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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biochemistry

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

Published: August 28th, 2018

DOI:

10.3791/58265

1Division of Cardiology, Department of Medicine, Zuckerberg San Francisco General and University of California San Francisco, 2Department of Cellular and Molecular Pharmacology and Howard Hughes Medical Institute, University of California San Francisco

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.

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.

PCSK9 targets the LDL receptor (LDL-R) for degradation, raising LDL cholesterol (LDL-C) and driving atherosclerotic heart disease1,2. Therapeutics targeting PCSK9 robustly lower LDL-C and improve cardiovascular outcomes for patients, even when added to an aggressive lipid-lowering therapy with statins3,4. Currently approved therapies are limited to antibody-based approaches, however, and suffer from a lack of cost-effectiveness5,6. To solve this problem, less costly therapeutic alternatives, a ....

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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 (Tm) calculator sp.......

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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.......

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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.......

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The authors thank the generous funding support from the NHLBI/NIH (K08 HL124068 and LRP HMOT1243), NCATS/NIH through the UCSF Clinical and Translational Science Institute Catalyst Program (UL1 TR000004), the UCSF Academic Senate, the Hellman Foundation, a Gilead Sciences Research Scholar Award, a Pfizer ASPIRE Cardiovascular Award (all to John S. Chorba) and the Howard Hughes Medical Institute (to Adri M. Galvan and Kevan M. Shokat).

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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

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