ABCMdb

ABCD3 p.Gly478Arg

Predicted by SNAP2:	A: D (95%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), H: 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, H: 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] Functional characterization of the adrenoleukodyst... Endocr Res. 2002 Nov;28(4):741-8. Gartner J, Dehmel T, Klusmann A, Roerig P
Functional characterization of the adrenoleukodystrophy protein (ALDP) and disease pathogenesis.
Endocr Res. 2002 Nov;28(4):741-8., [PMID:12530690]

Abstract [show]
X-linked adrenoleukodystrophy (X-ALD) is the most common peroxisomal disorder characterized by abnormal accumulation of saturated very long chain fatty acids in tissues and body fluids with predominance in brain white matter and adrenal cortex. The clinical phenotype is highly variable ranging from the severe childhood cerebral form to asymptomatic persons. The responsible ALD gene encodes the adrenoleukodystrophy protein (ALDP), a peroxisomal integral membrane protein that is a member of the ATP-binding cassette (ABC) transporter protein family. The patient gene mutations are heterogeneously distributed over the functional domains of ALDP. The extreme variability in clinical phenotype, even within one affected family, indicates that besides the ALD gene mutations other factors strongly influence the clinical phenotype. To understand the cell biology and function of mammalian peroxisomal ABC transporters and to determine their role in the pathogenesis of X-ALD we developed a system for expressing functional ABC protein domains in fusion with the maltose binding protein. Wild type and mutant fusion proteins of the nucleotide-binding fold were overexpressed, purified, and characterized by photoaffinity labeling with 8-azido ATP or 8-azido GTP and a coupled ATP regenerating enzyme assay for ATPase activity. Our studies provide evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter and that ALD gene mutations alter peroxisomal transport function. The established disease model will be used further to study the influence of possible disease modifier proteins on ALDP function.

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No. Sentence Comment
41 The mutant constructs included missense mutations of patients with X-ALD in the nucleotide binding fold regions Walker A and 19mer (ALDP-NBF-G512S, ALDP-NBF-Q544R, ALDP-NBF-P560L, ALDP-NBF-R591Q, ALDP-NBF-S606L, and ALDP-NBF-D629H) and corresponding mutations in another ABC transporter in the peroxisome membrane, the 70 kDa peroxisomal membrane protein (PMP70; PMP70-NBF-G478R, PMP70- NBF-S572I). Login to comment
63 The mutation S572I in the PMP70 gene reduces considerable ATPase activity where as the mutation G478R alters ATP-binding affinity. Login to comment

[hide] Characterization and functional analysis of the nu... FEBS Lett. 2001 Mar 9;492(1-2):66-72. Roerig P, Mayerhofer P, Holzinger A, Gartner J
Characterization and functional analysis of the nucleotide binding fold in human peroxisomal ATP binding cassette transporters.
FEBS Lett. 2001 Mar 9;492(1-2):66-72., [PMID:11248239]

Abstract [show]
The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.

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No. Sentence Comment
5 Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Login to comment
24 For the mutant constructs we selected X-ALD patient mutations in highly conserved residues in the Walker A and 19-mer region of the NBF of ALDP (G512S and S606L) and the corresponding PMP70 mutations (G478R and S572I). Login to comment
84 We mutated the central glycine in the Walker A motif of PMP70 and ALDP making the evolutionary severe substitutions G478R and G512S. Login to comment
85 Additionally, we changed the conserved serine in the 19-mer motif of PMP70 and ALDP to isoleucine (S572I) and leucine (S606L), respectively. Login to comment
88 The K-subunit of L-galactosidase (L-Protein Synthetic oligonucleotide Mutation ALDP 5P-CCCCAATGGCTGCAGCAAGAGCTCCC-3P G512S 5P-GGATCCGGACAGGGAGCTCTTGCTGCAGC-3P ALDP 5P-ACTGGAAGGACGTCCTGTTGGG-3P S606L 5P-CGCCACCCAACAGGACGTCCTTCC-3P PMP70 5P-GGCTGCAGAAAGAGTTCACTTTTCCG-3P G478R 5P-GGCCATAATTCACCAAGAACACGGAAA AGTGAACTCTTTCTG-3P PMP70 5P-GACGTACTCATTGGTGGAG-3P S572I 5P-CCACCAATGAGTACGTCCATCCAATCC-3P gal) in fusion with the MBP was used as a control. Login to comment
136 The S606L and G478R mutants have a decreased ATP binding a¤nity while the G512S and S572I mutants decrease the maximum velocity of ATPase activity. Login to comment
182 Previous studies on MDR suggest that the ATPase activity of the native protein Table 1 Kinetic parameters of ATPase activity in wild type and mutant ALDP and PMP70 NBF fusion proteins Fusion protein KM (WM) Vmax (nmol/Wmol NBF/min) Speci'c activity (1033 U/mg) ALDP (wild type) 11.5 þ 0.97 641.9 þ 10.7 10.0 þ 0.17 ALDP (G512S) 17.9 þ 1.23 279.3 þ 4.2 4.4 þ 0.06 ALDP (S606L) 45.6 þ 2.40 666.0 þ 8.7 10.4 þ 0.14 PMP70 (wild type) 8.2 þ 0.52 580.8 þ 6.7 9.0 þ 0.10 PMP70 (G478R) 161.8 þ 34.40 641.2 þ 28.2 10.0 þ 0.44 PMP70 (S572I) 9.9 þ 0.82 298.1 þ 4.7 4.6 þ 0.07 The kinetic data of all fusion proteins are mean values and standard deviations of 15^20 measurements at various protein concentrations using at least two distinct protein preparations. Login to comment
89 The K-subunit of L-galactosidase (LProtein Synthetic oligonucleotide Mutation ALDP 5P-CCCCAATGGCTGCAGCAAGAGCTCCC-3P G512S 5P-GGATCCGGACAGGGAGCTCTTGCTGCAGC-3P ALDP 5P-ACTGGAAGGACGTCCTGTTGGG-3P S606L 5P-CGCCACCCAACAGGACGTCCTTCC-3P PMP70 5P-GGCTGCAGAAAGAGTTCACTTTTCCG-3P G478R 5P-GGCCATAATTCACCAAGAACACGGAAA AGTGAACTCTTTCTG-3P PMP70 5P-GACGTACTCATTGGTGGAG-3P S572I 5P-CCACCAATGAGTACGTCCATCCAATCC-3P gal) in fusion with the MBP was used as a control. Login to comment
137 The S606L and G478R mutants have a decreased ATP binding a&#a4;nity while the G512S and S572I mutants decrease the maximum velocity of ATPase activity. Login to comment
183 Previous studies on MDR suggest that the ATPase activity of the native protein Table 1 Kinetic parameters of ATPase activity in wild type and mutant ALDP and PMP70 NBF fusion proteins Fusion protein KM (WM) Vmax (nmol/Wmol NBF/min) Speci'c activity (1033 U/mg) ALDP (wild type) 11.5 &#fe; 0.97 641.9 &#fe; 10.7 10.0 &#fe; 0.17 ALDP (G512S) 17.9 &#fe; 1.23 279.3 &#fe; 4.2 4.4 &#fe; 0.06 ALDP (S606L) 45.6 &#fe; 2.40 666.0 &#fe; 8.7 10.4 &#fe; 0.14 PMP70 (wild type) 8.2 &#fe; 0.52 580.8 &#fe; 6.7 9.0 &#fe; 0.10 PMP70 (G478R) 161.8 &#fe; 34.40 641.2 &#fe; 28.2 10.0 &#fe; 0.44 PMP70 (S572I) 9.9 &#fe; 0.82 298.1 &#fe; 4.7 4.6 &#fe; 0.07 The kinetic data of all fusion proteins are mean values and standard deviations of 15^20 measurements at various protein concentrations using at least two distinct protein preparations. Login to comment