huh7 cells  (CLS Cell Lines Service GmbH)

Journal: Journal of Lipid Research

Article Title: APOA2 increases cholesterol efflux capacity to plasma HDL by displacing the C-terminus of resident APOA1

doi: 10.1016/j.jlr.2024.100686

Figure Lengend Snippet: Effect of APOA2 on ABCA1-mediated cholesterol efflux to isolated HDL. A: size-exclusion profiles of UC-isolated HDL incubated with increasing concentration of exogenous APOA2 for 1 h and immediately separated on tandem Superdex 200 columns. Peaks eluting between 20-25 ml and 25–30 ml were HDL-bound (lipidated) and displaced (lipid-free) protein fractions, respectively. B: SDS-PAGE-based densitometry analysis of HDL-bound fractions shows the change in APOA1 and APOA2 content with increasing addition of exogenous APOA2. The APOA2:APOA1 band intensity ratios are indicated above each APOA2 data point. C: cholesterol efflux ability of HDL-bound fractions to mediate cholesterol efflux in BHK cells with and without ABCA1 expression. Data points and error bars represent the mean and SDs from three technical replicates, respectively.

Article Snippet: This was accomplished by inserting a second copy of the APOA1 coding sequence immediately after the first with T4 DNA ligase (New England Biolabs), per the manufacturer's instructions, terminating with a stop codon.

Techniques: Isolation, Incubation, Concentration Assay, SDS Page, Expressing

Journal: Journal of Lipid Research

Article Title: APOA2 increases cholesterol efflux capacity to plasma HDL by displacing the C-terminus of resident APOA1

doi: 10.1016/j.jlr.2024.100686

Figure Lengend Snippet: Ability of different scaffold proteins in reconstituted HDL particles to promote ABCA1-mediated cholesterol efflux. A: cholesterol efflux from BHK cells with ABCA1 expression for reconstituted HDL (rHDL) containing APOA1 alone, APOA2 alone, and APOA1/APOA2 showing that both APOA1 and APOA2 are required for enhanced ABCA1-mediated cholesterol efflux. Particles were compared at equal phospholipid concentration. B: native PAGE analysis of reconstituted HDL particles produced with POPC. Lanes 1, 2, and 3 show reconstituted HDL particles containing APOA1, APOA2, and APOA1/APOA2, respectively. C: SDS-PAGE analysis (nonreducing) of the rHDL particles showing APOA1 (28 kDa) and/or APOA2 (14 kDa). Lanes 1, 2, and 3 show reconstituted HDL particles containing APOA1, APOA2, and APOA1/APOA2 particles. All gels were loaded with equal protein and stained with Coomassie blue. Bars and error bars represent the mean and SDs from three technical replicates, respectively. Star (∗) represents a difference from the APOA2-only rHDL particle by Kruskal-Wallis and Dunn’s post-hoc test at P < 0.05.

Article Snippet: This was accomplished by inserting a second copy of the APOA1 coding sequence immediately after the first with T4 DNA ligase (New England Biolabs), per the manufacturer's instructions, terminating with a stop codon.

Techniques: Expressing, Concentration Assay, Clear Native PAGE, Produced, SDS Page, Staining

Journal: Journal of Lipid Research

Article Title: APOA2 increases cholesterol efflux capacity to plasma HDL by displacing the C-terminus of resident APOA1

doi: 10.1016/j.jlr.2024.100686

Figure Lengend Snippet: Structural changes in APOA1 in the presence of exogenous APOA2. Top bar shows cartoon with the 10 amphipathic alpha-helical repeats as reported by Segrest et al . as a reference for the structural barcodes. Residue-level structural changes in APOA1 in the presence of 20% ( middle ) and 40% ( bottom ) exogenous APOA2 are represented as 2-D structural barcodes of APOA1. The log2 fold changes of significant semi-tryptic peptides from five biological replicates ( P -value < 0.01) are distributed across the encompassing residues. Regions in red get more exposed to the nonspecific protease and the regions shown in the blue are shielded from the nonspecific protease upon the exogenous addition of APOA2.

Article Snippet: This was accomplished by inserting a second copy of the APOA1 coding sequence immediately after the first with T4 DNA ligase (New England Biolabs), per the manufacturer's instructions, terminating with a stop codon.

Techniques: Residue

Journal: Journal of Lipid Research

Article Title: APOA2 increases cholesterol efflux capacity to plasma HDL by displacing the C-terminus of resident APOA1

doi: 10.1016/j.jlr.2024.100686

Figure Lengend Snippet: Identified BS3 cross-links between APOA1 and APOA2

Article Snippet: This was accomplished by inserting a second copy of the APOA1 coding sequence immediately after the first with T4 DNA ligase (New England Biolabs), per the manufacturer's instructions, terminating with a stop codon.

Techniques:

Journal: Journal of Lipid Research

Article Title: APOA2 increases cholesterol efflux capacity to plasma HDL by displacing the C-terminus of resident APOA1

doi: 10.1016/j.jlr.2024.100686

Figure Lengend Snippet: Identified DSBU/DSSO cross-links between APOA1 and APOA2

Article Snippet: This was accomplished by inserting a second copy of the APOA1 coding sequence immediately after the first with T4 DNA ligase (New England Biolabs), per the manufacturer's instructions, terminating with a stop codon.

Techniques:

Journal: Journal of Lipid Research

Article Title: APOA2 increases cholesterol efflux capacity to plasma HDL by displacing the C-terminus of resident APOA1

doi: 10.1016/j.jlr.2024.100686

Figure Lengend Snippet: Cholesterol efflux to rHDL particles produced with various mutants of APOA1. Cholesterol efflux from BHK cells expressing ABCA1 is shown. All the particles were reconstituted with POPC at an approximate wt:wt ratio of 2.5 mg POPC/1.0 mg WT APOA1 or mutant. Gray bars show the rHDL particles containing the WT APOA1 or mutant indicated alone. Black bars show the same particle with added APOA2 (at about 20% of APOA1 mass). Data and error bars represent the mean and SDs from six technical replicates. The data from individual replicates is shown along each bar. ∗∗ P < 0.01 by Mann-Whitney test compared to corresponding non-APOA2 containing rHDL particle.

Article Snippet: This was accomplished by inserting a second copy of the APOA1 coding sequence immediately after the first with T4 DNA ligase (New England Biolabs), per the manufacturer's instructions, terminating with a stop codon.

Techniques: Produced, Expressing, Mutagenesis, MANN-WHITNEY

Journal: Journal of Lipid Research

Article Title: APOA2 increases cholesterol efflux capacity to plasma HDL by displacing the C-terminus of resident APOA1

doi: 10.1016/j.jlr.2024.100686

Figure Lengend Snippet: Cartoon showing mechanism of increase in ABCA-1-mediated cholesterol efflux induced by exogenous APOA2. Lipid-free APOA1 interacts with ABCA1 through its exposed C-termini and acts as an efficient acceptor of cholesterol. However, in mature HDL, the APOA1 termini are in close contact with the lipoprotein surface, hindering interaction in ABCA1. Driven by the structural and functional evidence at hand, we propose that APOA2 leads to the dislodgement of Helix 10 away from the lipid surface, making it available to form auxiliary interactions with ABCA1, thereby increasing the rate of apolipoprotein-mediated cholesterol efflux.

Article Snippet: This was accomplished by inserting a second copy of the APOA1 coding sequence immediately after the first with T4 DNA ligase (New England Biolabs), per the manufacturer's instructions, terminating with a stop codon.

Techniques: Functional Assay