E3 ubiquitin ligase Listerin regulates macrophage cholesterol efflux and atherosclerosis by targeting ABCA1

Abstract

Atherosclerosis arises from disrupted cholesterol metabolism, notably impaired macrophage cholesterol efflux leading to foam cell formation. Through single-cell and bulk RNA-Seq, we identified Listerin E3 ubiquitin protein ligase 1 (Listerin) as a regulator of macrophage cholesterol metabolism. Listerin expression increased during atherosclerosis progression in humans and rodents. Its deficiency suppressed cholesterol efflux, promoted foam cell formation, and exacerbated plaque features (macrophage infiltration, lipid deposition, necrotic cores) in macrophage-specific KO mice. Conversely, Listerin overexpression attenuated these atherosclerotic manifestations. Mechanistically, Listerin stabilizes ABCA1, a key cholesterol efflux mediator, by catalyzing K63-linked polyubiquitination at residues K1884/K1957, countering ESCRT-mediated lysosomal degradation of ABCA1 induced by oxidized LDL (oxLDL). ABCA1 agonist erythrodiol restored cholesterol efflux in Listerin-deficient macrophages, while KO of ABCA1 abolished Listerin's effects in Tsuchiya human monocytic leukemia line (THP-1) cells. This study establishes Listerin as a protective factor in atherosclerosis via posttranslational stabilization of ABCA1, offering a potential therapeutic strategy targeting ABCA1 ubiquitination to enhance cholesterol efflux.

Keywords: Atherosclerosis; Cardiology; Metabolism; Ubiquitin-proteosome system.

Figure 1. Listerin expression increases in human and mouse atherosclerotic plaque tissues.

(A) All cell types identified in atherosclerotic plaque samples (n = 8) and 28,687 cells were obtained after filtering, with 7 of the cell types shown. (B) The proportion of macrophages was specifically increased with the progression of plaques. UMAP, uniform manifold approximation and projection. (C) The number and expression level of Listerin in macrophages (Mφ) from atherosclerotic plaques at different time points. (D) Data on Listerin mRNA expression in human atherosclerotic plaques were obtained from the GEO database (GSE57691). (E) Data on Listerin mRNA expression in human PBMCs were obtained from the GEO database (GSE23749). (F) Immunofluorescence staining for Listerin (green particles) and CD68 (red particles) in pathological intimal thickening and fibroatheroma in human coronary artery atherosclerotic plaques. n = 5 per group. Scale bars: 500 μm and 200 μm (enlarged insets). (G) Immunofluorescence staining for Listerin (green particles) and MOMA2 (red particles) in early lesions (WD for 8 weeks) and advanced lesions (WD for 24 weeks) of male mice. n = 8 per group. Scale bar: 200 μm. (H) Immunoblot analysis of Listerin expression in whole aorta lysates from ApoE^–/– mice fed a WD for 0, 12, or 24 weeks. n = 8 per group. (I) Immunoblot analysis of Listerin expression in PMs after oxLDL (50 μg/mL) treatment. n = 5 per group. (J) Quantitative RT-PCR analysis of Listerin mRNA levels in PMs after oxLDL (50 μg/mL) treatment for the indicated durations. n = 6 per group. Data are presented as the mean ± SD. The Shapiro-Wilk method was used to test normal distributions, and statistical analysis was performed with an unpaired, 2-tailed Student’s t test (D and E), Mann-Whitney U test (C), and 1-way ANOVA with Dunnett’s post hoc test (H, I, and J). Adjusted P values are provided for multiple-group comparisons. NS, P > 0.05; *P < 0.05, **P < 0.01, ***P < 0.001, and ***P < 0.0001. Each experiment was conducted at least 3 times independently.

Figure 2. Macrophage Listerin KO inhibits cholesterol efflux and aggravates foam cell formation.

(A) Macrophage subtypes were classified into 4 different subpopulations. (B) The percentage of Listerin⁺ macrophages among the 4 subtypes of macrophages as the atherosclerosis progressed. (C) GO pathway analysis of differentially expressed genes (DEGs) in Listerin⁺ macrophages. (D) Oil Red O–stained images and quantitation of PMs from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice after incubation with or without oxLDL (50 μg/mL) for 24 hours. n = 5 per group. Scale bar: 100 μm. Ctrl, control. (E) Oil Red O–stained images and (F) quantitation of PMs incubated with or without oxLDL (50 μg/mL) for 24 hours after adenovirus-mediated overexpression of Flag-Listerin and Flag–Listerin-ΔRing. n = 5 per group. Scale bar: 100 μm. (G) Immunofluorescence images and quantitation of PMs from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice treated with Dil-oxLDL (40 μg/mL) at 37°C for 4 hours. n = 5 per group. Scale bar: 50 μm. (H) Immunofluorescence images and quantitation of PMs preloaded with the NBD cholesterol and then incubated with ApoA1 for 4 hours. n = 5 per group. Scale bar: 50 μm. (I) Time course of ApoA1-mediated cholesterol efflux assay of PMs from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice. n = 3 per group. (J) ApoA1-mediated cholesterol efflux assay of PMs infected with the indicated adenovirus. n = 6 per group. (K) Percentage of [3^H] cholesterol appearance in plasma, liver, and feces 48 hours after transplantation of cholesterol-loaded RAW264.7 macrophages transfected with either normal control or Listerin siRNA (n = 6 per group). Data are presented as the mean ± SD. The Shapiro-Wilk method was used to test normal distributions. Statistical analysis was performed using an unpaired, 2-tailed Student’s t test (D, G, and K), a Mann-Whitney U test (H), 2-way ANOVA followed by Šidák’s post hoc test (I), and 1-way ANOVA with Dunnett’s post hoc test (F and J). The adjusted P values are provided for multiple-group comparisons. NS, P > 0.05; *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3. Listerin KO promotes lipid accumulation and foam cell formation through downregulation of ABCA1.

(A) Diagram of 4D-FastDIA–based quantitative proteomics of PMs isolated from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice. (B) Immunoblot analysis of the indicated proteins in Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre PMs after oxLDL treatment. n = 5 per group. (C) Immunoblot and quantitative analyses of ABCA1 expression in PMs obtained from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice. The cells were treated with oxLDL for 12 hours and then treated with cycloheximide (CHX) (50 μg/mL) for various durations. (D) Immunoblot analysis of ABCA1 expression in MDMs (derived from PBMCs induced by M-CSF [50 ng/mL] for 7 days) after Listerin knockdown. n = 3 per group. (E) Immunoblot analysis of ABCA1 expression after adenovirus-mediated overexpression of Flag-Listerin or Flag-Listerin (ΔRing) in PMs. n = 5 per group. (F) ApoA1-mediated cholesterol efflux assay of PMs isolated from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice. PMs were preincubated with or without the ABCA1 agonist erythrodiol. n = 4 per group. (G) Oil Red O–stained images and quantitation of WT and ABCA1-KO THP-1 cells transfected with siCTL or siListerin. n = 5 per group. Scale bar: 25 μm. Data are presented as the mean ± SD. The Shapiro-Wilk method was used to test normal distributions. Significance was determined by 2-way ANOVA followed by Tukey’s post hoc test (F), 2-way ANOVA followed by Šidák’s post hoc test (B–D and G), and 1-way ANOVA with Dunnett’s post hoc test (F). The adjusted P values are provided for multiple-group comparisons. NS, P > 0.05; *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4. Listerin inhibits the degradation of ABCA1 through the ESCRT lysosome pathway.

(A and B) Immunoblot analysis of ABCA1 expression in PMs from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice treated for 12 hours with oxLDL (50 μg/mL) and proteasome/lysosome inhibitors (MG132, calpeptin, chloroquine, NH₄Cl, bafilomycin A1, 3-MA, wortmannin, DBeQ). n = 5 per group. (C) ABCA1 immunoblot in control/HRS-KO THP-1 cells with siCTL/Listerin silencing and oxLDL stimulation. n = 4 per group. (D) ApoA1-mediated cholesterol efflux in HRS-KO THP-1 cells transfected with siCTL/siListerin. n = 4 per group. (E) Immunoblotting for ABCA1 was performed in control/ATG5-KO THP-1 cells with siCTL/Listerin silencing. n = 4 per group. (F) Cholesterol efflux in ATG5-KO THP-1 cells transfected with siCTL/siListerin. n = 4 per group. (G) Co-IP of ABCA1-HRS interaction in oxLDL-treated PMs. An equal amount of nonspecific antibody was used as a negative control. (H) Confocal imaging of Flag-HRS/GFP-ABCA1 colocalization with/without His-Listerin overexpression. in HeLa cells. Scale bars: 10 μm and 2 μm (enlarged insets). (I) Flow cytometric analysis of membrane ABCA1 in PMs from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice. n = 5 per group. (J) Immunoblot analysis of membrane (MEM) and total ABCA1 in PMs from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice. n = 5 per group. (K) Confocal microscopic images of ABCA1 expression in PMs from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice. HRS was silenced in PMs, which were then stimulated with oxLDL (50 μg/mL). n = 5 per group. Scale bar: 20 μm. Data are presented as the mean ± SD. The Shapiro-Wilk method was used to test normal distributions. Data analysis was performed using 1-way ANOVA followed by Dunnett’s post hoc test (A and B), unpaired, 2-tailed Student’s t test (I), and 2-way ANOVA followed by Šidák’s post hoc test for the other panels. The adjusted P values are provided for multiple-group comparisons. NS, P > 0.05; *P < 0.05, **P < 0.01, and ***P < 0.001. Each experiment was repeated at least 3 times independently.

Figure 5. Listerin targets ABCA1.

(A) Co-IP assay of endogenous shows that Listerin interacted with ABCA1 in PMs treated with oxLDL (50 μg/mL) for the indicated durations. An equal amount of nonspecific antibody was used as a negative control. (B) Co-IP assay of Flag-Listerin with GFP-ABCA1, Myc-ABCG1, or Myc-SRB1 in HEK293T cells. (C) Confocal microscopic images and fluorescence intensity analysis for Listerin and ABCA1 in primary PMs after oxLDL incubation (50 μg/mL). Scale bar: 10 μm. (D) Expression of Listerin and ABCA1 in CD11b⁺ monocytes from PBMCs from healthy individuals (n = 19) and patients with ASCVD (n = 27). The graph shows the proportion of Listerin⁺ABCA1⁺CD11b⁺ monocytes among total CD11b⁺ monocytes. (E) Expression of Listerin and ABCA1 was measured in CD11b⁺ monocyte from PBMCs from healthy individuals (n = 19) and patients with AS (n = 27). (F) Immunofluorescence staining for Listerin (red particles), ABCA1 (pink particles), and CD68 (green particles) in pathological intimal thickening and fibroatheroma in human coronary artery atherosclerotic plaques. Scale bars: 50 μm and 10 μm (enlarged inset). (G) Fluorescence intensity analysis for Listerin (red particles), ABCA1(pink particles), and CD68 (green particles) in fibroatheroma in human coronary artery atherosclerotic plaques. (H and J) Topological diagrams of human ABCA1 and mutants. (I and K) Co-IP assay of the interaction of Flag-Listerin with GFP-ABCA1 (WT) and the ABCA1 truncation mutants in HEK293T cells. Data are presented as the mean ± SD. The Shapiro-Wilk method was used to test normal distributions. Data analysis was performed using the Mann-Whitney U test (D and E). The adjusted P values are provided for multiple-group comparisons. NS, P > 0.05; **P < 0.01 and ***P < 0.001. Each experiment was repeated at least 3 times independently.

Figure 6. Listerin catalyzes K63-linked polyubiquitination of ABCA1 at residues Lys1884 and Lys1957 to inhibit foam cell formation.

(A) Co-IP assay of ABCA1 polyubiquitination in HEK293T cells transfected with GFP-ABCA1, Flag-Listerin, HA-ubiquitin (WT), or HA-ubiquitin (K48 or k63). (B) Co-IP assay of ABCA1 polyubiquitination in HEK293T cells transfected with GFP-ABCA1, HA-ubiquitin (WT), HA-ubiquitin (K48 or K63), as well as a control vector, Flag-Listerin (WT), Flag-Listerin (C/A), or Flag-Listerin-ΔRing. (C) Co-IP assay of endogenous ABCA1 polyubiquitination in PMs from Listerin^fl/fl and Listerin^fl/fl Lyz2^Cre mice after being stimulated with oxLDL for 12 hours. (D) Co-IP assay of ABCA1 polyubiquitination after ubiquitin (TUBE) pull-downs in PMs. (E) LC-MS spectra analysis identified the ubiquitin modification of ABCA1 at lysine residues K1884 and K1957. (F) Co-IP analysis of the polyubiquitination of ABCA1 (WT) and its mutants in HEK293T cells transfected with GFP-ABCA1 (WT or mutants), Flag-Listerin, or HA-ubiquitin (K63). (G) Oil Red O–stained images and (H) quantitation analysis of RAW264.7 macrophages transfected with Flag-Listerin and GFP-ABCA1 (WT) or GFP-ABCA (K1884R and K1957R), and then incubated with oxLDL (50 μg/mL) for 24 hours. n = 6 per group. Scale bar: 20 μm. (I) Immunoblot analysis of GFP-ABCA1 and GFP-ABCA1(K1884 and K1957) expression in RAW246.7 macrophages. n = 5 per group. (J) ApoA1-mediated cholesterol efflux assay of RAW246.7 macrophages transfected with Flag-Listerin, GFP-ABCA1, or GFP-ABCA1(K1884 and K1957). n = 6 per group. Data are presented as the mean ± SD. The Shapiro-Wilk method was used to test normal distributions. Data analysis was performed with 2-way ANOVA followed by Šidák’s post hoc test. The adjusted P values are provided for multiple-group comparisons. NS, P > 0.05; **P < 0.01 and ***P < 0.001. Each experiment was repeated at least 3 times independently.

Figure 7. Listerin KO aggravates the development of atherosclerosis in vivo.

Male ApoE^–/– Listerin^fl/fl and ApoE^–/– Listerin^fl/fl Lyz2^Cre mice were fed a WD for 16 weeks. (A) The measurement of BW and serum levels of triglycerides (mmol/L), cholesterol (mmol/L), HDL-C (mmol/L), and LDL (mmol/L). n = 8 per group. (B) Representative images and quantitation of aortic arch regions containing white plaques (yellow arrowheads). (C) En face Oil Red O staining and (D) quantitation of atherosclerotic plaques in the whole aorta. n = 8 per group. (E) H&E staining of representative aortic root sections and quantification of lesion area and necrotic core area. n = 8 per group. Scale bar: 200 μm. (F) Oil Red O–stained cross-section images and analysis of atherosclerotic plaques in the aortic root. n = 8 per group. Scale bar: 200 μm. Immunofluorescence staining for (G) CD68 and (H) ABCA1 in the aortic root. n = 8 per group. Scale bars: 200 μm (G) and 100 um (H). (I) Immunoblot images and quantitative analysis of ABCA1 in whole-aorta lysates from ApoE^–/– Listerin^fl/fl and ApoE^–/– Listerin^fl/fl Lyz2^Cre mice. n = 8 per group. Data are presented as the mean ± SD. The Shapiro-Wilk method was used to test normal distributions. Statistical analysis was performed using an unpaired, 2-tailed Student’s t test. **P < 0.01 and ***P < 0.001.

Figure 8. Listerin overexpression ameliorates the development of atherosclerosis in vivo.

(A) Measurement of BW and serum levels of triglycerides (mmol/L), cholesterol (mmol/L), HDL-C (mmol/L), and LDL (mmol/L). n = 8 per group. (B) Representative images and quantitation of aortic arch regions containing white plaques (yellow arrowheads). (C) En face Oil Red O staining and (D) quantitation of atherosclerotic plaques in the whole aorta. n = 8 per group. (E) H&E staining of representative aortic root sections, quantification of lesions area and necrotic core area. n = 8 per group. Scale bar: 200 μm. (F) Oil Red O–stained cross section analysis of atherosclerotic plaques in the aortic root. n = 8 per group. Scale bar: 200 μm. Immunofluorescence staining for (G) CD68 and (H) ABCA1 in aortic root. n = 8 per group. Scale bars: 200 μm (G) and 100 μm (H). (I) Immunoblot images and quantitative analysis of ABCA1 in whole-aorta lysates from OE-CTR ApoE^–/– and OE-Listerin ApoE^–/– mice. n = 8 per group. Data are presented as the mean ± SD. The Shapiro-Wilk method was used to test normal distributions. For comparisons between the 2 groups, an unpaired, 2-tailed Student’s t test was used if the data were normally distributed, and the Mann-Whitney U test was performed if not. **P < 0.01 and ***P < 0.001.