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).

1. Site-directed Mutagenesis of Protease Vector
<ol>
	<li>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 specific to the polymerase of interest.</li>
	<li>Set up polymerase chain reactions (PCRs) for the site-directed mutagenesis on ice, as indicated in Table 2.</li>
	<li>Cycle the PCRs as indicated in Table 3.</li>
	<li>Run 2 - 5 &#181;L of each PCR on a 1% agarose gel and visualize the products to confirm a successful amplification. 
	Note: A successful PCR will show a single, prominent DNA band at the approximate size marker of the template (~7.8 kB).</li>
	<li>Add 0.5 &#181;L (per 25-&#181;L reaction) of DpnI and incubate the samples at 37 &#176;C for 1 h.</li>
	<li>Transform 2 &#181;L of the reaction into chemically competent Escherichia coli. 
	Note: A fast-growing E. coli strain will reduce incubation times, but any cloning strain will be acceptable.</li>
	<li>Plate the transformations onto Luria-Bertani (LB) agar containing 50 - 100 &#181;g/mL carbenicillin. Incubate the plates overnight at 37 &#176;C.</li>
	<li>Select 2 - 4 colonies to grow in a small-scale culture (2 - 5 mL of LB medium with 50 - 100 &#181;g/mL carbenicillin). Incubate the culture at 37 &#176;C at 220 rpm until it is turbid.</li>
	<li>Isolate the plasmid DNA from the cells using a plasmid DNA purification (i.e., &#8220;miniprep&#8221;) kit according to the manufacturer&#8217;s instructions. Quantify the concentration of eluted DNA using a microvolume spectrophotometer.</li>
	<li>Sequence the plasmid DNA extensively using a Sanger sequencing service (such as a core or commercial facility). Prepare the DNA samples according to the service&#8217;s specifications. Primers used successfully in prior sequencing reactions are noted in Table 4. 
	Note: Due to the PCR amplification of the entire plasmid, the sequencing of the entire PCSK9 coding region is recommended for each mutant.</li>
</ol>
2. High-throughput Luciferase-based Proteolysis Assay
<ol>
	<li>Cell plating (day 0) 
	<ol>
		<li>Use low-passage HEK293T cells for experiments and a culture in Dulbecco&#8217;s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS). Perform all cell work under sterile conditions in a tissue culture hood until ready to assay the luciferase activity. Estimate the number of wells and plates needed for the experiment, anticipating that transfections will be performed in triplicate for each condition tested.</li>
		<li>Dissociate HEK293T cells from a parent flask by treating them with a minimal volume of 0.05% trypsin-ethylenediaminetetraacetic acid (EDTA) to cover the cells. Inactivate the trypsin-EDTA with 2 volumes of DMEM supplemented with 10% FBS and transfer the cells to a sterile tube. Count the cells using an automated cell counter (and staining with trypan blue). Centrifuge the tube at 500 x g for 5 min to recover cells.</li>
		<li>Aspirate trypsin-containing medium and reconstitute the cells in the culture medium to a concentration of 2 x 105 cells/mL. Using a multichannel pipette, transfer 100 &#181;L of the cells to each well of a white (opaque-bottom) 96-well plate, which gives a final seeding concentration of 2 x 104 cells/well. 
		Note: For initial experiments, it may be useful to additionally seed a sister, clear-bottom 96-well plate, so as to monitor cell growth and adherence during the protocol and guide future troubleshooting.</li>
		<li>Incubate the cells at 37 &#176;C and 5% CO2 for 24 h.</li>
		<li>Prepare a master plate of plasmids in a 96-well format. Dilute each plasmid in elution buffer (Tris-HCl, pH 8.5) to 50 ng/&#181;L in an individual well of a 96-well plate.
		<ol>
			<li>Prepare 4 wells each for the positive control (WT) plasmid8, negative control (S386A) plasmid8, and plasmid-free buffer (for mock transfections). If drug or other cellular-based treatments are being planned, then prepare the master plate with the positive control (WT) plasmid in each well, along with 4 wells each of the negative control (S386A) plasmid and the plasmid-free buffer.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Transfection (day 1)
	<ol>
		<li>Prepare the transfection mixture in a 96-well plate format using deep-well 96-well plates. Perform the transfections in triplicate, being sure to prepare enough reagents to account for pipetting and transfer losses. 
		Note: The following calculations prepare enough reagent to run one 96-well plate in triplicate (estimated conservatively for 420 wells).
		<ol>
			<li>Make a master mix of a diluted lipid transfection reagent, adding 50.4 &#181;L of reagent to 2050 &#181;L of reduced-serum medium. Make a master mix of a DNA precomplexation reagent, adding 33.6 &#181;L of reagent into 1730 &#181;L of reduced-serum medium.</li>
			<li>Using a multichannel pipette, aliquot 16.8 &#181;L of the diluted DNA precomplexation reagent mixture into each well of the deep-well plate.</li>
			<li>Using a multichannel pipette, aliquot 3.2 &#181;L (160 ng) of each plasmid from the master plate into each well of the deep-well plate.</li>
			<li>Using a multichannel pipette, aliquot 20 &#181;L of the diluted lipid transfection reagent mixture to each well of the plate and mix the contents of each well using the multichannel pipette. Cover the plates and let them sit at room temperature (RT) for 10 - 15 min to form lipid:DNA complexes. 
			Note: Upon transfection, the final components per well will be as follows: 40 ng of DNA, 0.12 &#181;L of transfection reagent, and 0.08 &#181;L of DNA pre-complexation reagent, and the content of each well will be 10 &#181;L in total volume (reduced-serum medium).</li>
		</ol>
		</li>
		<li>Gently exchange the medium on the 293T cells in the 96-well plates using a multichannel pipette, taking care not to disrupt the cells. Replace the aspirated medium with 95 &#181;L of DMEM supplemented with 10% FBS. 
		Note: This would be an appropriate time to treat the cells with any drug of interest.</li>
		<li>Add 10 &#181;L of the transfection mixture to each appropriate well via a multichannel pipette. Gently swirl the plate to mix the contents in the wells. Incubate the plate at 37 &#176;C with 5% CO2 for 24 h. 
		Note: The final volume of the wells comes to 105 &#181;L, to account for evaporation over 24 h.</li>
	</ol>
	</li>
	<li>Assay (day 2)
	<ol>
		<li>Prepare a stock solution for coelenterazine reagents: 3 M sodium ascorbate [dissolved in phosphate-buffered saline (PBS), prepared fresh], 5 M NaCl, 10 mg/mL bovine serum albumin (BSA; dissolved in PBS, prepared fresh), and 2 mM coelenterazine (dissolved in acidified methanol containing 200 &#181;L of 3 N HCl per 10 mL). 
		Note: The 2 mM coelenterazine can be stored for 2 weeks when it is kept at -80 &#176;C and in the absence of light.</li>
		<li>Prepare 2x coelenterazine reagents for the luciferase readout, with a separate reagent each for the cells and medium, according to Table 5. Mix all reagents save the coelenterazine first, filter the mixture through a 0.22-&#181;m syringe filter, and then add the coelenterazine. Protect the reagents from light until they are ready to be added to the plates. 
		Note: Due to the loss of solution from filtration, make enough reagent to account for both the loss from filtration, as well as from transfers. Table 5 shows the final concentration of the 2x reagents (as well as the amount of stock solution to add to read out one 96-well plate with one reagent).</li>
		<li>Remove the cells from the incubator 24 h after the transfection. Using a multichannel pipette, transfer 50 &#181;L of conditioned medium from each well to a fresh, white-bottomed (opaque) 96-well plate.
		<ol>
			<li>Label the plates as to whether they contain medium or cells. If more than one 96-well plate was transfected, label the plates so as to ensure that each medium-containing plate is paired with its parent plate of cells.</li>
		</ol>
		</li>
		<li>Using a multichannel pipette, add 50 &#181;L of 2x non-lytic coelenterazine reagent to the plate containing only conditioned medium. Gently rock or shake the plate in the absence of light for 5 - 10 min at RT.</li>
		<li>Using a multichannel pipette, add 50 &#181;L of 2x lytic coelenterazine reagent to the plate containing the cells. Gently rock or shake the plate in the absence of light for 5 - 10 min at RT.</li>
		<li>After the incubation, read out the luminescence of the medium-only plate in a plate reader. Then, read out the luminescence of the cell-containing plate in the same plate reader.</li>
		<li>Discard the plates and save the files for data analysis.</li>
	</ol>
	</li>
</ol>
3. Data Analysis
<ol>
	<li>Perform an initial data analysis using spreadsheet software. Create a spreadsheet containing the results from the cell and the medium plates.</li>
	<li>Manually inspect the data from the cell plates to identify poorly transfected wells. Wells that show &#60; 5% - 10% of the readout of the negative control (S386A) plasmid should be considered as poorly transfected, making the interpretation of those data suspect.</li>
	<li>Calculate the average background luminescence of each plate from the mock-transfected wells. Subtract the background of each plate from the values of that plate. 
	Note: This value may be negligible depending on the plate reader used.</li>
	<li>Process the data by calculating the proportion of luciferase activity in the medium compared to the overall luciferase activity for each well. Because the cell plate contains both conditioned medium in addition to cells, and the medium-only plate contains the same amount of conditioned medium as the cell plate, it is appropriate to use the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/58265/58265eq1.jpg" /> 
	Here, RLU = relative luminescence units, the background-subtracted readout from the plate reader.</li>
	<li>Calculate the mean secreted luciferase of the positive control (WT) and the negative control (S386A) wells.</li>
	<li>Evaluate the overall quality of the experiment by calculating a Z-factor13: 
	<img alt="Equation 2" src="/files/ftp_upload/58265/58265eq2.jpg" /> 
	Note: The active values come from the positive control (WT) and the inactive values come from the negative control (S386A) wells. Values closer to 1 indicate a higher experimental quality. Consider repeating the experiment or optimizing the workflow if the value is below 0.</li>
	<li>Transfer the data from the spreadsheet program into scientific data analysis software.</li>
	<li>Normalize the secreted luciferase activity to the mean values of the positive control (WT) and the negative control (S386A), setting the positive control as 1 and the negative control as 0.</li>
	<li>Clean the data for outliers using the regression and outlier removal (ROUT) method, setting the maximum false discovery rate to 1%.</li>
	<li>Evaluate for statistically significant differences by comparing the data for each mutant condition (or mutant) to the mean of the WT activity (normalized to a value of 1). Perform multiple unpaired t-tests, correcting for multiple comparisons using the Holm-Sidak method and &#945; = 0.05.</li>
</ol>

<ol><li>Park, S. W., Moon, Y. A., Horton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Post-transcriptional+regulation+of+low+density+lipoprotein+receptor+protein+by+proprotein+convertase+subtilisin%2Fkexin+type+9a+in+mouse+liver%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisin/kexin type 9a in mouse liver.</a> Journal of Biological Chemistry. 279 (48), 50630-50638 (2004).</li>
<li>Cohen, J. C., Boerwinkle, E., Mosley, T. H., Hobbs, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sequence+variations+in+PCSK9%2C+low+LDL%2C+and+protection+against+coronary+heart+disease%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Sequence variations in PCSK9, low LDL, and protection against coronary heart disease.</a> New England Journal of Medicine. 354 (12), 1264-1272 (2006).</li>
<li>Ridker, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cardiovascular+Efficacy+and+Safety+of+Bococizumab+in+High-Risk+Patients%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Cardiovascular Efficacy and Safety of Bococizumab in High-Risk Patients.</a> New England Journal of Medicine. 376 (16), 1527-1539 (2017).</li>
<li>Sabatine, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolocumab+and+Clinical+Outcomes+in+Patients+with+Cardiovascular+Disease%5BTitle%5D)+AND+%22New+England+Journal+of+Medicine%22%5BJournal%5D)">Evolocumab and Clinical Outcomes in Patients with Cardiovascular Disease.</a> New England Journal of Medicine. 376 (18), 1713-1722 (2017).</li>
<li>Kazi, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cost-effectiveness+of+PCSK9+Inhibitor+Therapy+in+Patients+With+Heterozygous+Familial+Hypercholesterolemia+or+Atherosclerotic+Cardiovascular+Disease%5BTitle%5D)+AND+%22Journal+of+the+American+Medical+Association%22%5BJournal%5D)">Cost-effectiveness of PCSK9 Inhibitor Therapy in Patients With Heterozygous Familial Hypercholesterolemia or Atherosclerotic Cardiovascular Disease.</a> Journal of the American Medical Association. 316 (7), 743-753 (2016).</li>
<li>Kazi, D. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Updated+Cost-effectiveness+Analysis+of+PCSK9+Inhibitors+Based+on+the+Results+of+the+FOURIER+Trial%5BTitle%5D)+AND+%22Journal+of+the+American+Medical+Association%22%5BJournal%5D)">Updated Cost-effectiveness Analysis of PCSK9 Inhibitors Based on the Results of the FOURIER Trial.</a> Journal of the American Medical Association. 318 (8), (2017).</li>
<li>Pettersen, D., Fjellström, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecule+modulators+of+PCSK9+-+A+literature+and+patent+overview%5BTitle%5D)+AND+%22Bioorganic+%26+Medicinal+Chemistry+Letters%22%5BJournal%5D)">Small molecule modulators of PCSK9 - A literature and patent overview.</a> Bioorganic & Medicinal Chemistry Letters. 28 (7), 1155-1160 (2018).</li>
<li>Chorba, J. S., Galvan, A. M., Shokat, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stepwise+processing+analyses+of+the+single-turnover+PCSK9+protease+reveal+its+substrate+sequence+specificity+and+link+clinical+genotype+to+lipid+phenotype%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Stepwise processing analyses of the single-turnover PCSK9 protease reveal its substrate sequence specificity and link clinical genotype to lipid phenotype.</a> Journal of Biological Chemistry. 293 (6), 1875-1886 (2018).</li>
<li>Maxwell, K. N., Breslow, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adenoviral-mediated+expression+of+Pcsk9+in+mice+results+in+a+low-density+lipoprotein+receptor+knockout+phenotype%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (18), 7100-7105 (2004).</li>
<li>Benjannet, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NARC-1%2FPCSK9+and+its+natural+mutants%3A+zymogen+cleavage+and+effects+on+the+low+density+lipoprotein+%28LDL%29+receptor+and+LDL+cholesterol%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">NARC-1/PCSK9 and its natural mutants: zymogen cleavage and effects on the low density lipoprotein (LDL) receptor and LDL cholesterol.</a> Journal of Biological Chemistry. 279 (47), 48865-48875 (2004).</li>
<li>Cunningham, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+biophysical+studies+of+PCSK9+and+its+mutants+linked+to+familial+hypercholesterolemia%5BTitle%5D)+AND+%22Nature+Structural+%26+Molecular+Biology%22%5BJournal%5D)">Structural and biophysical studies of PCSK9 and its mutants linked to familial hypercholesterolemia.</a> Nature Structural & Molecular Biology. 14 (5), 413-419 (2007).</li>
<li>Liu, H., Naismith, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+efficient+one-step+site-directed+deletion%2C+insertion%2C+single+and+multiple-site+plasmid+mutagenesis+protocol%5BTitle%5D)+AND+%22BMC+Biotechnology%22%5BJournal%5D)">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC Biotechnology. 8, 91(2008).</li>
<li>Zhang, J., Chung, T., Oldenburg, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Simple+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays%5BTitle%5D)+AND+%22Journal+of+Biomolecular+Screening%22%5BJournal%5D)">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li>
<li>Hall, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineered+luciferase+reporter+from+a+deep+sea+shrimp+utilizing+a+novel+imidazopyrazinone+substrate%5BTitle%5D)+AND+%22ACS+Chemical+Biology%22%5BJournal%5D)">Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate.</a> ACS Chemical Biology. 7 (11), 1848-1857 (2012).</li>
<li>McNutt, M. C., Lagace, T. A., Horton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Catalytic+activity+is+not+required+for+secreted+PCSK9+to+reduce+low+density+lipoprotein+receptors+in+HepG2+cells%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Catalytic activity is not required for secreted PCSK9 to reduce low density lipoprotein receptors in HepG2 cells.</a> Journal of Biological Chemistry. 282 (29), 20799-20803 (2007).</li>
<li>Chorba, J. S., Shokat, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+proprotein+convertase+subtilisin%2Fkexin+type+9+%28PCSK9%29+active+site+and+cleavage+sequence+differentially+regulate+protein+secretion+from+proteolysis%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">The proprotein convertase subtilisin/kexin type 9 (PCSK9) active site and cleavage sequence differentially regulate protein secretion from proteolysis.</a> Journal of Biological Chemistry. 289 (42), 29030-29043 (2014).</li>
<li>Benjannet, S., Rhainds, D., Hamelin, J., Nassoury, N., Seidah, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+proprotein+convertase+%28PC%29+PCSK9+is+inactivated+by+furin+and%2For+PC5%2F6A%3A+functional+consequences+of+natural+mutations+and+post-translational+modifications%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">The proprotein convertase (PC) PCSK9 is inactivated by furin and/or PC5/6A: functional consequences of natural mutations and post-translational modifications.</a> Journal of Biological Chemistry. 281 (41), 30561-30572 (2006).</li>
<li>Zhao, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+characterization+of+loss-of-function+mutations+in+PCSK9+and+identification+of+a+compound+heterozygote%5BTitle%5D)+AND+%22American+Journal+of+Human+Genetics%22%5BJournal%5D)">Molecular characterization of loss-of-function mutations in PCSK9 and identification of a compound heterozygote.</a> American Journal of Human Genetics. 79 (3), 514-523 (2006).</li>
</ol>

The authors have nothing to disclose.

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...

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 means to identify patients likely to gain greater benefits, or both, are needed.
Small-molecule approaches could target intracellular PCSK9, provide an improved route of administration, and reduce costs, making them the &#34;Holy Grail&#34; in this area7. However, PCSK9 has proven difficult to drug by small molecules. As a protease, targeting PCSK9&#39;s proteolytic function is an attractive strategy, as self-proteolysis is the rate-limiting step of PCSK9 maturation8 and is required for its maximal effect on the LDL-R9. To date, however, this strategy has not been successful, likely due to PCSK9&#39;s unique biochemistry: PCSK9 cleaves only itself10, performing a single-turnover reaction, and after self-cleavage, the PCSK9 prodomain remains bound in the active site as an auto-inhibitor11, preventing the readout of any further protease activity.
This article presents a method to evaluate PCSK9 proteolytic function in high-throughput fashion8. Through site-directed mutagenesis, investigators can use this assay to probe the effects of coding SNPs found in the clinic to assess them for effects on proteolysis, the rate-limiting step of PCSK9 maturation. Additionally, this method will be useful in the design of high-throughput screens to identify modulators of PCSK9 proteolysis, which are anticipated to ultimately disrupt the presentation of PCSK9 to the LDL-R (and modulate PCSK9&#39;s hypercholesterolemic effect). Lastly, this protocol can be adapted to other proteases with intrinsically low activity, provided that i) a specific substrate-protease pair can be found, and ii) a suitable intracellular anchor can be established for the substrate.

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

a high-throughput luciferase assay to evaluate proteolysis of the single-turnover protease pcsk9

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.

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...

Watch this Scientific Journal Video about A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9 at JoVE.com

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

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, ...

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Research

JoVE Journal

Biochemistry

8.1K Views.  Zuckerberg San Francisco General and 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.

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

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

Cancer Research

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicine

A Simple Assay to Measure ER-Mitochondria Contacts for High-Throughput Screens

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer&#39;s disease, Parkinson&#39;s disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe&#160;a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

Author Spotlight: Regulation and Dysregulation of ER-Mitochondria Contacts &#8212; Implications for Neurodegenerative Disease Pathogenesis

We have established a split-luciferase reassembly assay to monitor the endoplasmic reticulum-mitochondria contacts in live cells. Using this assay, we describe a protocol to quantitatively measure the level of these inter-organelle couplings in HEK293T cells, under the condition of chemical treatment.

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid ...

Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript describes procedures that may be used to monitor &#947;-secretase-mediated cleavage of either APP-C99 or Notch, using a Gal4 promoter-driven firefly luciferase reporter system. These assays were established by stably co-transfecting HEK293 cells with the Gal4-driven luciferase reporter gene and either the Gal4/VP16-tagged C-terminal fragment of APP (APP-C99; CG cells), or the Gal4/VP16-tagged Notch-&#916;E (N&#916;E; NG cells). Using these reporter assays in parallel, we have demonstrated that an ErbB2 inhibitor, CL-387,785, can preferentially suppress &#947;-secretase cleavage of APP-C99 in CG cells, but not N&#916;E in NG cells. The differential responses exhibited by the CG and NG cells, when treated with CL-387,785, represent a preferred characteristic for &#947;-secretase modulators, and these responses are in stark contrast to the pan-inhibition of &#947;-secretase induced by DAPT. Our studies provide direct evidence that &#947;-secretase activities toward different substrates can be differentiated in a cellular context. These new assays may therefore be useful tools in drug discovery for improved AD therapies.

We have successfully generated two substrate-specific &#947;-secretase assays. Both cell-based assays presented here are designed to quantify &#947;-secretase enzymatic activities via the output of firefly luciferase reporters.

We have developed a pair of cell-based reporter gene assays to quantitatively measure &#947;-secretase cleavage of distinct substrates. This manuscript ...

Neuroscience

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Biology

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Measuring Relative Insulin Secretion using a Co-Secreted Luciferase Surrogate

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, depending on the specific assay. Secreted luciferase assays expedite results and lower the assay cost per sample substantially. Here we present a relatively underused approach to gauge insulin secretory activity from pancreatic &#946; cells by using Gaussia luciferase genetically inserted within the C-peptide. During proteolytic processing of proinsulin, the C-peptide is excised releasing the luciferase within the insulin secretory vesicle where it is co-secreted with insulin. Results can be obtained within minutes after sample collection because of the speed of luciferase assays. A limitation of the assay is that it is a relative measurement of insulin secretion and not an absolute quantitation. However, this protocol is economical, scalable, and can be performed using most standard luminescence plate readers. Analog and digital multichannel pipettes facilitate multiple steps of the assay. Many different experimental variations can be tested simultaneously. Once a focused set of conditions are decided upon, insulin concentrations should be measured directly using antibody-based assays with standard curves to confirm the luciferase assay results.

This protocol describes how to perform rapid low-cost luciferase assays at medium-throughput using an insulin-linked Gaussia luciferase as a proxy for insulin secretion from beta cells. The assay can be performed with most luminescence plate readers and multichannel pipettes.

Performing antibody-based assays for secreted insulin post-sample collection usually requires a few hours to a day of assay time and can be expensive, ...

A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Source: Jaimes, J. A., et al. <a href="https://app.jove.com/v/58892">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

1. Designing and Preparing the Peptides

	Acquire the sequence of the fusion protein of interest from a public database such as NCBI ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

The Luciferase Assay: A High-Throughput Assay to Measure Luminescent Signals Using Engineered Nanoluciferase System-Based Bioreporters

Source: Rezaei, R., et al. <a href="https://app.jove.com/v/62488">Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter.</a> J. Vis. Exp. (2021).
The video demonstrates the luciferase assay used to quantify luciferase-tagged proteins. The assay uses an engineered luciferase enzyme composed of a large fragment that binds with the small fragment, which is conjugated with a protein of interest. The bound subunits form an active enzyme that, in the presence of the substrate, releases a luminescent signal. This signal is then quantified using a luminometer.

Source: Rezaei, R., et al. Detection of SARS-CoV-2 Receptor-Binding Domain Antibody using a HiBiT-Based Bioreporter. J. Vis. Exp. (2021).
The video ...

Biological Techniques

Assay Techniques

Biochemical assays

Luciferase Assay for Measuring &#947;-Secretase Activity in Human Cells

Source: Wang, B., et al., <a href="https://app.jove.com/v/56795">Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms.</a> J. Vis. Exp. (2018).
This video demonstrates the procedure of measuring &#947;-secretase activity in genetically modified human cells using a tetracycline-inducible system. The process involves inducing APP gene expression with tetracycline, generating amyloid-&#946; peptides, and activating luciferase production. The bioluminescence resulting from the luciferase-substrate reaction is then measured, reflecting &#947;-secretase activity.

Source: Wang, B., et al., Quantitative Measurement of &#947;-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase ...

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

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