Sirtuin-1 directly binds and deacetylates hepatic PCSK9 to inhibit low-density lipoprotein receptor degradation

Abstract

Abstract Background Low-density lipoprotein cholesterol (LDL-C) is causally involved in atherosclerotic cardiovascular disease (ASCVD). We have previously shown that pharmacological activation of Sirtuin-1 (SIRT1), a NAD+-dependent deacetylase, reduces plasma LDL-C levels by decreasing PCSK9 and increasing hepatic LDLR expression, thus exerts atheroprotective effects in mice; however, the mechanism of SIRT1 action remains elusive. Purpose We found diminished levels of circulating SIRT1 in atherosclerotic mice compared to wild-type healthy controls. We hypothesized that this decrease in SIRT1 levels may promote dysregulated lipid metabolism and ASCVD in humans. Herein, we aimed to restore the reduced systemic Sirtuin-1 plasma levels in ASCVD by injecting recombinant murine Sirtuin-1 (rmSIRT1) in atherosclerotic mice. Methods Twelve-week-old apolipoprotein E-deficient (ApoE-/-) male mice fed a high-cholesterol diet (1.25% w/w) were randomized to receive rmSIRT1 (n=6; 0.3mg/kg BW i.p.) or vehicle treatment (n=6) every third day over 4-weeks. Mice were euthanized and organs were harvested to study lipid metabolism and ASCVD progression. Human hepatocytes (Huh7) cells were used to perform radioactive assays to study cellular uptake of radioiodinated LDL, surface plasmon resonance to study binding and site-directed mutagenesis to dissect the underlying molecular mechanism exerted by circulating SIRT1. Results Boosting circulating SIRT1 levels increased hepatic LDLR expression, reduced plasma LDL-C levels and progression of plaques in ApoE-/- mice (Figure 1). rmSIRT1 treatment did not change hepatic expression of total PCSK9 but increased its deacetylated status. Mechanistically, rmSIRT1 directly bound to hepatic PCSK9 and deacetylated PCSK9 at 3 sites: Lys243, Lys421 and Lys506 as shown by Surface Plasmon Resonance and mass spectrometry, respectively. In vitro mutagenesis to triple deacetylation mimetic (3KR) in hepatocytes (Huh7 cells) reduced SIRT1-induced PCSK9 activity, as evidenced by increased cellular binding and association of 125I-LDL through LDLR in Huh7cells. Finally, plasma levels of SIRT1 and PCSK9 were assessed at baseline in patients with acute coronary syndromes (ACS) where plasma SIRT1 showed an inverse correlation with PCSK9 and conferred a reduced risk of major adverse cardiovascular events. Conclusion SIRT1 directly binds PCSK9 and decreases its activity by deacetylation, thereby enhancing LDL-C clearance by hepatic LDLR upregulation. Increased circulating SIRT1 exerts atheroprotective effects and is associated with improved prognosis in patients with ACS.Figure 1