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

Summary

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

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.

Introduction

PCSK9 targets the LDL receptor (LDL-R) for degradation, raising LDL cholesterol (LDL-C) and driving atherosclerotic heart disease^1,2. Therapeutics targeting PCSK9 robustly lower LDL-C and improve cardiovascular outcomes for patients, even when added to an aggressive lipid-lowering therapy with statins^3,4. Currently approved therapies are limited to antibody-based approaches, however, and suffer from a lack of cost-effectiveness^5,6. To solve this problem, less costly therapeutic alternatives, a ....

Protocol

1. Site-directed Mutagenesis of Protease Vector

1. Design and order custom-synthesized oligonucleotides to install a mutation of interest using a modification of standard site-directed mutagenesis protocols¹². Standard desalted primers (without additional purification) are perfectly acceptable.
   Note: A general approach to primer designs involves creating partially overlapping primers as indicated in Table 1, using a melting temperature (T_m) calculator specific to the polymerase of interest.
2. Set up polymerase chain reactions (PCRs) for the site-directed mutagenesis on ice, as indicated in

Results

The high-throughput proteolysis assay relies upon overcoming three major challenges. First, to overcome the intrinsically low output of a single-turnover PCSK9 protease, a PCSK9 protease lacking the inhibitory prodomain is used, with the cleavage sequence at the tail of the prodomain linked to a luciferase that can be secreted¹⁴. Second, to satisfy the need for the protease to fold in complex with its inhibitory prodomain, the two polypeptides are coexpressed i.......

Discussion

The experimental procedures described above present a method to overcome the intrinsically low activity of the single-turnover protease PCSK9 and evaluate its proteolytic function in a robust manner. The key concept of the assay relies upon converting a single-turnover event into an enzymatically amplified readout. The strengths of the assay include the relatively short time-frame and ease of use of the luciferase reporter, as well as its scalability to high-throughput approaches. In addition, the assay evaluates proteol.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PCR Tubes	USA Scientific	1402-2900	For PCR
Q5 Hot Start	New England Biolabs	M0493L	High-fidelity DNA Polymerase
Deoxynucleotide Solution Mix	New England Biolabs	N0447L	dNTPs (for PCR)
pPCSK9-NLucProteaseAssay-WT	Authors	n/a	Available from authors
pPCSK9-NLucProteaseAssay-S386A	Authors	n/a	Available from authors
Agarose LE	Gold Biotechnology	A-201-100	For DNA gels
E-Gel Imager System with Blue Light Base	ThermoFisher Scientific	4466612	For imaging DNA gels
SYBR Safe DNA Gel Stain	ThermoFisher Scientific	S33102	For DNA gels
Tris Base	ThermoFisher Scientific	BP152-1	For DNA gel running buffer
Glacial acetic acid	ThermoFisher Scientific	A38-500	For DNA gel running buffer
Ethylenediaminetetraacetic acid solution	Millipore Sigma	3690	EDTA, for DNA gel running buffer
1 kb DNA ladder	Gold Biotechnology	D010	DNA ladder
DpnI	New England Biolabs	R0176S	Restriction enzyme
LB Agar plates with 100 µg/mL carbenicillin	Teknova	L1010	LB-Carb plates
One Shot Mach1 T Phage-Resistent Chemically Competent E. coli	ThermoFisher Scientific	C862003	Chemically competent cells
LB Broth, Miller	ThermoFisher Scientific	BP1426-2	LB
Carbenicillin	Gold Biotechnology	C-103-5	Selective antibiotic
E.Z.N.A. Plasmid Mini Kit I	Omega BioTek	D6942-02	DNA Purification Miniprep kit
NanoDrop 2000 Spectrophotomer	ThermoFisher Scientific	ND-2000C	Spectrophotometer
293T Cells	American Tissue Culture Collection (ATCC)	CRL-3216	HEK 293T cells
DMEM, high glucose, pyruvate	ThermoFisher Scientific	11995065	DMEM, mammalian cell media
Fetal Bovine Sera	Axenia Biologix	F001	FBS
Trypsin-EDTA (0.05%), phenol red	ThermoFisher Scientific	25300062	Trypsin, for cell dissociation
Phosphate buffered saline (PBS)	ThermoFisher Scientific	10010023	PBS
Countess automated cell counter	ThermoFisher Scientific	C10227	Automated cell counting
Countess cell counting chamber slides	ThermoFisher Scientific	C10228	Slides for cell counting
CELLSTAR Tissue Culture Plates, White, White-Bottom, with Lid	Grenier Bio-One	655083	White, white-bottom 96 well plate
TempPlate non-skirted 96-well PCR plate, natural	USA Scientific	1402-9596	96 well plate for master plasmid plate
Nunc 2.0mL DeepWell Plates	ThermoFisher Scientific	278743	96 well deep well plate
Lipofectamine 3000	ThermoFisher Scientific	L3000008	Lipid transfection reagent, Lf3K
P3000 Reagent	ThermoFisher Scientific	L3000008	DNA pre-complexation reagent, provided with Lf3K
OptiMEM I Reduced Serum Medium	ThermoFisher Scientific	31985062	Reduced serum medium for transfection
(+)-Sodium L-ascorbate	Millipore Sigma	A4034	Sodium ascorbate
Sodium chloride	Millipore Sigma	S9888	NaCl
Albumin, Bovine Serum, Fraction V, Low Heavy Metals	Millipore Sigma	12659	BSA
Methanol (HPLC)	ThermoFisher Scientific	A4524	MeOH
Hydrochloric acid	VWR	JT9535-2	Concentrated HCl
Coelenterazine	Gold Biotechnology	CZ2.5	Luciferase substrate
Syringe Filter, Sterile	ThermoFisher Scientific	09-720-3	Sterile filter, PVDF, 0.22 µm pore
Pipet-Lite Multi Pipette L12-200XLS+	Rainin	17013810	Multichannel pipette
Pipet-Lite Multi Pipette L12-20XLS+	Rainin	17013808	Multichannel pipette
Pipet-Lite Multi Pipette L12-10XLS+	Rainin	17013807	Multichannel pipette
Reagent reservoir	Corning	4870	Trough for reagents
Centrifuge tubes, 15 mL	ThermoFisher Scientific	05-539-12	15 mL tubes
Centrifuge tubes, 50 mL	Corning	430829	50 mL tubes
Spark Microplate Reader	Tecan	N/a	Plate Reader
Excel	Microsoft	2016 for Mac	Spreadsheet software
Prism	GraphPad Software	v7	Scientific data analysis software