Asparagine endopeptidase cleaves apolipoprotein A1 and accelerates pathogenesis of atherosclerosis
Mengmeng Wang, Bowei Li, Shuke Nie, Xin Meng, Guangxing Wang, Menghan Yang, Wenxin Dang, Kangning He, Tucheng Sun, Ping Xu, Xifei Yang, Keqiang Ye
Mengmeng Wang, Bowei Li, Shuke Nie, Xin Meng, Guangxing Wang, Menghan Yang, Wenxin Dang, Kangning He, Tucheng Sun, Ping Xu, Xifei Yang, Keqiang Ye
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Research Article Cardiology Vascular biology

Asparagine endopeptidase cleaves apolipoprotein A1 and accelerates pathogenesis of atherosclerosis

Abstract

Atherosclerosis is a slowly progressing inflammatory disease characterized with cholesterol disorder and intimal plaques. Asparagine endopeptidase (AEP) is an endolysosomal protease that is activated under acidic conditions and is elevated substantially in both plasma and plaques of patients with atherosclerosis. However, how AEP accelerates atherosclerosis development remains incompletely understood, especially from the view of cholesterol metabolism. This project aims to reveal the crucial substrate of AEP during atherosclerosis plaque formation and to lay the foundation for developing novel therapeutic agents for Atherosclerosis. Here, we show that AEP is augmented in the atherosclerosis plaques obtained from patients and proteolytically cuts apolipoprotein A1 (APOA1) and impairs cholesterol efflux and high-density lipoprotein (HDL) formation, facilitating atherosclerosis pathologies. AEP is activated in the liver and aorta of apolipoprotein E–null (APOE-null) mice, and deletion of AEP from APOE–/– mice attenuates atherosclerosis. APOA1, an essential lipoprotein in HDL for cholesterol efflux, is cleaved by AEP at N208 residue in the liver and atherosclerotic macrophages of APOE–/– mice. Blockade of APOA1 cleavage by AEP via N208A mutation or its specific inhibitor, #11a, substantially diminishes atherosclerosis in both APOE–/– and LDLR–/– mice. Hence, our findings support that AEP disrupts cholesterol metabolism and accelerates the development of atherosclerosis.

Authors

Mengmeng Wang, Bowei Li, Shuke Nie, Xin Meng, Guangxing Wang, Menghan Yang, Wenxin Dang, Kangning He, Tucheng Sun, Ping Xu, Xifei Yang, Keqiang Ye

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Figure 3

AEP cleaves APOA1 at N208 and severely impairs cholesterol efflux and HDL formation.

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AEP cleaves APOA1 at N208 and severely impairs cholesterol efflux and HD...
(A) HEK293 cells lysates overexpressing GST-APOA1 were incubated with AEP for 5 minutes, 10 minutes, and 20 minutes. Western blot showing the cleavage of APOA1 by recombinant AEP in a time-dependent manner. (B) Cells cotransfected with GST-APOA1 and myc-AEP WT or myc-AEP C189S. Western blot showing that WT AEP but not C189S mutant AEP cleaved GST-APOA1. (C) AEP activity was diminished by the C189S mutant of AEP. (D) Cleavage of APOA1 was blocked by AEP inhibitor #11a. (E) AEP activity was inhibited by #11a. (F) Cleavage of purified GST-APOA1 analyzed by immunoblotting (left panel) or Coomassie blue staining (right panel). (G) Proteomic analysis of APOA1 recombinant proteins processed by AEP. The detected peptide sequences indicate that N208 is the main cleavage site with the shed bands of molecular weight (MW) 50 kDa. (H) Cell lysates overexpressing GST-APOA1 WT, APOA1 mutant (N67A, N73A, N98A, N208A) were incubated with AEP. Western blot showing that the N208A mutant blocked the cleavage. (I) DMPC multilamellar vesicles were incubated with BSA alone (control, purple), APOA1 (blue), APOA1 N208Al (green), APOA1 1-208 (orange)or APOA1 209-276 (red). Ability to solubilize DMPC was determined. (J) Representative fluorescent images of the NBD-cholesterol burden in RAW264.7 obtained in the indicated group after incubation with APOA1, APOA1 N208A, APOA1 1–208, or APOA1 209–276 for 4 hours. Original maginification, ×20. (K) Quantification of NBD-cholesterol efflux. All data are presented as the mean ± SEM from 3 independent experiments. 1-way ANOVA with Tukey’s post hoc test (C, D, and K).*P < 0.05; **P < 0.01; ***P < 0.001.