and H667D) might be degraded after translation.
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ABCD1 p.His667Asp 17542813:125:55
status: NEW128 In contrasts, His-ALDPs (Y174C and H667D) appear to be mislocated to other organelles.
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ABCD1 p.His667Asp 17542813:128:35
status: NEW135 In this experiment, we chose R104C, G116R, and S606P (with normal localization in peroxisomes), Y174C (mislocalization), and S606L and H667D (degradation).
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ABCD1 p.His667Asp 17542813:135:135
status: NEW141 In contrast, mutant ALDP-GFP (S606L and H667D) was not detected in any subcellular fractions although PMP70 and catalase were recovered in fractions 2 and 3.
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ABCD1 p.His667Asp 17542813:141:40
status: NEW150 The fragments were not extractable with 0.1 mol/L sodium carbonate, indicating (a) (c) (b) (d) 400 140 120 100 Expressionratio(%) 80 60 40 His-ALDP R104C G116R Y174C S342P Q544R S606P S606L R617H H667D Catalase 20 0 100 Expressionratio(%) 80 60 40 20 0 350 300 250 200 150 pmol/h/mgprotein 100 50 0 Normal (139T) X-ALD (163T) M ock M ock W ild W ild N one S606L His-ALDP GFP Catalase R 617H H 667D R 104CG 116RY174C S342PQ 544RS606PS606LR 617HH 667D M ock W ildR 104CG 116RY174CS342PQ 544RS606PS606LR 617HH 667D Fig. 3 Expression of wild type and mutant His-adrenoleukodystrophy proteins (ALDPs) in X-linked adrenoleukodystrophy (X-ALD) fibroblasts (163T).
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ABCD1 p.His667Asp 17542813:150:196
status: NEW159 (c) Co-expression of wild or mutant His-ALDP (S606L, R617H, or H667D) with green fluorescence proteins (GFP).
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ABCD1 p.His667Asp 17542813:159:63
status: NEW169 Effect of proteasome inhibitors on mutant ALDP The transient and stable expression experiments of mutant ALDPs suggest that mutant ALDPs such as S606L, R617H, H667D, and R104C are degraded by proteases.
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ABCD1 p.His667Asp 17542813:169:159
status: NEW170 As CHO cells stably expressed wild type ALDP and mutant ALDP-GFP (H667D) did not show any immunodetectable proteins corresponding to ALDP or ALDP-GFP, we attempted to inhibit the degradation of ALDP-GFP (H667D) by treatment with several protease inhibitors.
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ABCD1 p.His667Asp 17542813:170:66
status: NEWX
ABCD1 p.His667Asp 17542813:170:204
status: NEW175 Furthermore, we analyzed where ALDP-GFP (H667D) was located in the cells after treatment with MG132.
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ABCD1 p.His667Asp 17542813:175:41
status: NEW177 In the absence of MG132, CHO cells expressing mutant ALDP-GFP (H667D) did not show any fluorescent dots after the permeabilization by digitonin (data not shown).
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ABCD1 p.His667Asp 17542813:177:63
status: NEW178 As in Fig. 6, in the presence of MG132, immunofluorescent dots of mutant ALDP-GFP (H667D) were detected and the dots were superimposable on the pattern of PMP70.
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ABCD1 p.His667Asp 17542813:178:83
status: NEW179 Newly synthesized ALDP-GFP (H667D) might escape from degradation in cytosol and target peroxisomes or localize on peroxisomal membranes after targeting to peroxisomes.
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ABCD1 p.His667Asp 17542813:179:28
status: NEW180 Degradation of mutant ALDP (S606L) was similarly inhibited by lactacystin, but not by leupeptin, wild R104C G116R ALDP-GFP ALDP PMP70 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 1 5 10 Non specific ALDP-GFP ALDP PMP70 Non specific ALDP-GFP ALDP PMP70 Non specific Y174C H667D S606P S606L Fig. 4 Subcellular localization of wild type and mutant adrenoleukodystrophy protein (ALDP) -green fluorescence proteins (GFP) in CHO cells.
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ABCD1 p.His667Asp 17542813:180:268
status: NEW181 The mitochondrial and light mitochondrial fraction from CHO cells expressing wild type ALDP and ALDP-GFP or each mutant ALDP-GFP (R104C, G116R, Y174C, S606P, S606L, or H667D) was fractionated by equilibrium density centrifugation on sucrose.
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ABCD1 p.His667Asp 17542813:181:168
status: NEW191 These results suggest that missense mutations, including R617H and H667D, were also degraded by proteasomes.
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ABCD1 p.His667Asp 17542813:191:67
status: NEW195 Some mutations in the first extracellular domain of ABCA1 have been shown to cause impaired localization of the mutant ABCA1 to plasma membranes from the ALDP-GFP(H667D) ALDP 100 Lactacystin MG132 kDa kDa 0 100 75 2.5 5 10 10 20 kDa 0 100 75 2.5 5 10 20 ALDP-GFP(H667D) ALDP ALDP-GFP(H667D) ALDP µmol/L h 75 70 (a) (b) (c) Leupeptin AEBSF E-64-dLactacystin None PMP70 Fig. 5 Effect of protease inhibitors on the stability of adrenoleukodystrophy protein (ALDP)-green fluorescence proteins (GFPs) (H667D) and wild type ALDP in CHO cells.
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ABCD1 p.His667Asp 17542813:195:163
status: NEWX
ABCD1 p.His667Asp 17542813:195:263
status: NEWX
ABCD1 p.His667Asp 17542813:195:284
status: NEWX
ABCD1 p.His667Asp 17542813:195:502
status: NEW196 (a) CHO cells co-expressing wild type ALDP and ALDP-GFP (H667D) were cultured with leuteptin (50 lmol/L), AEBSF (300 lmol/L), E64-d (10 lmol/L), or lactacystin (10 lmol/L) for 20 h. Cell homogenates (100 lg of protein) were analyzed by immunoblotting using anti-ALDP and anti-PMP70 antibodies.
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ABCD1 p.His667Asp 17542813:196:57
status: NEW197 (b) CHO cells co-expressing wild type ALDP and ALDP-GFP (H667D) were incubated with different concentrations of lactacystin or MG132 for 20 h and subjected to immunoblot analysis as in (a).
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ABCD1 p.His667Asp 17542813:197:57
status: NEW198 (c) CHO cells co-expressing wild type ALDP and ALDP-GFP (H667D) were incubated with 10 lmol/L MG132 for 0-20 h. ALDP-GFP (a) (b) (c) (d) (e) (f) PMP70 MERGE Fig. 6 Distribution of mutant adrenoleukodystrophy protein (ALDP) -green fluorescence proteins (GFPs) (H667D) in CHO cells incubated with MG132.
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ABCD1 p.His667Asp 17542813:198:57
status: NEWX
ABCD1 p.His667Asp 17542813:198:260
status: NEW199 CHO cells co-expressing wild type ALDP and mutant ALDP-GFP (H667D) were incubated with 10 lmol/L MG132 for 20 h. They were then incubated with or without digitonin (25 lg/mL) for 10 min followed by immunostaining.
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ABCD1 p.His667Asp 17542813:199:60
status: NEW200 CHO cells expressing mutant ALDP-GFP (H667D).
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ABCD1 p.His667Asp 17542813:200:38
status: NEW[hide] Decreased expression of ABCD4 and BG1 genes early ... Hum Mol Genet. 2005 May 15;14(10):1293-303. Epub 2005 Mar 30. Asheuer M, Bieche I, Laurendeau I, Moser A, Hainque B, Vidaud M, Aubourg P
Decreased expression of ABCD4 and BG1 genes early in the pathogenesis of X-linked adrenoleukodystrophy.
Hum Mol Genet. 2005 May 15;14(10):1293-303. Epub 2005 Mar 30., [PMID:15800013]
Abstract [show]
Childhood cerebral adrenoleukodystrophy (CCER), adrenomyeloneuropathy (AMN) and AMN with cerebral demyelination (AMN-C) are the main phenotypic variants of X-linked adrenoleukodystrophy (ALD). It is caused by mutations in the ABCD1 gene encoding a half-size peroxisomal transporter that has to dimerize to become functional. The biochemical hallmark of ALD is the accumulation of very-long-chain fatty acids (VLCFA) in plasma and tissues. However, there is no correlation between the ALD phenotype and the ABCD1 gene mutations or the accumulation of VLCFA in plasma and fibroblast from ALD patients. The absence of genotype-phenotype correlation suggests the existence of modifier genes. To elucidate the mechanisms underlying the phenotypic variability of ALD, we studied the expression of ABCD1, three other peroxisomal transporter genes of the same family (ABCD2, ABCD3 and ABCD4) and two VLCFA synthetase genes (VLCS and BG1) involved in VLCFA metabolism, as well as the VLCFA concentrations in the normal white matter (WM) from ALD patients with CCER, AMN-C and AMN phenotypes. This study shows that: (1) ABCD1 gene mutations leading to truncated ALD protein are unlikely to cause variation in the ALD phenotype; (2) accumulation of saturated VLCFA in normal-appearing WM correlates with ALD phenotype and (3) expression of the ABCD4 and BG1, but not of the ABCD2, ABCD3 and VLCS genes, tends to be correlated with the severity of the disease, acting early in the pathogenesis of ALD.
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No. Sentence Comment
76 Mutation Amino acid alteration Type of mutation at the protein level Tissue sample CCER1 521A.G Y174C Missense CCER2 1414insC fsE471 Frame shift CCER3 Unknown Unknown Unknown Fibroblast CCER4 411G.A W137X Nonsense CCER5 1961T.C L654P Missense CCER6 529C.T Q177X Nonsense CCER7 901-1G.A fsE300 Frame shift CCER8 796G.A G266R Missense CCER9 1822G.A G608S Missense Brain CCER10 1390C.A R464X Nonsense CCER11 253-254insC fsP84 Frame shift CCER12 619_627del S207_A209del Deletion AMN-C1 1414-1415insC fsE471 Frame shift AMN-C2 1661G.A R554H Missense AMN-C3 1585delG fsG528 Frame shift Fibroblast AMN-C4 1661G.A R554H Missense AMN-C5 1825G.A E609K Missense AMN-C6 919C.T Q307X Nonsense AMN-C7 1850G.A R617H Missense AMN-C8 887A.G Y296C Missense AMN-C9 965T.C L322P Missense Brain AMN-C10 1390C.T R464X Nonsense AMN-C11 [1165C.T;1224 þ 1GT.TG] [R389C;fSE408] Missense; frame shift AMN-C12 1661G.A R554H Missense AMN-C13 [1997A.C;2007C.G] [Y666S;H669Q] Missense AMN-C14 1755delG fsH586 Frame shift AMN1 529C.T Q177X Nonsense AMN2 1999C.G H667D Missense AMN3 1415delAG fsE471 Frame shift Fibroblast AMN4 337delC fsA112 Frame shift AMN5 310C.T R104C Missense AMN6 919C.T Q307X Nonsense AMN7 323C.T S108L Missense Brain All mutation designations conform to the nomenclature described by Antonarakis and den Dunnen (30,31).
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ABCD1 p.His667Asp 15800013:76:1034
status: NEW[hide] [Adrenoleukodystrophy: structure and function of A... Yakugaku Zasshi. 2007 Jan;127(1):163-72. Takahashi N, Morita M, Imanaka T
[Adrenoleukodystrophy: structure and function of ALDP, and intracellular behavior of mutant ALDP with naturally occurring missense mutations].
Yakugaku Zasshi. 2007 Jan;127(1):163-72., [PMID:17202797]
Abstract [show]
Adrenoleukodystrophy (ALD) is an inherited disorder characterized by progressive demyelination of the central nervous system and adrenal dysfunction. The biochemical characterization is based on the accumulation of pathgnomonic amounts of saturated very long-chain fatty acid (VLCFA; C>22) in all tissues, including the brain white matter, adrenal glands, and skin fibroblasts, of the patients. The accumulation of VLCFA in ALD is linked to a mutation in the ALD (ABCD1) gene, an ABC subfamily D member. The ALD gene product, so-called ALDP (ABCD1), is thought to be involved in the transport of VLCFA or VLCFA-CoA into the peroxisomes. ALDP is a half-sized peroxisomal ABC protein and it has 745 amino acids in humans. ALDP is thought to be synthesized on free polysomes, posttranslationally transported to peroxisomes, and inserted into the membranes. During this process, ALDP interacts with Pex19p, a chaperone-like protein for intracellular trafficking of peroxisomal membrane protein (PMP), the complex targets Pex3p on the peroxisomal membranes, and ALDP is inserted into the membranes. After integration into the membranes, ALDP is thought to form mainly homodimers. Here, we chose nine arbitrary mutations of human ALDP with naturally occurring missense mutations and examined the intracellular behavior of their ALDPs. We found that mutant ALDP (S606L, R617H, and H667D) was degraded together with wild-type ALDP by proteasomes. These results suggest that the complex of mutant and wild-type ALDP is recognized as misfolded proteins and degraded by the protein quality control system associated with proteasomes. Further, we found fragmentation of mutant ALDP (R104C) on peroxisomes and it was not inhibited by proteasomes inhibitors, suggesting that an additional protease(s) is also involved in the quality control of mutant ALDP. In addition, mutation of ALDP (Y174C) suggests that a loop between transmembrane domains 2 and 3 is important for the targeting of ALDP to peroxisomes.
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8 We found that mutant ALDP (S606L, R617H, and H667D) was degraded together with wild-type ALDP by proteasomes.
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ABCD1 p.His667Asp 17202797:8:45
status: NEW20 R617H and H667D are located in Walker B region and in COOH-terminal region, respectively.
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ABCD1 p.His667Asp 17202797:20:10
status: NEW28 ミスセンス変異を持つ ALDP の細胞内動態 ―一過性発現による解析 ALD 患者の持つ変異 ALDP のڥf;能,細胞内局在 性,細胞内における安定性を解析することは, ALDP の各ドメインの機能を知る上で有用な情報 を提供すると思われる．特にミスセンス変異は,た った 1 つのアミノ酸変異による異常であるので特に 興味深い．われわれは ALD 患者で報告されている ミスセンス変異の中から,TMD から 4 つ(R104C, G116R, Y174C, S342P),NBD から 4 つ(Q544R, S606P, S606L, R617H),C 末端部位から 1 つ (H667D)を任意に選び(Fig. 1),その機能と細胞 内動態を解析した．これらの実験は,大学院シンポ ジウムで報告したので,詳しく述べたいと思う． ALDP はペルオキシソームにおける極長鎖脂肪 酸の b 酸化に関与していることが知られている． 実際に ALD 患者由来の繊維芽細胞では極長鎖脂肪 酸の b 酸化活性が正常な線維芽細胞と比べて約 50 ―70%程度減少している．そこで野生型及び変異型 ALDP の機能を確認するため,ALDP を発現して いない ALD 患者由来線維芽細胞に,N 末端に His タグを付加した野生型と変異型 ALDP を一過性に 発現し,［1-14 C]lignoceric acid を基質として極長鎖 脂肪酸 b 酸化活性の測定を行った．その結果, ALDP 欠損線維芽細胞の極長鎖脂肪酸 b 酸化活性 は,正常細胞の約 50%まで減少していたが,野生 型 His-ALDP を発現させると正常と同程度にまで 活性が回復した．このことから発現させた野生型 His-ALDP は ALDP と同等の機能を持つことが確 認された．一方,9 種類のミスセンス変異 ALDP を発現した線維芽細胞では極長鎖脂肪酸 b 酸化活 性の増加は認められなかった．よって,これらのミ スセンス変異 ALDP は機能を欠くことが確認され た． ついで,野生型及び変異型 His-ALDP を発現し た ALD 患者線維芽細胞を回収し,変異型 ALDP の発現量を immunoblotting により定量化し解析し た(Table 1)．なお ALDP の発現量は,ペルオキ シソームの指標酵素であるカタラーゼの発現量で補 正した．その結果,変異型 ALDP(R104C, G116R, Y174C, S342P, Q544R, S606P)は,野生型とほぼ 同程度の発現量を示した．一方,変異型 ALDP (S606L, R617H, H667D)では発現量が野生型の発 167 Table 1.
X
ABCD1 p.His667Asp 17202797:28:304
status: NEWX
ABCD1 p.His667Asp 17202797:28:1465
status: NEWX
ABCD1 p.His667Asp 17202797:28:5964
status: NEW29 Expression and Localization of Missense ALDPs Mutant Transient Stable Expression Localization b-Oxidation Expression Localization Wild Z Px ＋ Z Px R104C, G116R S342P, Q544R S606P Z Px － Z Px Y174C Z mis － Z mis S606L ＋ Px － － － R617H ± － － na na H667D ＋ － － － － Wild and Mutant His-ALDPs or ALDP-GFPs were transiently expressed in X-ALD ˆbroblasts and stably expressed in CHO cells, respectively.
X
ABCD1 p.His667Asp 17202797:29:314
status: NEW36 1 現量と比べて約 50%程度減少していた．なお,各 ALDP ポジティブの細胞は約 30%程度であり,各 細胞間での発現効率に有意な差は認められなかっ た．このことから,ALDP の発現量が減少してい た 3 つの変異 ALDP は細胞内での安定性が低下し ていると推察された．また興味深いことに S606P と S606L は同じ部位の変異にも係わらず,置換し たアミノ酸によって発現量には差が認められた． ついで,変異型 His-ALDP の細胞内局在を蛍光 抗体法で確認した．変異型 ALDP(R104C, G116R, S342P, Q544R, S606P, S606L)では ALDP がカタ ラーゼの局在と一致したことから,正常にペルオキ シソームへ局在していることが確認された．一方, 変異型 ALDP(Y174C, H667D)では局在が一致せ ず,ALDP が他の細胞内小器官へ間違って輸送さ れていると考えられた．変異型 ALDP(R617H) では ALDP の発現が認められなかった．変異型 ALDP(R104C, G116R, S342P, Q544R, S606P)で は野生型とほぼ同程度のタンパク量が発現し,ペル オキシソームへの局在も確認されたので,これらの 変異型 ALDP は合成されたのちに正常にペルオキ シソームに運ばれるが,ペルオキシソーム膜におい てその機能(ATP 結合・加水分解若しくは基質輸 送)に異常を持つことが推察された．特に R104C, G116R, S342P は TMD に存在することから ALDP の基質輸送能が変化していると考えられる．一方, NBD に存在する Q544R, S606P は ATP 結合・加水 分解に影響を与えている可能性が考えられる．また S606P, S606L は変異が同じ部位でも構造的に安定 性が異なっていた．Roerig らは S606L の変異型 ALDP は,ATP との親和性が低下している一方で ATP 加水分解は正常に行われていると報告してい る．29) このことは ALDP と ATP の親和性が ALDP の安定性にも影響を及ぼしている可能性を示してい る．S606L と S606P の安定性の違いと機能の関係 は ALDP の機能を知る上でも興味深い点であり, 今後さらに検討を行う必要がある．一方,Y174C の変異型 ALDP は正常に発現するにも係わらず, ペルオキシソームへ局在せず他の細胞内小器官へミ スターゲッティングした．これまでにペルオキシ ソームへの局在化シグナルを欠くペルオキシソーム 膜タンパク質は,非特異的にミトコンドリアや小胞 体に移行することが知られている．30,31) よって, ALDP の TMD2―3 の間のループは,ペルオキシ ソームへの局在化に重要な役割を果たしている可能 性が推察される．Pex19p 存在化での in vitro タン パク質翻訳系において,ALDP(Y174C)は Pex19p に結合できるので,ALDP の N 末端 67―164 に存在するペルオキシソーム移行に係わる領域が ALDP の何らかの構造変化によってマスクされる のかもしれない． 5.
X
ABCD1 p.His667Asp 17202797:36:206
status: NEWX
ABCD1 p.His667Asp 17202797:36:1985
status: NEW38 Subcellular Localization of Wild Type and Mutant ALDP-GFP in CHO ML fraction (mitochondria and light mitochondrial fraction) from CHO cells expressing wild type ALDP and each mutant ALDP-GFP (R104C, G116R, H667D) were fractionated by equilibrium density centrifugation on sucrose.
X
ABCD1 p.His667Asp 17202797:38:206
status: NEW42 Dots in the electrophoretogram in R104C shows the band corresponding to ALDP-GFP fragments.
X
ABCD1 p.His667Asp 17202797:42:64
status: NEWX
ABCD1 p.His667Asp 17202797:42:156
status: NEW43 168 Vol. 127 (2007) ALDP については ALDP の C 末端に GFP(green ‰uorescent protein)を融合させた変異 ALDP-GFP を発現させるとともに,野生型ヒト ALDP を CHO 細胞に共 発現させた(CHO 細胞にも 内在性の ALDP が発現しているが,本実験に用いた抗体が 交差しないため,ヒト ALDP を発現させた)．GFP 融合タンパク質は内在性のタンパク質との区別が容 易であること,安定発現細胞の取得が容易にできる ことなどの利点がある．この実験では,ペルオキシ ソームに正常に輸送される変異型 ALDP(R104C, G116R, S606P),ペルオキシソームに局在しない変 異型 ALDP(Y174C),発現量が低下している変異 型 ALDP(H667D)を選んだ．これら安定過剰発 現細胞における ALDP-GFP の細胞内局在性をみる と,その分布は一過性発現させた His-ALDP と同 様であった． 得られた安定発現細胞よりオルガネラ粗分画を調 製し,ショ糖密度勾配遠心分離法により各フラクシ ョンに分けたのち,SDS-PAGE 及び immunoblotting により変異 ALDP の局在について解析を行っ た(Fig. 3)．ペルオキシソームマーカーとしてペ ルオキシソーム膜タンパク質である PMP70 とペル オキシソームの主要なマトリックスタンパク質であ るカタラーゼを用いた．野生型 ALDP を安定過剰 発現している細胞において,カタラーゼ活性並びに PMP70 が主としてフラクション 3 及び 4 に存在す ることより,この分画にペルオキシソームが回収さ れたことが示唆された．また約 110 kDa の分子サ イズを持つ ALDP-GFP 並びに 83 kDa の野生型 ALDP は,ペルオキシソームマーカーとほぼ同じ 分布を示していたことからペルオキシソームに局在 していることが示唆された．また変異型 ALDP-GFP(G116R, S606P)も同様の分布を示した． 一方,変異型 ALDP-GFP(H667D)を安定過剰 発現している細胞の場合は,ALDP-GFP のバンド は検出されなかった(Fig. 3)．興味深いことに, PMP70 は 検 出 さ れ た が , 共 発 現 さ せ た 野 生 型 ALDP のバンドも検出されなかった．また変異型 ALDP(S606L)についても同様であった．これら の 結 果 は , 変 異 型 ALDP ( H667D, S606L ) は PMP70 とではなく,野生型 ALDP と複合体を形成 し,両者が分解される可能性を示唆している． ABC タンパク質の機能発現に重要である TMD や NBD 以外の C 末端部位での変異がタンパク質の安 定性に影響を及ぼすことは興味深い．ALDP の C 末端部位である 600―700 アミノ酸での変異が X-ALD を引き起こす頻度が高いことから,ALDP の C 末端部位はタンパク質の安定性に重要な役割を担 っている可能性がある．Liu らは ALDP のダイ マー化には C 末端部位(AA.631―745)が重要で あると報告している．13) H667D や S606L のような 変異は,それ自身あるいは野生型 ALDP とミスフ ォールドしたダイマーを形成し,異常タンパク質と して認識され分解されると考えられる．一方,変異 型 ALDP-GFP(R104C)はペルオキシソーム分画 に回収されるものの,フラグメント化していること 169 Fig. 4.
X
ABCD1 p.His667Asp 17202797:43:1741
status: NEWX
ABCD1 p.His667Asp 17202797:43:4866
status: NEWX
ABCD1 p.His667Asp 17202797:43:5712
status: NEWX
ABCD1 p.His667Asp 17202797:43:7305
status: NEW44 EŠect of Protease Inhibitors on the Stability of ALDP-GFP (H667D) and Wild Type ALDP in CHO Cells CHO cells co-expressing wild type ALDP and ALDP-GFP (H667D) were cultured with each protease inhibitors.
X
ABCD1 p.His667Asp 17202797:44:65
status: NEWX
ABCD1 p.His667Asp 17202797:44:157
status: NEW49 変異型 ALDP の分解過程の解析 新生タンパク質が正しいフ0a9;ールディングを受け ることは,そのタンパク質の正常な機能発現のため に必須である．遺伝子変異などが存在すると,タン パク質がミスフォールディングされる．このミスフ ォールドタンパクが細胞外へ分泌されたり,細胞内 に蓄積したりすると生体にとって極めて有害になる ため,このようなタンパクはプロテアソーム,リソ ソーム等によって迅速に分解される．ちなみに,嚢 胞性線維症の原因タンパク質 CFTR は細胞膜イオ ンチャネルとして機能する ABC タンパク質である が,変異 CFTR は小胞体膜からプロテアソームに リクルートされ分解されることが報告されてい る．32,33) しかしながら,変異型 ALDP を始めとし て,ペルオキシソーム膜タンパク質についての解析 はほとんど行われていない． 変異型 ALDP の一過性発現と安定過剰発現実験 より,ALDP(S606L, R617H, H667D, R104C)は, プロテアーゼにより分解されていると推定された． そこで,ALDP-GFP(H667D)を発現している CHO 細胞に各種プロテアーゼ阻害剤を処理し,解 析を行った．その結果,プロテアソーム阻害剤であ る lactacystin を処理した細胞では ALDP-GFP 及び ALDP の バ ン ド が 出 現 し た ( Fig. 4 )． 一 方 , leupeptin, AEBSF, E64d には効果がなかった．ま た他のプロテアソーム阻害剤である MG132 も有効 であった．さらにプロテアソーム阻害剤により分解 を逃れた変異型 ALDP-GFP(H667D)の細胞内局 在を蛍光抗体法で観察すると,ペルオキシソームに 局在していることが確認された．一方,変異型 ALDP(R104C)のフラグメント化は上記プロテアー ゼ処理では阻害されなかった． さらに ALD 患者由来細胞の内因性変異 ALDP の分解とプロテアソーム分解系の関与について確認 するため,変異型 ALDP(R617H)を持つ患者由 来線維芽細胞を用いてタンパク分解の阻害実験を行 った．その結果,lactacystin と MG132 処理により, ALDP のバンドが出現した．以上の結果より,ペ ルオキシソーム膜上にはミスフォールドしたタンパ ク質を認識する仕組みが存在し,プロテアソーム及 び他のプロテアーゼを介して排除していることが示 唆された． 一方,山田らは ALD 患者線維芽細胞を［35 S］メチ オニンでパルスチェイスすることにより,変異型 ALDP(G512S, R660W)の分解が E-64 と leupepu- tin により抑制されることを報告している．34) 彼ら の実験ではプロテアソーム阻害剤については実験し ていないので,プロテアソームの関与は不明である が,変異型 ALDP の分解には,複数のプロテアー ゼが関与している可能性がある． 7.
X
ABCD1 p.His667Asp 17202797:49:191
status: NEW52 Some mutant ALDPs (R104C, G116R, S342P, Q544R and S606P) are normally inserted into the peroxisomal membrane, and others were mislocalized (Y174C) or degraded by proteasome (S606L, R617H and H667D).
X
ABCD1 p.His667Asp 17202797:52:191
status: NEW53 170 Vol. 127 (2007) その結果より,ミスセンス変異 ALDP は以下に 示すように 4 種類の細胞内動態を持つことが示され た(Fig. 5)．1) 野生型と同様にペルオキシソーム に 局 在 す る が そ の 機 能 が 阻 害 さ れ て い る 変 異 (R104C, G116R, S342P, Q544R, S606P),2) ペル オキシソームへの局在化に障害がある変異(Y174C), 3) 変異によりタンパク質の安定性が低下しプロテ アソームでの分解を受けるが,一部ペルオキシソー ムに局在する変異(S606L),4) 変異によりタンパ ク質の安定性が低下しプロテアソームで選択的に分 解を受け,細胞内でほとんど確認できない変異 (R617H, H667D)の 4 種類のパターンである． 発現量も局在化も正常な変異では,ABC タンパ ク質としての機能に直接関与している機能ドメイン の障害が起こっていると推察される．この中で G116R, S342P は TMD に位置しており,基質の認 識や輸送に障害があると推察される．また Q544R, S606P は ATP と の 結 合 ・ 加 水 分 解 に 関 与 す る NBD に位置している．このような変異は,ALDP の ABC タンパク質としての機能を解析するために 有益と考えられる． 発現量は正常だが局在化に異常が認められた Y174C は,TMD2 と 3 の間のループ 2 に位置して おり,この領域が ALDP のペルオキシソームへの ターゲッティングに必要であることを示している． ALDP のターゲッティングに必要な領域は 67―164 番目のアミノ酸に存在することが報告されてい る．20) このことから,Y174C の変異による構造変化 のため,ターゲッティングシグナルがマスクされて いるのかもしれない．このタイプの変異は ALDP のペルオキシソームへの局在化を調べる上で重要と 考えられる． ALDP の変異で最も多いミスセンス変異ではそ の多くが細胞内で分解を受けている．R617H 及び H667D では発現量の著しい低下が認められる．特 に安定発現した CHO では immunoblot で検出でき なかった．ミスフォールドタンパク質の分解システ ムの 1 つにプロテアソームによる分解系がある．こ のタンパク質分解は,生物の様々な高次機能の制御 や環境ストレスに応答した恒常性の維持(ストレス 応答,タンパク質の品質管理など)に必須な役割を 担っている．しかし,小胞体を経由して合成される 分泌タンパク質や膜タンパク質に比べて,小胞体を 経由しない細胞内タンパク質の品質管理機構はあま り報告されていない．ALDP は遊離のポリソーム から直接ペルオキシソームに輸送されるが,この過 程でどのように R617H, H667D などの変異が認識 され,プロテアソーム系が働いているか興味深い． 171171No.
X
ABCD1 p.His667Asp 17202797:53:1734
status: NEWX
ABCD1 p.His667Asp 17202797:53:5115
status: NEWX
ABCD1 p.His667Asp 17202797:53:7237
status: NEW27 df; b9; bb; f3; b9;᜕ᶒఔᢝ௸ ALDP IJe;d30;Pde;ᑁ4d5;ɦb; ߟe00;Έe;ឋ˿a;Ife;IJb;ఐĴb;Ye3;᪆ ALD <a3;ὅIJe;ᢝ௸᜕ᶒ ALDP IJe;a5f;Pfd;,d30;Pde;ᑁc40;ᙠ ឋ,d30;Pde;ᑁIJb;İa;௫Ĵb;b89;b9a;ឋఔYe3;᪆௳Ĵb;௭IJf;, ALDP IJe;ᔜ c9; e1; a4; f3;IJe;a5f;Pfd;ఔMe5;Ĵb;e0a;ᨵᵨIJa;<c5;ᛇ ఔ?d0;f9b;௳Ĵb;əd;Ĵf;Ĵc;Ĵb;&#ff0e;ᱯIJb; df; b9; bb; f3; b9;᜕ᶒIJf;,ıf; ௷ıf; 1 ௸IJe; a2; df; ce;⏚᜕ᶒIJb;ఐĴb;ᶒe38;Ĵb;IJe;ᱯIJb; ‐ᕡdf1;&#ff0e;Ĵf;Ĵc;Ĵf;Ĵc;IJf; ALD <a3;ὅᛇȠa;௯Ĵc;௺Ĵb; df; b9; bb; f3; b9;᜕ᶒIJe;e2d;İb;,TMD İb; 4 ௸(R104C, G116R, Y174C, S342P) ,NBD İb; 4 ௸(Q544R, S606P, S606L, R617H) ,C ʠb;aef;Ze8;f4d;İb; 1 ௸ (H667D)ఔefb;ɢf;IJb;⍶ఁ(Fig. 1) ,ıd;IJe;a5f;Pfd;d30;Pde; ᑁ4d5;ɦb;ఔYe3;᪆௱ıf;&#ff0e;௭Ĵc;IJe;b9f; a13;IJf;,ᜧb66;▾ b7; f3; dd; b8; a6; e0;ᛇȠa;௱ıf;IJe;,a73;௱İf;ff0;ఇıf;əd;௦&#ff0e; ALDP IJf; da; eb; aa; ad; b7; bd; fc; e0;IJb;İa;௫Ĵb;ᬿ╩⒴ᾦPaa; ⏚IJe; b ⏚ᓄIJb;_a2;e0e;௱௺Ĵb;௭İc;Me5;Ĵc;௺Ĵb;&#ff0e; b9f;ωb;IJb; ALD <a3;ὅᵫᩭIJe;e4a;dad;Rbd;d30;Pde;IJf;ᬿ╩⒴ᾦPaa; ⏚IJe; b ⏚ᓄd3b;ឋİc;b63;e38;IJa;dda;dad;Rbd;d30;Pde;bd4;ఇ௺d04; 50 ߟ70%a0b;ea6;e1b;c11;௱௺Ĵb;&#ff0e;ıd;௭[ce;˯f;ɂb;5ca;ఁ᜕ᶒɂb; ALDP IJe;a5f;Pfd;ఔNba;a8d;௳Ĵb;ıf;ఉ,ALDP ఔ˿a;Ife;௱௺ IJa; ALD <a3;ὅᵫᩭdda;dad;Rbd;d30;Pde;IJb;,N ʠb;aef;IJb; His bf; b0;ఔed8;4a0;௱ıf;[ce;˯f;ɂb;᜕ᶒɂb; ALDP ఔe00;Έe;ឋIJb; ˿a;Ife;௱, &#ff3b;1-14 C]lignoceric acid ఔ9fa;cea;௱௺ᬿ╩⒴ ᾦPaa;⏚ b ⏚ᓄd3b;ឋIJe;e2c;b9a;ఔʹc;௷ıf;&#ff0e;ıd;IJe;d50;ʧc;, ALDP b20;ʀd;dda;dad;Rbd;d30;Pde;IJe;ᬿ╩⒴ᾦPaa;⏚ b ⏚ᓄd3b;ឋ IJf;,b63;e38;d30;Pde;IJe;d04; 50%ije;e1b;c11;௱௺ıf;İc;,[ce;˯f; ɂb; His-ALDP ఔ˿a;Ife;௯ıb;Ĵb;b63;e38;Ȝc;a0b;ea6;IJb;ije; d3b;ឋİc;8de;fa9;௱ıf;&#ff0e;௭IJe;௭İb;˿a;Ife;௯ıb;ıf;[ce;˯f;ɂb; His-ALDP IJf; ALDP Ȝc;b49;IJe;a5f;Pfd;ఔᢝ௸௭İc;Nba; a8d;௯Ĵc;ıf;&#ff0e;e00;Ab9;,9 a2e;ϙe;IJe; df; b9; bb; f3; b9;᜕ᶒ ALDP ఔ˿a;Ife;௱ıf;dda;dad;Rbd;d30;Pde;IJf;ᬿ╩⒴ᾦPaa;⏚ b ⏚ᓄd3b; ឋIJe;ᜉ4a0;IJf;a8d;ఉĴc;IJa;İb;௷ıf;&#ff0e;ఐ௷௺,௭Ĵc;IJe; df; b9; bb; f3; b9;᜕ᶒ ALDP IJf;a5f;Pfd;ఔb20;İf;௭İc;Nba;a8d;௯Ĵc; ıf;&#ff0e; ௸,[ce;˯f;ɂb;5ca;ఁ᜕ᶒɂb; His-ALDP ఔ˿a;Ife;௱ ıf; ALD <a3;ὅdda;dad;Rbd;d30;Pde;ఔ8de;5ce;௱,᜕ᶒɂb; ALDP IJe;˿a;Ife;[cf;ఔ immunoblotting IJb;ఐĴa;b9a;[cf;ᓄ௱Ye3;᪆௱ ıf;(Table 1) &#ff0e;IJa;İa; ALDP IJe;˿a;Ife;[cf;IJf;, da; eb; aa; ad; b7; bd; fc; e0;IJe;ᢣa19;⏗d20;Ĵb; ab; bf; e9; fc; bc;IJe;˿a;Ife;[cf;Xdc; b63;௱ıf;&#ff0e;ıd;IJe;d50;ʧc;,᜕ᶒɂb; ALDP(R104C, G116R, Y174C, S342P, Q544R, S606P)IJf;,[ce;˯f;ɂb;ijb;ijc; Ȝc;a0b;ea6;IJe;˿a;Ife;[cf;ఔ̙a;௱ıf;&#ff0e;e00;Ab9;,᜕ᶒɂb; ALDP (S606L, R617H, H667D)IJf;˿a;Ife;[cf;İc;[ce;˯f;ɂb;IJe;˿a; 167 Table 1.
X
ABCD1 p.His667Asp 17202797:27:1299
status: NEWX
ABCD1 p.His667Asp 17202797:27:5277
status: NEW34 167 No. 1 Ife;[cf;bd4;ఇ௺d04; 50%a0b;ea6;e1b;c11;௱௺ıf;&#ff0e;IJa;İa;,ᔜ ALDP dd; b8; c6; a3; d6;IJe;d30;Pde;IJf;d04; 30%a0b;ea6;Ĵa;,ᔜ d30;Pde;╹IJe;˿a;Ife;4b9;᳛IJb;ᨵɢf;IJa;dee;IJf;a8d;ఉĴc;IJa;İb;௷ ıf;&#ff0e;௭IJe;௭İb;,ALDP IJe;˿a;Ife;[cf;İc;e1b;c11;௱௺ ıf; 3 ௸IJe;᜕ᶒ ALDP IJf;d30;Pde;ᑁIJe;b89;b9a;ឋİc;f4e;e0b;௱ ௺Ĵb;?a8;bdf;௯Ĵc;ıf;&#ff0e;ije;ıf;‐ᕡdf1;௭IJb; S606P S606L IJf;Ȝc;௲Ze8;f4d;IJe;᜕ᶒIJb;ఊfc2;Ĵf;ıa;,f6e;?db;௱ ıf; a2; df; ce;⏚IJb;ఐ௷௺˿a;Ife;[cf;IJb;IJf;dee;İc;a8d;ఉĴc;ıf;&#ff0e; ௸,᜕ᶒɂb; His-ALDP IJe;d30;Pde;ᑁc40;ᙠఔVcd;ᐝ ᢙf53;cd5;Nba;a8d;௱ıf;&#ff0e;᜕ᶒɂb; ALDP(R104C, G116R, S342P, Q544R, S606P, S606L)IJf; ALDP İc; ab; bf; e9; fc; bc;IJe;c40;ᙠe00;Qf4;௱ıf;௭İb;,b63;e38;IJb; da; eb; aa; ad; b7; bd; fc; e0;ఆc40;ᙠ௱௺Ĵb;௭İc;Nba;a8d;௯Ĵc;ıf;&#ff0e;e00;Ab9;, ᜕ᶒɂb; ALDP(Y174C, H667D)IJf;c40;ᙠİc;e00;Qf4;ıb; ıa;,ALDP İc;ed6;IJe;d30;Pde;ᑁc0f;ᘤb98;ఆ╹⍟௷௺f38;〈௯ Ĵc;௺Ĵb;ὃ௨Ĵc;ıf;&#ff0e;᜕ᶒɂb; ALDP(R617H) IJf; ALDP IJe;˿a;Ife;İc;a8d;ఉĴc;IJa;İb;௷ıf;&#ff0e;᜕ᶒɂb; ALDP(R104C, G116R, S342P, Q544R, S606P) IJf;[ce;˯f;ɂb;ijb;ijc;Ȝc;a0b;ea6;IJe; bf; f3; d1; af;[cf;İc;˿a;Ife;௱, da; eb; aa; ad; b7; bd; fc; e0;ఆIJe;c40;ᙠఊNba;a8d;௯Ĵc;ıf;IJe;,௭Ĵc;IJe; ᜕ᶒɂb; ALDP IJf;ᔠᡂ௯Ĵc;ıf;IJe;௵IJb;b63;e38;IJb; da; eb; aa; ad; b7; bd; fc; e0;IJb;Έb;Ĵc;Ĵb;İc;, da; eb; aa; ad; b7; bd; fc; e0;̳c;IJb;İa; ௺ıd;IJe;a5f;Pfd;(ATP d50;ᔠ fb;4a0;c34;ᑖYe3;Re5;௱İf;IJf;9fa;cea;f38; 〈)IJb;ᶒe38;ఔᢝ௸௭İc;?a8;bdf;௯Ĵc;ıf;&#ff0e;ᱯIJb; R104C, G116R, S342P IJf; TMD IJb;b58;ᙠ௳Ĵb;௭İb; ALDP IJe;9fa;cea;f38;〈Pfd;İc;᜕ᓄ௱௺Ĵb;ὃ௨Ĵc;Ĵb;&#ff0e;e00;Ab9;, NBD IJb;b58;ᙠ௳Ĵb; Q544R, S606P IJf; ATP d50;ᔠ fb;4a0;c34; ᑖYe3;IJb;f71;aff;ఔe0e;௨௺Ĵb;5ef;Pfd;ឋİc;ὃ௨Ĵc;Ĵb;&#ff0e;ije;ıf; S606P, S606L IJf;᜕ᶒİc;Ȝc;௲Ze8;f4d;ఊEcb;⌼ḄIJb;b89;b9a; ឋİc;ᶒIJa;௷௺ıf;&#ff0e;Roerig IJf; S606L IJe;᜕ᶒɂb; ALDP IJf;,ATP IJe;Yaa;Ȥc;ឋİc;f4e;e0b;௱௺Ĵb;e00;Ab9; ATP 4a0;c34;ᑖYe3;IJf;b63;e38;IJb;ʹc;Ĵf;Ĵc;௺Ĵb;ᛇȠa;௱௺ Ĵb;&#ff0e; 29) ௭IJe;௭IJf; ALDP ATP IJe;Yaa;Ȥc;ឋİc; ALDP IJe;b89;b9a;ឋIJb;ఊf71;aff;ఔ5ca;ijc;௱௺Ĵb;5ef;Pfd;ឋఔ̙a;௱௺ Ĵb;&#ff0e;S606L S606P IJe;b89;b9a;ឋIJe;⍟a5f;Pfd;IJe;_a2;fc2; IJf; ALDP IJe;a5f;Pfd;ఔMe5;Ĵb;e0a;ఊ‐ᕡdf1;Fb9;Ĵa;, eca;f8c;௯IJb;ʳc;a0e;ఔʹc;௦fc5;⌕İc;Ĵb;&#ff0e;e00;Ab9;,Y174C IJe;᜕ᶒɂb; ALDP IJf;b63;e38;IJb;˿a;Ife;௳Ĵb;IJb;ఊfc2;Ĵf;ıa;, da; eb; aa; ad; b7; bd; fc; e0;ఆc40;ᙠıb;ıa;ed6;IJe;d30;Pde;ᑁc0f;ᘤb98;ఆ df; b9; bf; fc; b2; c3; c6; a3; f3; b0;௱ıf;&#ff0e;௭Ĵc;ije;IJb; da; eb; aa; ad; b7; bd; fc; e0;ఆIJe;c40;ᙠᓄ b7; b0; ca; eb;ఔb20;İf; da; eb; aa; ad; b7; bd; fc; e0; ̳c; bf; f3; d1; af;cea;IJf;,Ϗe;ᱯᶒḄIJb; df; c8; b3; f3; c9; ea; a2;ఌc0f;Pde; f53;IJb;Ofb;ʹc;௳Ĵb;௭İc;Me5;Ĵc;௺Ĵb;&#ff0e; 30,31) ఐ௷௺, ALDP IJe; TMD2ߟ3 IJe;╹IJe; eb; fc; d7;IJf;, da; eb; aa; ad; b7; bd; fc; e0;ఆIJe;c40;ᙠᓄIJb;[cd;⌕IJa;f79;ᒘఔʧc;ıf;௱௺Ĵb;5ef;Pfd; ឋİc;?a8;bdf;௯Ĵc;Ĵb;&#ff0e;Pex19p b58;ᙠᓄIJe; in vitro bf; f3; d1; af;cea;ffb;a33;cfb;IJb;İa;௺,ALDP(Y174C)IJf; Pex19p IJb;d50;ᔠİd;Ĵb;IJe;,ALDP IJe; N ʠb;aef; 67ߟ164 IJb;b58;ᙠ௳Ĵb; da; eb; aa; ad; b7; bd; fc; e0;Ofb;ʹc;IJb;fc2;Ĵf;Ĵb;♚9df;İc; ALDP IJe;f55;İb;IJe;Ecb;⌼᜕ᓄIJb;ఐ௷௺ de; b9; af;௯Ĵc;Ĵb; IJe;İb;ఊ௱Ĵc;IJa;&#ff0e; 5.
X
ABCD1 p.His667Asp 17202797:34:1762
status: NEW41 168 Vol. 127 (2007) ALDP IJb;௸௺IJf; ALDP IJe; C ʠb;aef;IJb; GFP(green ߮uorescent protein)ఔͮd;ᔠ௯ıb;ıf;᜕ᶒ ALDP-GFP ఔ˿a;Ife;௯ıb;Ĵb;ఊIJb;,[ce;˯f;ɂb; d2; c8; ALDP ఔ CHO d30;Pde;IJb;ᐳ ˿a;Ife;௯ıb;ıf;(CHO d30;Pde;IJb;ఊ ᑁᙠឋIJe; ALDP İc;˿a;Ife;௱௺Ĵb;İc;,ʠc;b9f; a13;IJb;ᵨıf;ᢙf53;İc; ea4;dee;௱IJa;ıf;ఉ, d2; c8; ALDP ఔ˿a;Ife;௯ıb;ıf;) &#ff0e;GFP ͮd;ᔠ bf; f3; d1; af;cea;IJf;ᑁᙠឋIJe; bf; f3; d1; af;cea;IJe;ȕa;ᑩİc;bb9; ᧕Ĵb;௭,b89;b9a;˿a;Ife;d30;Pde;IJe;5d6;f97;İc;bb9;᧕IJb;İd;Ĵb; ௭IJa;IJe;ᑭFb9;İc;Ĵb;&#ff0e;௭IJe;b9f; a13;IJf;, da; eb; aa; ad; b7; bd; fc; e0;IJb;b63;e38;IJb;f38;〈௯Ĵc;Ĵb;᜕ᶒɂb; ALDP(R104C, G116R, S606P) , da; eb; aa; ad; b7; bd; fc; e0;IJb;c40;ᙠ௱IJa;᜕ ᶒɂb; ALDP(Y174C) ,˿a;Ife;[cf;İc;f4e;e0b;௱௺Ĵb;᜕ᶒ ɂb; ALDP(H667D)ఔ⍶క௴&#ff0e;௭Ĵc;b89;b9a;Έe;ᒖ˿a; Ife;d30;Pde;IJb;İa;௫Ĵb; ALDP-GFP IJe;d30;Pde;ᑁc40;ᙠឋఔijf;Ĵb; ,ıd;IJe;ᑖe03;IJf;e00;Έe;ឋ˿a;Ife;௯ıb;ıf; His-ALDP Ȝc; Ed8;௷ıf;&#ff0e; f97;Ĵc;ıf;b89;b9a;˿a;Ife;d30;Pde;ఐĴa; aa; eb; ac; cd; e9;c97;ᑖ˱b;ఔabf; Xfd;௱, b7; e7;cd6;bc6;ea6;4fe;Βd;⍤fc3;ᑖ`e2;cd5;IJb;ఐĴa;ᔜ d5; e9; af; b7; e7; f3;IJb;ᑖ௫ıf;IJe;௵,SDS-PAGE 5ca;ఁ immunoblotting IJb;ఐĴa;᜕ᶒ ALDP IJe;c40;ᙠIJb;௸௺Ye3;᪆ఔʹc;௷ ıf;(Fig. 3) &#ff0e; da; eb; aa; ad; b7; bd; fc; e0; de; fc; ab; fc;௱௺ da; eb; aa; ad; b7; bd; fc; e0;̳c; bf; f3; d1; af;cea;Ĵb; PMP70 da; eb; aa; ad; b7; bd; fc; e0;IJe;e3b;⌕IJa; de; c8; ea; c3; af; b9; bf; f3; d1; af;cea; Ĵb; ab; bf; e9; fc; bc;ఔᵨıf;&#ff0e;[ce;˯f;ɂb; ALDP ఔb89;b9a;Έe;ᒖ ˿a;Ife;௱௺Ĵb;d30;Pde;IJb;İa;௺, ab; bf; e9; fc; bc;d3b;ឋe26;ఁIJb; PMP70 İc;e3b;௱௺ d5; e9; af; b7; e7; f3; 3 5ca;ఁ 4 IJb;b58;ᙠ௳ Ĵb;௭ఐĴa;,௭IJe;ᑖ˱b;IJb; da; eb; aa; ad; b7; bd; fc; e0;İc;8de;5ce;௯ Ĵc;ıf;௭İc;̙a;ᖂ௯Ĵc;ıf;&#ff0e;ije;ıf;d04; 110 kDa IJe;ᑖb50; b5; a4; ba;ఔᢝ௸ ALDP-GFP e26;ఁIJb; 83 kDa IJe;[ce;˯f;ɂb; ALDP IJf;, da; eb; aa; ad; b7; bd; fc; e0; de; fc; ab; fc;ijb;ijc;Ȝc;௲ ᑖe03;ఔ̙a;௱௺ıf;௭İb; da; eb; aa; ad; b7; bd; fc; e0;IJb;c40;ᙠ ௱௺Ĵb;௭İc;̙a;ᖂ௯Ĵc;ıf;&#ff0e;ije;ıf;᜕ᶒɂb; ALDP-GFP(G116R, S606P)ఊȜc;Ed8;IJe;ᑖe03;ఔ̙a;௱ıf;&#ff0e; e00;Ab9;,᜕ᶒɂb; ALDP-GFP(H667D)ఔb89;b9a;Έe;ᒖ ˿a;Ife;௱௺Ĵb;d30;Pde;IJe;ᛊᔠIJf;,ALDP-GFP IJe; d0; f3; c9; IJf;ʳc;3fa;௯Ĵc;IJa;İb;௷ıf;(Fig. 3) &#ff0e;‐ᕡdf1;௭IJb;, PMP70 IJf; ʳc; 3fa; ௯ Ĵc; ıf; İc; , ᐳ ˿a; Ife; ௯ ıb; ıf; [ce; ˯f; ɂb; ALDP IJe; d0; f3; c9;ఊʳc;3fa;௯Ĵc;IJa;İb;௷ıf;&#ff0e;ije;ıf;᜕ᶒɂb; ALDP(S606L)IJb;௸௺ఊȜc;Ed8;௷ıf;&#ff0e;௭Ĵc; IJe; d50; ʧc; IJf; , ᜕ ᶒ ɂb; ALDP ( H667D, S606L ) IJf; PMP70 IJf;IJa;İf;,[ce;˯f;ɂb; ALDP ⋋ᔠf53;ఔf62;ᡂ ௱,e21;ὅİc;ᑖYe3;௯Ĵc;Ĵb;5ef;Pfd;ឋఔ̙a;ᖂ௱௺Ĵb;&#ff0e; ABC bf; f3; d1; af;cea;IJe;a5f;Pfd;˿a;Ife;IJb;[cd;⌕Ĵb; TMD ఌ NBD ee5;IJe; C ʠb;aef;Ze8;f4d;IJe;᜕ᶒİc; bf; f3; d1; af;cea;IJe;b89; b9a;ឋIJb;f71;aff;ఔ5ca;ijc;௳௭IJf;‐ᕡdf1;&#ff0e;ALDP IJe; C ʠb;aef;Ze8;f4d;Ĵb; 600ߟ700 a2; df; ce;⏚IJe;᜕ᶒİc; X-ALD ఔf15;İd;d77;௭௳ϗb;ea6;İc; ad8;௭İb;,ALDP IJe; C ʠb;aef;Ze8;f4d;IJf; bf; f3; d1; af;cea;IJe;b89;b9a;ឋIJb;[cd;⌕IJa;f79;ᒘఔ>c5; ௷௺Ĵb;5ef;Pfd;ឋİc;Ĵb;&#ff0e;Liu IJf; ALDP IJe; c0; a4; de; fc;ᓄIJb;IJf; C ʠb;aef;Ze8;f4d;(AA.631ߟ745)İc;[cd;⌕ Ĵb;ᛇȠa;௱௺Ĵb;&#ff0e; 13) H667D ఌ S606L IJe;ఐ௦IJa; ᜕ᶒIJf;,ıd;Ĵc;Qea;eab;Ĵb;IJf;[ce;˯f;ɂb; ALDP df; b9; d5; a9; fc; eb; c9;௱ıf; c0; a4; de; fc;ఔf62;ᡂ௱,ᶒe38; bf; f3; d1; af;cea; ௱௺a8d;b58;௯Ĵc;ᑖYe3;௯Ĵc;Ĵb;ὃ௨Ĵc;Ĵb;&#ff0e;e00;Ab9;,᜕ᶒ ɂb; ALDP-GFP(R104C)IJf; da; eb; aa; ad; b7; bd; fc; e0;ᑖ˱b; IJb;8de;5ce;௯Ĵc;Ĵb;ఊIJe;IJe;, d5; e9; b0; e1; f3; c8;ᓄ௱௺Ĵb;௭ 169 Fig. 4.
X
ABCD1 p.His667Asp 17202797:41:1547
status: NEWX
ABCD1 p.His667Asp 17202797:41:4305
status: NEWX
ABCD1 p.His667Asp 17202797:41:5057
status: NEWX
ABCD1 p.His667Asp 17202797:41:6466
status: NEW[hide] Functional hot spots in human ATP-binding cassette... Protein Sci. 2010 Nov;19(11):2110-21. Kelly L, Fukushima H, Karchin R, Gow JM, Chinn LW, Pieper U, Segal MR, Kroetz DL, Sali A
Functional hot spots in human ATP-binding cassette transporter nucleotide binding domains.
Protein Sci. 2010 Nov;19(11):2110-21., [PMID:20799350]
Abstract [show]
The human ATP-binding cassette (ABC) transporter superfamily consists of 48 integral membrane proteins that couple the action of ATP binding and hydrolysis to the transport of diverse substrates across cellular membranes. Defects in 18 transporters have been implicated in human disease. In hundreds of cases, disease phenotypes and defects in function can be traced to nonsynonymous single nucleotide polymorphisms (nsSNPs). The functional impact of the majority of ABC transporter nsSNPs has yet to be experimentally characterized. Here, we combine experimental mutational studies with sequence and structural analysis to describe the impact of nsSNPs in human ABC transporters. First, the disease associations of 39 nsSNPs in 10 transporters were rationalized by identifying two conserved loops and a small alpha-helical region that may be involved in interdomain communication necessary for transport of substrates. Second, an approach to discriminate between disease-associated and neutral nsSNPs was developed and tailored to this superfamily. Finally, the functional impact of 40 unannotated nsSNPs in seven ABC transporters identified in 247 ethnically diverse individuals studied by the Pharmacogenetics of Membrane Transporters consortium was predicted. Three predictions were experimentally tested using human embryonic kidney epithelial (HEK) 293 cells stably transfected with the reference multidrug resistance transporter 4 and its variants to examine functional differences in transport of the antiviral drug, tenofovir. The experimental results confirmed two predictions. Our analysis provides a structural and evolutionary framework for rationalizing and predicting the functional effects of nsSNPs in this clinically important membrane transporter superfamily.
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No. Sentence Comment
50 Disease-associated nsSNPs at Three Structural Hotspots in Human ABC Transporter NBDs Gene Disease Position ARA motif ABCB11 BRIC2 A570T ABCD1 X-ALD A616V CFTR CF A559T ABCC6 PXE R765Q ABCC8 HHF1 R841G ABCC8 HHF1 R1493Q ABCC8 HHF1 R1493W ABCD1 X-ALD R617C ABCD1 X-ALD R617G ABCD1 X-ALD R617H CFTR CF R560K CFTR CF R560S CFTR CF R560T ABCA1 HDLD1 A1046D ABCB4 ICP A546D C-loop 1 motif ABCC8 HHF1 D1471H ABCC8 HHF1 D1471N CFTR CBAVD G544V ABCC8 HHF1 G1478R C-loop2 motif ABCA4 STGD1 H2128R ABCC8 HHF1 K889T ABCD1 X-ALD R660P ABCD1 X-ALD R660W ABCA1 HDLD2 M1091T ABCA4 STGD1 E2131K ABCA12 LI2 E1539K ABCA4 STGD1 and CORD3 E1122K CFTR CF L610S ABCC8 HHF1 L1543P ABCA1 Colorectal cancer sample; somatic mutation A2109T ABCC9 CMD1O A1513T ABCD1 X-ALD H667D CFTR CF A613T ABCA1 HDLD2 D1099Y ABCD1 X-ALD T668I CFTR CF D614G ABCA4 STGD1 R2139W ABCA4 STGD1 R1129C ABCA4 ARMD2, STGD1, and FFM R1129L Disease abbreviations are as follows: BRIC2, benign recurrent intrahepatic cholestasis type 2; X-ALD, X-linked adrenoleukodystrophy; CF, cystic fibrosis; PXE, Pseudoxanthoma elasticum; HHF1, familial hyperinsulinemic hypoglycemia-1; HDLD1, high density lipoprotein deficiency type 1; ICP, intrahepatic cholestasis of pregnancy; CBAVD, congenital bilateral absence of the vas deferens; STGD1, Stargardt disease type 1; HDLD2, high density lipoprotein deficiency type 2; LI2, ichthyosis lamellar type 2; CORD3, cone-rod dystrophy type 3; CMD1O, cardiomyopathy dilated type 1O; ARMD2, age-related macular degeneration type 2; FFM, fundus flavimaculatus.
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ABCD1 p.His667Asp 20799350:50:744
status: NEW