Low Cholesterol level in CAP1-Knockout mice: CAP1 is an essential binding partner of PCSK9 to degrade LDLR

Abstract

Introduction: Proproteinconvertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) expression on liver cells mediating its lysosomal degradation. Therefore, PCSK9 has emerged as an important target of lowering LDL-cholesterol and also treatment of atherosclerotic cardiovascular diseases. However, the precise mechanism how PCSK9 mediates LDLR internalization and degradation has not yet been elucidated. Interestingly, the fully folded C-terminal Cysteine-histidine rich domain (CHRD) structure of PCSK9 has a distinct structural similarity to the resistin homotrimer. CHRD of PCSK9 does not interact directly with the LDL receptor, but the CHRD is nevertheless required for PCSK9 mediated LDLR degradation. Previously, we identified adenylyl cyclase-associated protein 1 (CAP1) as a functional receptor for human resistin and clarified its intracellular signaling pathway to modulate inflammatory action of monocytes. We showed PCSK9 directly interacts with CAP1 in vitro and in vivo. Furthermore, interaction between PCSK9 CHRD and CAP1 SH3 domain is crucial for LDLR degradation. High-fed mice deficient CAP1 (CAP1-/+) significantly display decreased low-density lipoprotein cholesterol levels in serum and increased LDLR protein in liver without significant change in its mRNA level. We also found that binding between CAP1 and PCSK9 occurs in lipid-raft on 30 minutes after treatment of PCSK9. Caveolin 1 knockdown in HepG2 cells did not show a decrease in LDLR. In contrast, clathrin knockdown cells caused the degradation of LDLR in a treatment of PCSK9 dose-dependent manner. Taken together, we suggested that PCSK9/CAP1 axis stimulates LDLR internalization for degradation in caveolae lipid raft dependent pathway, distinct from clathrin-dependent pathway for LDLR recycling. Methods: In this study, we analyzed that CAP1 dependent PCSK9-LDLR degradation, endocytosis mechanism in transient CAP1 knockdown human hepatic cells (HepG2) and TALEN-mediated CAP1-/+ mouse. Results: CAP1-SH3 domain binds to PCSK9 directly. CAP1-PCSK9 complex increased LDLR degradation through lysosomal pathway. CAP1 targeting siRNA in HepG2 cells resulted in abolished LDLR degradation. These pathways are mainly showed caveolin 1 rich lipid-raft. Although caveolin 1 knockdown HepG2 cells abolished LDLR degradation treated PCSK9. In addition, lipoprofile analysis in CAP1-/+ mouse showed that LDL cholesterol physiological level in high-fed mice was decreased than wild type mice. Supportive results were obtained with an in vitro, in vivo system. Conclusions: The sorting of PCSK9 to the CAP1-LDLR is required for caveolae lipid raft to promote lysosomal LDLR degradation. This study provides valuable insights into mechanism regulating cholesterol homeostasis and LDLR degradation.