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Updated: Jun 6, 2026
A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9
Published on: August 28, 2018
In vivo evidence that furin from hepatocytes inactivates PCSK9
Rachid Essalmani1, Delia Susan-Resiga, Ann Chamberland
1Laboratory of Biochemical Neuroendocrinology, Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, Canada.
Abstract:
The proprotein convertase PCSK9 plays a key role in cholesterol homeostasis by binding the LDL receptor and targeting it toward degradation. PCSK9 is strongly expressed in the liver and is found in human and mouse plasma as mature (∼ 62 kDa) and inactivated (∼ 55 kDa) forms. Ex vivo data showed that human PCSK9 is inactivated by cleavage at Arg(218)↓ by the overexpressed convertases furin and PC5/6A. Analysis of the plasma of human heterozygotes for R218S and F216L mutations revealed a ∼ 50% reduction in the levels of the ∼ 55-kDa form. To identify the convertase(s) responsible for cleavage at Arg(218) in vivo, we inactivated the genes of furin and/or PC5/6 specifically in hepatocytes. The PCSK9-inactivated form was strongly reduced in mice lacking furin in hepatocytes (Fur-hKO) and only slightly reduced in PC5/6-hKO plasma. In agreement with a key role of furin in regulating PCSK9 activity in vivo, we observed an overall 26% drop in the LDL receptor protein levels of Fur-hKO livers, likely due to the compound effects of a 35% increase in PCSK9 mRNA levels and the loss of PCSK9 cleavage, suggesting a higher activity of PCSK9 in these mice. Overexpression of PCSK9 in primary hepatocytes obtained from these mice revealed that only full-length, membrane-bound, but not soluble, furin is the cognate convertase. We conclude that in hepatocytes furin regulates PCSK9 mRNA levels and is the key in vivo-inactivating protease of circulating PCSK9.
Insights
Furin, a key enzyme in the liver, inactivates circulating proprotein convertase PCSK9 by cleaving it. This regulation impacts cholesterol homeostasis by influencing LDL receptor levels.
Area of Science:
- Biochemistry
- Molecular Biology
- Cholesterol Metabolism
Background:
- Proprotein convertase PCSK9 is crucial for cholesterol homeostasis, regulating LDL receptor degradation.
- PCSK9 exists in plasma as mature (62 kDa) and inactivated (55 kDa) forms, with inactivation linked to cleavage at Arg(218).
- Furin and PC5/6A were previously shown ex vivo to cleave PCSK9 at Arg(218).
Purpose of the Study:
- To identify the specific convertase responsible for in vivo PCSK9 inactivation at Arg(218).
- To elucidate the role of furin and PC5/6 in regulating PCSK9 activity and cholesterol levels in vivo.
Main Methods:
- Hepatocyte-specific gene inactivation of furin and/or PC5/6 in mice (Fur-hKO, PC5/6-hKO).
- Analysis of PCSK9 mature and inactivated forms in plasma.
- Quantification of LDL receptor protein levels in liver.
- Overexpression studies of PCSK9 in primary hepatocytes.
Main Results:
- PCSK9 inactivation was significantly reduced in Fur-hKO mice, with only a minor reduction in PC5/6-hKO mice.
- Fur-hKO mice showed decreased LDL receptor protein levels, increased PCSK9 mRNA, and evidence of higher PCSK9 activity.
- Full-length, membrane-bound furin, but not soluble furin, was identified as the cognate convertase for PCSK9.
Conclusions:
- Furin is the primary protease responsible for inactivating PCSK9 in hepatocytes in vivo.
- Furin regulates PCSK9 mRNA levels and is critical for controlling circulating PCSK9 activity.
- Furin-mediated inactivation of PCSK9 influences LDL receptor levels and cholesterol homeostasis.
