Format

Send to

Choose Destination
J Vis Exp. 2018 Aug 28;(138). doi: 10.3791/58265.

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9.

Author information

1
Division of Cardiology, Department of Medicine, Zuckerberg San Francisco General and University of California San Francisco; John.Chorba@ucsf.edu.
2
Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco.

Abstract

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

PMID:
30222160
PMCID:
PMC6231911
[Available on 2019-08-28]
DOI:
10.3791/58265
[Indexed for MEDLINE]

Supplemental Content

Full text links

Icon for MyJove Corporation
Loading ...
Support Center