ABCMdb

ABCA1 p.Lys1952Met

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Publications

PMID: 21787882 [PubMed] Bocer T et al: "The mammalian ABC transporter ABCA1 induces lipid-dependent drug sensitivity in yeast."

No. Sentence Comment

39 Two additional vectors, bearing the nonfunctional mutant, pTB1A1MM and pTB2A1MM were generated by replacing the central fragment of ABCA1 gene by a fragment containing K939M and K1952M mutations in the Walker A motif in both nucleotide binding domains [2,4]. Login to comment

PMID: 22028339 [PubMed] Nagao K et al: "ATP hydrolysis-dependent conformational changes in the extracellular domain of ABCA1 are associated with apoA-I binding."

No. Sentence Comment

7 Chambenoit et al. reported that substituting the Walker A lysine residue in either NBD with methionine (K939M or K1952M) abolishes the ability of apoA-I to bind to ABCA1-expressing cells and inhibits cholesterol secretion to apoA-I (12). Login to comment
26 Plasmids The expression vectors for wild-type ABCA1, ABCA1-K939M, ABCA1-K1952M, and ABCA1-K939M,K1952M that were tagged with GFP at the C terminus were generated as previously described (7, 13). Login to comment
88 HEK293 cells stably expressing wild-type (wt) ABCA1 or the Walker A lysine mutants ABCA1-K939M, ABCA1-K1952M, or ABCA1-K939M,K1952M were incubated for 24 h in DMEM containing 0.02% BSA with or without 5 ␮g/ml apoA-I. Login to comment
119 However, cholesterol efflux was impaired in cells expressing ABCA1-K939M,K1952M, a mutant in which the Walker A lysines in both NBDs are replaced with methionines, or the single Walker A lysine mutants ABCA1-K939M or ABCA1-K1952M, in which the Walker A lysine in NBD1 or NBD2 is replaced with methionine (Fig. 3B). Login to comment
135 We consistently observed stronger ATP binding to NBD2 in ABCA1-K939M,K1952M-TEV-GFP, although the reason for this higher-affinity interaction is unknown. Login to comment
143 On the other hand, the K1952M mutation drastically decreased the photoaffinity labeling by approximately 95%. Login to comment
144 Interestingly, the addition of vanadate significantly (1.7-fold) enhanced the photoaffinity labeling of ABCA1-K1952M, suggesting that vanadate stabilizes the nucleotide at NBD1 after ATP hydrolysis. Login to comment
145 There was no photoaffinity-labeled band with ABCA1-K939M,K1952M. Login to comment
147 These results suggest that the occluded nucleotide in the NBD of wild-type ABCA1, ABCA1-K939M, and ABCA1-K1952M is not ATP but ADP, that both NBDs occlude ADP after ATP hydrolysis, and that the features of the two NBDs of ABCA1 are quite different. Login to comment
162 In addition, there were no detectable nucleotide occlusions at either NBD in the K1952M mutant. Login to comment
27 Plasmids The expression vectors for wild-type ABCA1, ABCA1-K939M, ABCA1-K1952M, and ABCA1-K939M,K1952M that were tagged with GFP at the C terminus were generated as previously described (7, 13). Login to comment
89 HEK293 cells stably expressing wild-type (wt) ABCA1 or the Walker A lysine mutants ABCA1-K939M, ABCA1-K1952M, or ABCA1-K939M,K1952M were incubated for 24 h in DMEM containing 0.02% BSA with or without 5 òe;g/ml apoA-I. Login to comment
120 However, cholesterol efflux was impaired in cells expressing ABCA1-K939M,K1952M, a mutant in which the Walker A lysines in both NBDs are replaced with methionines, or the single Walker A lysine mutants ABCA1-K939M or ABCA1-K1952M, in which the Walker A lysine in NBD1 or NBD2 is replaced with methionine (Fig. 3B). Login to comment
137 We consistently observed stronger ATP binding to NBD2 in ABCA1-K939M,K1952M-TEV-GFP, although the reason for this higher-affinity interaction is unknown. Login to comment
146 Interestingly, the addition of vanadate significantly (1.7-fold) enhanced the photoaffinity labeling of ABCA1-K1952M, suggesting that vanadate stabilizes the nucleotide at NBD1 after ATP hydrolysis. Login to comment
149 These results suggest that the occluded nucleotide in the NBD of wild-type ABCA1, ABCA1-K939M, and ABCA1-K1952M is not ATP but ADP, that both NBDs occlude ADP after ATP hydrolysis, and that the features of the two NBDs of ABCA1 are quite different. Login to comment
165 In addition, there were no detectable nucleotide occlusions at either NBD in the K1952M mutant. Login to comment

PMID: 19202195 [PubMed] Nagao K et al: "Sodium taurocholate-dependent lipid efflux by ABCA1: effects of W590S mutation on lipid translocation and apolipoprotein A-I dissociation."

No. Sentence Comment

45 Cell culture Human embryonic kidney (HEK293) cells and WI-38 fibroblasts were grown in a humidified incubator (5% CO2) at 37°C in DMEM supplemented with 10% heat-inactivated FBS. Plasmids The expression vectors for wild-type ABCA1, ABCA1-W590S, and ABCA1-K939M,K1952M (MM), fused to GFP at their C termini, were generated as described previously (3, 18). Login to comment
43 Plasmids The expression vectors for wild-type ABCA1, ABCA1-W590S, and ABCA1-K939M,K1952M (MM), fused to GFP at their C termini, were generated as described previously (3, 18). Login to comment

PMID: 19258317 [PubMed] Hozoji M et al: "Formation of two intramolecular disulfide bonds is necessary for ApoA-I-dependent cholesterol efflux mediated by ABCA1."

No. Sentence Comment

90 ApoA-I bound to cells expressing wild-type ABCA1 but not to cells expressing ABCA1-MM (ABCA1(K939M/K1952M)), a mutant in which the Walker A lysines in both nucleotide-binding domains are replaced by methionines, although the expression level (data not shown) and surface expression (Fig. 4A) of the mutant were comparable with those of the wild-type protein, as reported (24). Login to comment
88 ApoA-I bound to cells expressing wild-type ABCA1 but not to cells expressing ABCA1-MM (ABCA1(K939M/K1952M)), a mutant in which the Walker A lysines in both nucleotide-binding domains are replaced by methionines, although the expression level (data not shown) and surface expression (Fig. 4A) of the mutant were comparable with those of the wild-type protein, as reported (24). Login to comment

PMID: 19170766 [PubMed] Azuma Y et al: "Retroendocytosis pathway of ABCA1/apoA-I contributes to HDL formation."

No. Sentence Comment

197 DNA construction We constructed plasmids for expressing human wild-type ABCA1, ABCA1(W590S) and ABCA1(K939M,K1952M)(MM) bearing an insertion of the influenza virus hemagglutinin (HA) epitope sequence between residues 207 and 208 (within the first extracellular loop), using the bicistronic expression vector pHaMAIRESneo. Login to comment

PMID: 16611739 [PubMed] Tam SP et al: "ABCA1 mediates high-affinity uptake of 25-hydroxycholesterol by membrane vesicles and rapid efflux of oxysterol by intact cells."

No. Sentence Comment

158 A: immunoblots of total membrane proteins (15 ␮g) from vesicles containing wild-type ABCA1 (KK) and mutant proteins containing Met substitutions of the highly conserved Walker A Lys of nucleotide binding domain (NBD1; K939M, MK), NBD2 (K1952M, KM), or both NBDs (K939M/K1952M, MM), as well as control vesicles of beta-glucuronidase (beta-gus). Login to comment

PMID: 16500904 [PubMed] Takahashi K et al: "Purification and ATPase activity of human ABCA1."

No. Sentence Comment

33 2 The abbreviations used are: ABCA1, ATP-binding cassette protein A1; ABCA1 MM, ABCA1 K939M-K1952M; MDR, multidrug resistance; MRP, multidrug resistance-related protein; NBF, nucleotide-binding fold; apo, apolipoprotein; HDL, high density lipoprotein; FC, free cholesterol; PL, phospholipid; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SM, sphingomyelin; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; DPPC, 1,2-dipalmitoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; DPPE, 1,2- dipalmitoylphosphatidylethanolamine; DPPG, 1,2-dipalmitoylphosphatidylglycerol; Sf9, Spodoptera frugiperda 9; HEK, human embryo kidney; NGF, N-glycosidase F; DDM, n-dodecyl-beta-D-maltoside; NTA, nitrilotriacetic acid. Login to comment
117 ABCA1 K939M-K1952M protein, expressed and purified in the same procedure, was as pure as the wild type (Figs. Login to comment
149 To confirm that ATPase activity is derived from purified ABCA1, a baculovirus for ABCA1 K939M-K1952M mutant, in which lysine 939 and lysine 1952 in the Walker A motif of nucleotide binding folds were replaced by methionines, was constructed. Login to comment
151 Purified ABCA1 K939M-K1952M protein showed little ATPase activity, less than 10 nmol/min/mg of protein (Fig. 5). Login to comment
166 E, purified ABCA1 K939M-K1952M mutant, 0.5 ␮g (Coomassie Brilliant Blue R-250 staining). Login to comment
214 A, ATPase activity was measured in the presence of various concen- trationsofMgATP.F,wildtype;E,K939M-K1952M mutant. Login to comment
32 2 The abbreviations used are: ABCA1, ATP-binding cassette protein A1; ABCA1 MM, ABCA1 K939M-K1952M; MDR, multidrug resistance; MRP, multidrug resistance-related protein; NBF, nucleotide-binding fold; apo, apolipoprotein; HDL, high density lipoprotein; FC, free cholesterol; PL, phospholipid; PC, phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; SM, sphingomyelin; PG, phosphatidylglycerol; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; DPPC, 1,2-dipalmitoylphosphatidylcholine; POPS, 1-palmitoyl-2-oleoylphosphatidylserine; DPPE, 1,2- dipalmitoylphosphatidylethanolamine; DPPG, 1,2-dipalmitoylphosphatidylglycerol; Sf9, Spodoptera frugiperda 9; HEK, human embryo kidney; NGF, N-glycosidase F; DDM, n-dodecyl-beta-D-maltoside; NTA, nitrilotriacetic acid. Login to comment
114 ABCA1 K939M-K1952M protein, expressed and purified in the same procedure, was as pure as the wild type (Figs. 1B and 2E). Login to comment
145 To confirm that ATPase activity is derived from purified ABCA1, a baculovirus for ABCA1 K939M-K1952M mutant, in which lysine 939 and lysine 1952 in the Walker A motif of nucleotide binding folds were replaced by methionines, was constructed. Login to comment
147 Purified ABCA1 K939M-K1952M protein showed little ATPase activity, less than 10 nmol/min/mg of protein (Fig. 5). Login to comment
162 E, purified ABCA1 K939M-K1952M mutant, 0.5 òe;g (Coomassie Brilliant Blue R-250 staining). Login to comment
208 A, ATPase activity was measured in the presence of various concen- trationsofMgATP.F,wildtype;E,K939M-K1952M mutant. Login to comment

PMID: 23479619 [PubMed] Nagata KO et al: "ABCA1 dimer-monomer interconversion during HDL generation revealed by single-molecule imaging."

No. Sentence Comment

131 Human ABCA1 and ABCA1-MM (with two point mutations of K939M and K1952M) were fused with EGFP or monomeric (m)EGFP at their carboxyl termini, where an mEGFP mutant was modified from EGFP by introducing a mutation corresponding to an A206K monomeric mutant (33). Login to comment

PMID: 24097981 [PubMed] Quazi F et al: "Differential phospholipid substrates and directional transport by ATP-binding cassette proteins ABCA1, ABCA7, and ABCA4 and disease-causing mutants."

No. Sentence Comment

66 Corresponding ABCA4 mutations determined by amino acid alignment with ABCA1 included S100P, W605S, F608L, T959I, N965S, C1502R, T1537M, R2107P, and P2180L. Login to comment
67 ABCA1-MM was constructed to harbor the Walker A-motif lysine-to-methionine mutations K939M/K1952M by the nested PCR method; ABCA4-MM had the corresponding K969M/ K1969M Walker A mutations (37), and ABCA7-MM had the Lipid Transport Activity of ABCA Transporters NOVEMBER 29, 2013ߦVOLUME 288ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 34415 at SEMMELWEIS UNIV OF MEDICINE on December 3, K847M/K1833M Walker A mutations. Login to comment
69 ABCA1-MM was constructed to harbor the Walker A-motif lysine-to-methionine mutations K939M/K1952M by the nested PCR method; ABCA4-MM had the corresponding K969M/ K1969M Walker A mutations (37), and ABCA7-MM had the Lipid Transport Activity of ABCA Transporters NOVEMBER 29, 2013ߦVOLUME 288ߦNUMBER 48 JOURNAL OF BIOLOGICAL CHEMISTRY 34415 at SEMMELWEIS UNIV OF MEDICINE on December 3, K847M/K1833M Walker A mutations. Login to comment