ABCMdb

PMID: 23087442

Sorrenson B, Suetani RJ, Williams MJ, Bickley VM, George PM, Jones GT, McCormick SP
Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.
J Lipid Res. 2013 Jan;54(1):55-62. doi: 10.1194/jlr.M027193. Epub 2012 Oct 20., [PubMed]

Sentences

No. Mutations Sentence Comment

16 ABCA1 p.Asn1800His ABCA1 p.Arg1068His ABCA1 p.Thr1512Met ABCA1 p.Arg2004Lys ABCA1 p.Ala2028Val ABCA1 p.Ile659Val ABCA1 p.Ala594Thr ABCA1 p.Gln2239Asn ABCA1 p.Tyr1767Asp Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Login to comment 18 ABCA1 p.Asn1800His ABCA1 p.Arg1068His In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Login to comment 53 ABCA1 p.Asn1800His ABCA1 p.Arg1068His ABCA1 p.Thr1512Met ABCA1 p.Arg2004Lys ABCA1 p.Ala2028Val ABCA1 p.Ile659Val The Pearson`s correlation coefficient between the GFP and AlexaFluor594 of the ABCA1 mutations were previously identified in low HDL-C subjects and included three uncharacterized mutations, p.I659V, p.R2004K, and p.A2028V (18) and three variants, p.R1068H, p.T1512M, and p.N1800H, known to have reduced localization and cholesterol efflux (19, 20). Login to comment 54 ABCA1 p.Ala594Thr ABCA1 p.Gln2239Asn ABCA1 p.Tyr1767Asp We identified a further three novel mutations, p.A594T, p.Y1767D, and p.Q2239N, and these were also included in this study. We hypothesized that efflux function would be improved for mutants that are dysfunctional as a result of protein mislocation. Login to comment 73 ABCA1 p.Ala594Thr ABCA1 p.Gln2239Asn ABCA1 p.Tyr1767Asp Both media were RESULTS ABCA1 mutants with impaired localization have reduced cholesterol efflux function We identified three novel ABCA1 variants, p.A594T, p.Y1767D, and p.Q2239N, in heterozygote form in three individuals with HDL-C levels of 0.61, 0.17, and 0.37 mmol/L, respectively. Login to comment 74 ABCA1 p.Asn1800His ABCA1 p.Tyr1767Asp The individual heterozygote for p.Y1767D was also heterozygote for the p.N1800H ABCA1 mutation. Login to comment 76 ABCA1 p.Arg2004Lys ABCA1 p.Ala2028Val ABCA1 p.Ile659Val We first characterized the three novel ABCA1 mutants and three previously identified but uncharacterized mutants (p.I659V, p.R2004K, and p.A2028V) in HEK293 cells, which lack the endogenous ABCA1 protein (see supplementary Fig. I). Login to comment 77 ABCA1 p.Arg2004Lys ABCA1 p.Ala2028Val ABCA1 p.Ile659Val ABCA1 p.Ala594Thr ABCA1 p.Tyr1767Asp Investigation of the six uncharacterized mutations in transfected HEK293 cells showed the p.A594T, p.I659V, p.Y1767D, p.R2004K, and p.A2028V mutants to have various degrees of mislocalization (Fig. 2A). Login to comment 79 ABCA1 p.Arg2004Lys ABCA1 p.Tyr1767Asp Areas of mislocalized ABCA1-GFP are indicated by arrows in Fig. 2A with the p.Y1767D and R2004K mutants being the most affected. Login to comment 80 ABCA1 p.Tyr1767Asp The mislocalization of mutant ABCA1s was associated with a reduced cholesterol efflux function compared with wild-type-GFP ABCA1 (Fig. 2B) with the p.Y1767D mutant being the most affected (30.3% the efflux of wild-type). Login to comment 81 ABCA1 p.Gln2239Asn The p.Q2239N mutation did not affect plasma localization or cholesterol efflux function. Login to comment 85 ABCA1 p.Asn1800His ABCA1 p.Arg1068His ABCA1 p.Thr1512Met 4-PBA rescues mutant ABCA1 localization and improves cholesterol efflux function in transfected HEK293 cells 4-PBA treatment was applied to the six uncharacterized ABCA1 mutants as well as to three mutants that we have previously shown to have reduced cholesterol efflux function, p.R1068H (19), p.T1512M (20), and p.N1800H (20, Fig. 1). Login to comment 109 ABCA1 p.Asn1800His ABCA1 p.Arg1068His ABCA1 p.Thr1512Met ABCA1 p.Arg2004Lys ABCA1 p.Ala2028Val ABCA1 p.Tyr1767Asp Upon 4-PBA treatment, efflux function was significantly increased relative to the untreated level for the p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, and p.A2028V mutants (Fig. 3B). Login to comment 112 ABCA1 p.Asn1800His ABCA1 p.Arg1068His ABCA1 p.Thr1512Met ABCA1 p.Arg2004Lys ABCA1 p.Ala594Thr ABCA1 p.Tyr1767Asp Treatment with 4-PBA induced a significant increase in colocalization for the p.A594T, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, and p.R2004K mutants. Treatment with 4-PBA did not affect the colocalization of the wild-type ABCA1-GFP protein (supplementary Fig. II). Login to comment 121 ABCA1 p.Asn1800His 4-PBA improves mutant ABCA1 efflux function independent of protein level in primary fibroblasts We assessed the effect of 4-PBA treatment in the con- textofthenativeABCA1promoterusingavailablep.R1068H, p.N1800H, and wild-type primary fibroblast cell lines (19, 20). Login to comment 122 ABCA1 p.Asn1800His ABCA1 p.Arg1068His Fibroblasts were obtained from two p.R1068H heterozygote carriers (RH1 and RH2), two p.N1800H heterozygote carriers (NH1 and NH2), and two ABCA1 wild-type subjects (WT1 and WT2). Login to comment 124 ABCA1 p.Asn1800His We had previously determined that subject NH2 was also heterozygous for the p.C978fsX988 mutation, which encodes a truncated protein that is functionally null and in this individual was present on a seperate allele to the p.N1800H mutation (20). Login to comment 125 ABCA1 p.Asn1800His ABCA1 p.Tyr1767Asp mislocated mutants, p.Y1767D, and p.N1800H, showed a restored efflux function that was equivalent to wild-type untreated cells. Login to comment 126 ABCA1 p.Arg1068His However, the cholesterol efflux of the p.R1068H mutant was unable to be restored to wild-type levels. Login to comment 141 ABCA1 p.Asn1800His ABCA1 p.Arg1068His In contrast, there was a clear trend for increased efflux in the mutants fibroblast cells with statistically significant increases for the p.R1068H heterozygote RH1 (p < 0.05) and the p.N1800H heterozygote NH2 (p < 0.001). Login to comment 143 ABCA1 p.Asn1800His It was noted that the increase in cholesterol efflux promoted by 4-PBA in the p.N1800H fibroblasts was not as striking as that seen in HEK293 cells transfected with this mutant where 4-PBA promoted cholesterol efflux back up to untreated wild-type levels. Login to comment 156 ABCA1 p.Asn1800His ABCA1 p.Arg1068His Fibroblast cultures established from wild-type (WT1, WT2), p.R1068H carriers (RH1, RH2) and p.N1800H carriers (NH1, NH2) were treated with 10 mM 4-PBA for 24 h. Login to comment 161 ABCA1 p.Gln597Arg Plasma membrane localization of the p.Q597R protein was promoted with thapsigargin, an inducer of ER stress, whereas 4-PBA is known to alleviate ER stress (14) by acting as a chaperone itself (13) as well as inducing the expression of endogenous chaperone proteins (15). Login to comment 164 ABCA1 p.Arg1068His ABCA1 p.Tyr1767Asp For example, the p.Y1767D mutant showed a much enhanced localization and dramatic increase in cholesterol efflux after 4-PBA treatment whereas the efflux function for the p.R1068H mutant remained low despite showing a similar enhancement in localization. Login to comment 165 ABCA1 p.Arg1068His The p.R1068H mutant, located in the first ATP binding domain, has been shown to be defective in oligomerisation (19) and it is likely that although localization was improved for this mutant with 4-PBA treatment, the oligomerisation and therefore efflux function remained defective. Login to comment 185 ABCA1 p.Arg1068His The magnitude by which 4-PBA rescued function was mutant-dependent as restored localization did not always result in a restoration of function, as was the case for the p.R1068H mutant. Login to comment 188 ABCA1 p.Asn1800His ABCA1 p.Arg1068His For two of the most dramatically affected mutants, p.R1068H and p.N1800H, a functional improvement with 4-PBA treatment was also confirmed ex vivo using primary fibroblast cells. Login to comment 193 ABCA1 p.Asn1800His ABCA1 p.Asn1800His The NH2 fibroblasts, which harbor a p.N1800H allele and a null allele, show a significant improvement in function, yet the NH1 fibroblasts, which harbor a p.N1800H allele and a wild-type allele, show no significant functional improvement. Login to comment 195 ABCA1 p.Gln597Arg Improved positioning of ABCA1 at the plasma membrane has been shown previously for a p.Q597R mutant under conditions of induced ER stress; however, in that instance, no improvement in efflux activity was seen (23). Login to comment 196 ABCA1 p.Gln597Arg ABCA1 p.Ala594Thr This is in contrast with our results where the majority of the mutants showed significant improvements in function, including the p.A594T mutant, which is of a similar location to p.Q597R. Login to comment 201 ABCA1 p.Asn1800His ABCA1 p.Arg1068His The authors thank the p.R1068H and p.N1800H family members for their participation in this study. We are grateful to Professor Christiane Albrecht for kindly providing the pCIneo-ABCA1-GFP expression vector. Login to comment