ABCMdb

PMID: 18218626

Hassan HH, Bailey D, Lee DY, Iatan I, Hafiane A, Ruel I, Krimbou L, Genest J
Quantitative analysis of ABCA1-dependent compartmentalization and trafficking of apolipoprotein A-I: implications for determining cellular kinetics of nascent high density lipoprotein biogenesis.
J Biol Chem. 2008 Apr 25;283(17):11164-75. Epub 2008 Jan 24., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg Such endocytosis was impaired by naturally occurring mutations of ABCA1 (Q597R and C1477R). Login to comment 35 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg EXPERIMENTAL PROCEDURES Patient Selection-For this study, we selected fibroblasts from three normal control subjects and two patients with TD (homozygous for Q597R at the ABCA1 gene and compound heterozygous for C1477R as described previously (12)). Login to comment 36 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg EXPERIMENTAL PROCEDURES Patient Selection-For this study, we selected fibroblasts from three normal control subjects and two patients with TD (homozygous for Q597R at the ABCA1 gene and compound heterozygous for C1477R as described previously (12)). Login to comment 71 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg Cellular Compartmentalization and Trafficking of ApoA-I/ABCA1 APRIL 25, 2008•VOLUME 283•NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11165 Dissociation of 125 I-ApoA-I from Intact Cells-BHK-ABCA1, normal human fibroblasts, or fibroblasts with ABCA1 mutations (Q597R and C1477R) from Tangier disease subjects were used. Login to comment 73 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg Cellular Compartmentalization and Trafficking of ApoA-I/ABCA1 APRIL 25, 2008ߦVOLUME 283ߦNUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 11165 at SEMMELWEIS UNIV OF MEDICINE on December 3, Dissociation of 125 I-ApoA-I from Intact Cells-BHK-ABCA1, normal human fibroblasts, or fibroblasts with ABCA1 mutations (Q597R and C1477R) from Tangier disease subjects were used. Login to comment 87 ABCA1 p.Gln597Arg To examine the specificity of the biotinylation reaction, a 30-fold excess of unlabeled apoA-I, absence of ABCA1 stimulation with 22OH/9CRA, and ABCA1 mutant fibroblasts (Q597R) were used as controls. Login to comment 88 ABCA1 p.Gln597Arg As shown in Fig. 1B, the presence of excess unlabeled apoA-I, the absence of stimulation with 22OH/9CRA, or utilization of an ABCA1 mutant (Q597R) 0.25 m g 0.5 m g 1 m g 2 m g 3 m g 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribut (ng/µgcellprotein) Biotin mg/mL ion A B 0.25 m g 0.5 m g 1 m g 2 m g 3 m g 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribut (ng/µgcellprotein) Biotin mg/mL ion C ontrol Excess apoA -I -22O H /9C R A A B C A 1(Q 597R ) 0.0 0.1 0.2 0.3 0.4 PM ICC 125 I-apoA-ICellDistribution (ng/µgcellprotein) Hsp-70 ICCPM Tubulin 150- 70- KDa 50- C Hsp-70 ICCPM Tubulin 150- 70- KDa 50- Hsp-70 ICCPM Tubulin 150- 70- KDa 50- VLA-2 FIGURE 1. Login to comment 90 ABCA1 p.Gln597Arg To examine the specificity of the biotinylation reaction, a 30-fold excess of unlabeled apoA-I, absence of ABCA1 stimulation with 22OH/9CRA, and ABCA1 mutant fibroblasts (Q597R) were used as controls. Login to comment 91 ABCA1 p.Gln597Arg As shown in Fig. 1B, the presence of excess unlabeled apoA-I, the absence of stimulation with 22OH/9CRA, or utilization of an ABCA1 mutant (Q597R) 0 . Login to comment 94 ABCA1 p.Gln597Arg B, normal fibroblasts stimulated with 22OH/9CRA in the presence or absence of 30-fold excess of unlabeled apoA-I, unstimulated normal fibroblasts, and ABCA1 mutant (Q597R) were incubated with 10 ␮g/ml 125 I-apoA-I for 45 min at 37 °C. Surface proteins were biotinylated with0.5mg/mlsulfo-NHS-biotin,and125 I-apoA-IassociatedwiththePMandICCs was determined as described in A. Login to comment 97 ABCA1 p.Gln597Arg We have previously reported that the Q597R- ABCA1 mutant does not bind apoA-I but is expressed normally and localizes to the cell surface (19). Login to comment 101 ABCA1 p.Gln597Arg B, normal fibroblasts stimulated with 22OH/9CRA in the presence or absence of 30-fold excess of unlabeled apoA-I, unstimulated normal fibroblasts, and ABCA1 mutant (Q597R) were incubated with 10 òe;g/ml 125 I-apoA-I for 45 min at 37 &#b0;C. Surface proteins were biotinylated with0.5mg/mlsulfo-NHS-biotin,and125 I-apoA-IassociatedwiththePMandICCs was determined as described in A. Login to comment 104 ABCA1 p.Gln597Arg We have previously reported that the Q597R- ABCA1 mutant does not bind apoA-I but is expressed normally and localizes to the cell surface (19). Login to comment 174 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg Similar results were obtained with normal fibroblasts but not TD mutants (Q597R and C1477R). Login to comment 175 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg The ratio of ABCA1 over activin receptor type II in 22OH/9CRA-stimulated normal fibroblasts showed increased internalization of ABCA1 (Fig. 5F), whereas ABCA1 mutant fibroblasts (Q597R) associated with TD showed no significant internalization in the presence of apoA-I. A representative experiment of internalized ABCA1 in normal and Q597R fibroblasts is shown (Fig. 5, D and E). Login to comment 176 ABCA1 p.Cys1477Arg Furthermore, we found that apoA-I-mediated ABCA1 internalization was impaired in the ABCA1 mutant (C1477R) associated with TD (data not shown). Login to comment 182 ABCA1 p.Gln597Arg ABCA1 p.Cys1477Arg Similar results were obtained with normal fibroblasts but not TD mutants (Q597R and C1477R). Login to comment 183 ABCA1 p.Gln597Arg The ratio of ABCA1 over activin receptor type II in 22OH/9CRA-stimulated normal fibroblasts showed increased internalization of ABCA1 (Fig. 5F), whereas ABCA1 mutant fibroblasts (Q597R) associated with TD showed no significant internalization in the presence of apoA-I. Login to comment 184 ABCA1 p.Gln597Arg A representative experiment of internalized ABCA1 in normal and Q597R fibroblasts is shown (Fig. 5, D and E). Login to comment 185 ABCA1 p.Cys1477Arg Furthermore, we found that apoA-I-mediated ABCA1 internalization was impaired in the ABCA1 mutant (C1477R) associated with TD (data not shown). Login to comment 199 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg In light of this, we hypothesized that PM-associated apoA-I could be released by treatment with phosphatidylcholine-specific phospholipase C (PC- BHK-ABCA1 (+ ApoA-I) A Endocytosed ABCA1 - 250 kDa - 150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa B C Endocytosed Integrin α4 Endocytosed ABCA1 Endocytosed Integrin α4 Endocytosed ABCA1 Endocytosed ActRII - 250 kDa - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 BHK-ABCA1(Basal) ABCA1 (Q597R) (+ ApoA-I)E F Normal Fibroblasts (+ ApoA-I) D Total ABCA1 - 250 kDa Total ABCA1 - 250 kDa - 250 kDa Total ABCA1 Total ABCA1 - 250 kDa 0 20 40 60 80 100 120 0 3 6 9 12 Normal Q597R Time of Incubation (min) RatioofEndocytosedABCA1ActRII 0 20 40 0 3 6 9 12 + ApoA-I Basal 60 80 100 120 Time of Incubation (min) RatioofEndocytosedABCA1/Intα4 FIGURE 5. Login to comment 202 ABCA1 p.Gln597Arg Confluent normal fibroblasts (D) and ABCA1 mutant (Q597R) fibroblasts (E) were stimulated with 22OH/9CRA. Login to comment 208 ABCA1 p.Gln597Arg ABCA1 p.Gln597Arg In light of this, we hypothesized that PM-associated apoA-I could be released by treatment with phosphatidylcholine-specific phospholipase C (PC- BHK-ABCA1 (+ ApoA-I) A Endocytosed ABCA1 - 250 kDa - 150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa - 250 kDa Time (min) 0 5 10 20 45 60 120 -150 kDa B C Endocytosed Integrin b1;4 Endocytosed ABCA1 Endocytosed Integrin b1;4 Endocytosed ABCA1 Endocytosed ActRII - 250 kDa - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Endocytosed ABCA1 - 250 kDa Endocytosed ActRII - 50 kDa Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 Time (min) 0 5 10 20 45 60 120 BHK-ABCA1(Basal) ABCA1 (Q597R) (+ ApoA-I) E F Normal Fibroblasts (+ ApoA-I) D Total ABCA1 - 250 kDa Total ABCA1 - 250 kDa - 250 kDa Total ABCA1 Total ABCA1 - 250 kDa 0 20 40 60 80 100 120 0 3 6 9 12 Normal Q597R Time of Incubation (min) Ratio of Endocytosed ABCA1ActRII 0 20 40 0 3 6 9 12 + ApoA-I Basal 60 80 100 120 Time of Incubation (min) Ratio of Endocytosed ABCA1/Int b1; 4 FIGURE 5. Login to comment 211 ABCA1 p.Gln597Arg Confluent normal fibroblasts (D) and ABCA1 mutant (Q597R) fibroblasts (E) were stimulated with 22OH/9CRA. Login to comment