The authors thank the Core Facility of the Institute of Cellular and Organismic Biology, Academia Sinica, for technical support. This study was supported by the Ministry of Science and Technology, Taiwan (MOST 103-2320-B-001-016-MY3 to Y.-F. L.), the Program for Translational Innovation of Biopharmaceutical Development &#8211; Technology Supporting Platform Axis Scheme (NP7 to Y.-F.L.), and Academia Sinica (to Y.-F.L.).

1. Measurement of Luciferase Reporter Signals, which Correspond to &#947;-Secretase Cleavage of APP-C99 or N&#916;E
NOTE: Please refer to the previous publications for detailed descriptions of the generation of CG and NG cells20,21.
<ol>
	<li>Seed NG or CG cells (20, 000 cells/well) onto 96-well microplates at a final volume of 200 &#956;L/well in growth medium that is composed of Dulbecco&#39;s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum (FBS), 5 &#956;g/mL blasticidin, 250 &#956;g/mL antibiotic (e.g., zeocin), and 200 &#956;g/mL hygromycin B.
	<ol>
		<li>Count cells with a hemocytometer.</li>
		<li>Transfer the microplates to a humidified CO2 incubator and incubate at 37 &#176;C overnight.</li>
	</ol>
	</li>
	<li>Remove 100 &#956;L of growth medium from each well.</li>
	<li>Add 100 &#956;L/well of growth medium containing 2 &#956;g/mL tetracycline with either 0.2% dimethyl sulfoxide (DMSO), 2 &#956;M CL-387,785, 2 &#956;M N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT), or other related ErbB1/ErbB2 inhibitors.</li>
	<li>Incubate treated CG or NG cells in microplates at 37 &#176;C for 24 h.</li>
	<li>Remove 150 &#956;L/well growth medium from microplates.</li>
	<li>Add 50 &#956;L/well luciferase assay reagent (please see Table of Materials).</li>
	<li>Transfer the microplates to a luminescence microplate reader.
	<ol>
		<li>Maintain the microplates at room temperature for 5 min with gentle agitation.</li>
	</ol>
	</li>
	<li>Determine the Firefly luciferase (FL)-emitted luminescence using a pre-defined program stored in the luminescence microplate reader.
	<ol>
		<li>Open the instrument control software (see Table of Materials for instrument details).</li>
		<li>From the &#39;Tool&#39; menu, select &#39;Luminometry&#39;.
		<ol>
			<li>Under the &#39;Parameters&#39; settings, choose &#39;No filter&#39; for Emission Filter, tick &#39;Normal&#39; for Emission Aperture, enter &#39;1&#39; for Counting Time, and click on &#39;OK&#39; to set up the luminescence reading.</li>
		</ol>
		</li>
		<li>Under the &#39;Instrument control&#39; tab, scroll through the list of active protocols, and select the pre-stored protocol for reading luminescence.</li>
		<li>Under the &#39;Live display&#39; tab, define the Scale from the pull-down list, and select &#39;Logarithm&#39; or &#39;Linear&#39; for the Type.</li>
		<li>Under the &#39;Temperature&#39; tab, select &#39;Off&#39; for Plate heating.</li>
		<li>Click the &#39;Start&#39; button to perform the luminescence reading.</li>
		<li>As needed, export results into a spreadsheet format using the &#39;Explorer&#39; embedded within the control software.</li>
	</ol>
	</li>
</ol>
2. Quantification of &#947;-Secretase Activity
<ol>
	<li>Define a luminescence signal emitted by CG or NG cells treated with 0.1% DMSO in growth medium containing 1 &#956;g/mL tetracycline alone as 100% relative &#947;-secretase activity.
	<ol>
		<li>Estimate background luminescence from CG or NG cells by treating cells with growth medium that does not include tetracycline.</li>
	</ol>
	</li>
	<li>Normalize luciferase signals from CG or NG cells that are cultured with growth medium containing 1 &#956;g/mL tetracycline and either 1 &#956;M CL-387,785 or 1 &#956;M of DAPT with the luciferase signal emitted by DMSO-treated CG or NG cells (100% relative &#947;-secretase activity, as defined in 2.1).</li>
</ol>

<ol>
	<li>Selkoe, D. J., Hardy, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease+at+25+years.">The amyloid hypothesis of Alzheimer's disease at 25 years.</a> EMBO Mol Med. 8 (6), 595-608 (2016).</li><li>Wolfe, M. S., Guenette, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=APP+at+a+glance.">APP at a glance.</a> J Cell Sci. 120 (Pt 18), 3157-3161 (2007).</li><li>Karran, E., Mercken, M., De Strooper, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+cascade+hypothesis+for+Alzheimer's+disease:+an+appraisal+for+the+development+of+therapeutics.">The amyloid cascade hypothesis for Alzheimer's disease: an appraisal for the development of therapeutics.</a> Nat Rev Drug Discov. 10 (9), 698-712 (2011).</li><li>Citron, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+strategies+for+disease+modification.">Alzheimer's disease: strategies for disease modification.</a> Nat Rev Drug Discov. 9 (5), 387-398 (2010).</li><li>Vassar, R., Kovacs, D. M., Yan, R., Wong, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beta-secretase+enzyme+BACE+in+health+and+Alzheimer's+disease:+regulation,+cell+biology,+function,+and+therapeutic+potential.">The beta-secretase enzyme BACE in health and Alzheimer's disease: regulation, cell biology, function, and therapeutic potential.</a> J Neurosci. 29 (41), 12787-12794 (2009).</li><li>Parks, A. L., Curtis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presenilin+diversifies+its+portfolio.">Presenilin diversifies its portfolio.</a> Trends Genet. 23 (3), 140-150 (2007).</li><li>Kukar, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate-targeting+gamma-secretase+modulators.">Substrate-targeting gamma-secretase modulators.</a> Nature. 453 (7197), 925-929 (2008).</li><li>Kounnas, M. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+gamma-secretase+reduces+beta-amyloid+deposition+in+a+transgenic+mouse+model+of+Alzheimer's+disease.">Modulation of gamma-secretase reduces beta-amyloid deposition in a transgenic mouse model of Alzheimer's disease.</a> Neuron. 67 (5), 769-780 (2010).</li><li>Thathiah, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orphan+G+protein-coupled+receptor+3+modulates+amyloid-beta+peptide+generation+in+neurons.">The orphan G protein-coupled receptor 3 modulates amyloid-beta peptide generation in neurons.</a> Science. 323 (5916), 946-951 (2009).</li><li>Flajolet, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Alzheimer's+disease+amyloid-beta+formation+by+casein+kinase+I.">Regulation of Alzheimer's disease amyloid-beta formation by casein kinase I.</a> Proc Natl Acad Sci U S A. 104 (10), 4159-4164 (2007).</li><li>Eisele, Y. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gleevec+increases+levels+of+the+amyloid+precursor+protein+intracellular+domain+and+of+the+amyloid-beta+degrading+enzyme+neprilysin.">Gleevec increases levels of the amyloid precursor protein intracellular domain and of the amyloid-beta degrading enzyme neprilysin.</a> Mol Biol Cell. 18 (9), 3591-3600 (2007).</li><li>He, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gamma-secretase+activating+protein+is+a+therapeutic+target+for+Alzheimer's+disease.">Gamma-secretase activating protein is a therapeutic target for Alzheimer's disease.</a> Nature. 467 (7311), 95-98 (2010).</li><li>Haapasalo, A., Kovacs, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+substrates+of+presenilin/gamma-secretase.">The many substrates of presenilin/gamma-secretase.</a> J Alzheimers Dis. 25 (1), 3-28 (2011).</li><li>Gillman, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+Evaluation+of+BMS-708163,+a+Potent,+Selective+and+Orally+Bioavailable+gamma-Secretase+Inhibitor.">Discovery and Evaluation of BMS-708163, a Potent, Selective and Orally Bioavailable gamma-Secretase Inhibitor.</a> ACS Med Chem Lett. 1 (3), 120-124 (2010).</li><li>Hopkins, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACS+chemical+neuroscience+molecule+spotlight+on+Begacestat+(GSI-953).">ACS chemical neuroscience molecule spotlight on Begacestat (GSI-953).</a> ACS Chem Neurosci. 3 (1), 3-4 (2012).</li><li>Yang, T., Arslanova, D., Gu, Y., Augelli-Szafran, C., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+gamma-secretase+modulation+differentiates+inhibitor+compound+selectivity+between+two+substrates+Notch+and+amyloid+precursor+protein.">Quantification of gamma-secretase modulation differentiates inhibitor compound selectivity between two substrates Notch and amyloid precursor protein.</a> Mol Brain. 1, 15 (2008).</li><li>McKee, T. D., Loureiro, R. M., Dumin, J. A., Zarayskiy, V., Tate, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+cell-based+method+for+determining+the+gamma-secretase+enzyme+activity+against+both+Notch+and+APP+substrates.">An improved cell-based method for determining the gamma-secretase enzyme activity against both Notch and APP substrates.</a> J Neurosci Methods. 213 (1), 14-21 (2013).</li><li>Basi, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+precursor+protein+selective+gamma-secretase+inhibitors+for+treatment+of+Alzheimer's+disease.">Amyloid precursor protein selective gamma-secretase inhibitors for treatment of Alzheimer's disease.</a> Alzheimers Res Ther. 2 (6), 36 (2010).</li><li>Liao, Y. F., Tang, Y. C., Chang, M. Y., Wang, B. J., Hu, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+small+molecular+(D)-leucinamides+as+potent,+Notch-sparing+gamma-secretase+modulators.">Discovery of small molecular (D)-leucinamides as potent, Notch-sparing gamma-secretase modulators.</a> Eur J Med Chem. 79, 143-151 (2014).</li><li>Liao, Y. F., Wang, B. J., Cheng, H. T., Kuo, L. H., Wolfe, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+necrosis+factor-alpha,+interleukin-1beta,+and+interferon-gamma+stimulate+gamma-secretase-mediated+cleavage+of+amyloid+precursor+protein+through+a+JNK-dependent+MAPK+pathway.">Tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma stimulate gamma-secretase-mediated cleavage of amyloid precursor protein through a JNK-dependent MAPK pathway.</a> J Biol Chem. 279 (47), 49523-49532 (2004).</li><li>Wang, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ErbB2+regulates+autophagic+flux+to+modulate+the+proteostasis+of+APP-CTFs+in+Alzheimer's+disease.">ErbB2 regulates autophagic flux to modulate the proteostasis of APP-CTFs in Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 114 (15), E3129-E3138 (2017).</li><li>Sevigny, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+antibody+aducanumab+reduces+Abeta+plaques+in+Alzheimer's+disease.">The antibody aducanumab reduces Abeta plaques in Alzheimer's disease.</a> Nature. 537 (7618), 50-56 (2016).</li><li>Golde, T. E., Koo, E. H., Felsenstein, K. M., Osborne, B. A., Miele, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gamma-Secretase+inhibitors+and+modulators.">gamma-Secretase inhibitors and modulators.</a> Biochim Biophys Acta. 1828 (12), 2898-2907 (2013).</li><li>Coric, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Prodromal+Alzheimer+Disease+With+Avagacestat:+A+Randomized+Clinical+Trial.">Targeting Prodromal Alzheimer Disease With Avagacestat: A Randomized Clinical Trial.</a> JAMA Neurol. 72 (11), 1324-1333 (2015).</li><li>Mitani, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+between+gamma-secretase+inhibitors+and+modulators+on+cognitive+function+in+amyloid+precursor+protein-transgenic+and+nontransgenic+mice.">Differential effects between gamma-secretase inhibitors and modulators on cognitive function in amyloid precursor protein-transgenic and nontransgenic mice.</a> J Neurosci. 32 (6), 2037-2050 (2012).</li><li>Penninkilampi, R., Brothers, H. M., Eslick, G. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+Agents+Targeting+gamma-Secretase+Increase+Risk+of+Cancer+and+Cognitive+Decline+in+Alzheimer's+Disease+Patients:+A+Systematic+Review+and+Meta-Analysis.">Pharmacological Agents Targeting gamma-Secretase Increase Risk of Cancer and Cognitive Decline in Alzheimer's Disease Patients: A Systematic Review and Meta-Analysis.</a> J Alzheimers Dis. 53 (4), 1395-1404 (2016).</li></ol>

The authors have nothing to disclose.

The promising outcomes from a phase 1b trial of aducanumab immunotherapy for AD have firmly established the critical role of A&#946; in the pathogenesis of AD22 and suggest that anti-A&#946; approaches are still a viable strategy for the development of anti-AD drugs. Here, we describe cell-based assays for quantifying &#947;-secretase-catalyzed proteolysis of APP-C99 and N&#916;E using firefly luciferase reporter systems. APP-C99 and N&#916;E are the two most well-studied &#947;-secretase substrat...

Oligomeric forms of amyloid-&#946; (A&#946;) are believed to be the primary cause of neurodegeneration in the brains of patients suffering from Alzheimer&#39;s disease (AD)1. A&#946; peptides are produced by the stepwise cleavage of amyloid precursor protein (APP), first by &#946;-secretase and then by &#947;-secretase2. In the past decade, therapeutic approaches toward treatment of AD have focused on the prevention of A&#946; production3. The majority of studies have focused on either the augmentation of &#945;-secretase activity, which can preclude &#947;-secretase cleavage of APP and thereby decrease the production of A&#946;, or the inhibition of &#946;-and/or &#947;-secretase activities4. Unfortunately, non-selective inhibition of &#946;- or &#947;-secretases results in unavoidable side effects that are due to interference with the functioning of other physiological substrates of &#946;- and &#947;-secretase5,6. With regard to &#947;-secretase inhibitors, recent studies have reported the discovery of a number of chemical and genetic modulators that can regulate A&#946; production while exerting negligible effects on the essential &#947;-secretase-mediated processing of Notch7,8,9,10,11,12, however, successful therapeutics have not yet been developed. Thus, further systematic screens are warranted to discover novel genetic and chemical modifiers that may selectively modulate &#947;-secretase-mediated APP processing.
&#947;-Secretase is known to cleave more than 90 different membrane-anchored proteins. Among these substrates is Notch, whose activity is critical for cell fate determination and differentiation during development13. Selectively modulating &#947;-secretase-catalyzed APP processing in the absence of affecting Notch processing will be essential for minimizing Notch-related side effects of &#947;-secretase inhibitors, and this selectivity is thought to be a primary factor that will dictate the biological efficacy of potential &#947;-secretase modulators for the chronic treatment of AD. There have been a number of arylsulfonamide derivatives, such as GSI-953 (begacestat) and BMS-708163 (avagacestat), that have been found to exhibit potent and selective inhibition of &#947;-secretase14,15. A previous study has demonstrated that the differential inhibition of &#947;-secretase cleavage of APP and Notch can be observed using a quantitative ELISA-based assay in combination with in vivo validation using zebrafish16. Furthermore, cell-based assay paradigms have been established to address the potential substrate selectivity of &#947;-secretase toward APP and Notch17,18. Using protocols similar to those described herein, we have recently discovered a novel class of (D)-leucinamides that potently modulate &#947;-secretase cleavage of APP with Notch-sparing selectivity19. Together, this collection of studies provides a proof-of-concept supporting the notion that the substrate selectivity/availability of &#947;-secretase can be subject to chemical modulation and experimental verification. However, these assay platforms often rely on relatively low-throughput readouts and labor-intensive approaches that might not meet industrial standards for drug discovery programs.
We have recently generated cell-based luciferase reporter gene assays that can quantitatively determine the catalytic activity of &#947;-secretase toward two distinct substrates, the 99-amino acid C-terminal fragment of APP (APP-C99) and the extracellular domain-deleted Notch peptide (N&#916;E). Both APP-C99 and N&#916;E have been widely used as direct substrates of &#947;-secretase in various assays. To generate homogeneous and consistent luciferase reporter gene assays for the &#947;-secretase cleavage of either APP-C99 or N&#916;E, we generated one HEK-derived stable cell line (CG) that constitutively expresses a Gal4 promoter-driven firefly luciferase reporter (Gal4-Luc) and Gal4/VP16-tagged APP-C99 fusion protein (C99-GV). In addition, we generated another HEK-derived stable cell line (NG) that constitutively expresses Gal4-Luc and Gal4/VP16-tagged N&#916;E (N&#916;E-GV). Using these novel substrate-specific &#947;-secretase assays, we now provide a method to quantify and distinguish the catalytic activity of &#947;-secretase toward distinct substrates (APP-C99 in CG cells, or N&#916;E in NG cells). Moreover, the substrate-specific &#947;-secretase assays were designed to be conducive to high-content screening platforms. These newly generated &#947;-secretase assays could pave the way for the discovery of novel &#947;-secretase modulators that could improve cognitive function by suppressing A&#946; production without eliciting undesirable Notch inhibition for the next-generation treatment of AD.

<table><tbody><tr><td>VictorLight luminescence plate reader</td><td>PerkinElmer</td><td>2030-0010</td><td></td></tr><tr><td>Class II, Type 2 Biological Safety Cabinet</td><td>Thermo Scientific</td><td>1300 Series A2</td><td></td></tr><tr><td>CL-387,785</td><td>EMD Calbiochem</td><td>233100-1MG</td><td></td></tr><tr><td>DAPT</td><td>Merck</td><td>565770</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium (DMEM)</td><td>Thermo Fisher</td><td>12100046</td><td>main compnent of growth medium</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Fisher</td><td>16000044</td><td></td></tr><tr><td>Blasticidin</td><td>Thermo Fisher</td><td>A1113903</td><td></td></tr><tr><td>Zeocin</td><td>Thermo Fisher</td><td>R25005</td><td></td></tr><tr><td>Hygromycin B</td><td>Gibco</td><td>10687010</td><td></td></tr><tr><td>Hemocytomer</td><td>Sigma-Aldrich</td><td>BR717805 Aldrich</td><td></td></tr><tr><td>96-well microplate</td><td>Nunc</td><td>156545</td><td></td></tr><tr><td>Tetracycline</td><td>Sigma</td><td>T7660</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>Steady-Glo luciferase assay reagent</td><td>Promega</td><td>E2510</td><td></td></tr><tr><td>Humidified CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Revco</td><td>Ultima II</td><td></td></tr><tr><td>T-REx293 cell line</td><td>Invitrogen</td><td>R71007</td><td></td></tr><tr><td>Wallac 1420 software version 3.0</td><td>PerkinElmer</td><td></td><td>instrument control software of the luminescence microplate reader</td></tr></tbody></table>

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.

Inhibition of ErbB2 by CL-387,785 can differentially promote the processing of APP-C99 by &#947;-secretase without affecting Notch cleavage 
To establish a cell-based assay that can quantitatively measure the proteolytic cleavage of a particular &#947;-secretase substrate, we generated the HEK293-derived CG cell line by stable co-transfection of a tetracycline-inducible C-terminally Gal4/VP16-tagged APP-C99 and a Gal4 promoter-driven...

Watch this Scientific Journal Video about Quantitative Measurement of Î³-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms at JoVE.com

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

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 ...

quantitative-measurement-secretase-mediated-amyloid-precursor-protein

Research

JoVE Journal

Neuroscience

6.7K Views.  Academia Sinica. We hebben met succes twee substraat-specifieke γ-secretase tests gegenereerd. Zowel cel-gebaseerde testen hier gepresenteerd zijn ontworpen om het kwantificeren van de enzymatische activiteiten γ-secretase via de uitgang van firefly luciferase verslaggevers.

Video: Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

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.

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, ...

Biochemistry

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

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Developmental Biology

Quantifying DNA Double Strand Break Repair with Nanoluciferase Reporter Assays

Assessment of DNA Double Strand Break Repair Activity Using High-throughput and Quantitative Luminescence-Based Reporter Assays

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is mediated through several mechanisms: homologous recombination (HR), non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and single strand annealing (SSA). Cellular assays are essential to measure the proficiency and modulation of these pathways in response to various stimuli.
Here, we present a suite of extrachromosomal reporter assays that each measure the reconstitution of a nanoluciferase reporter gene by one of the four major DSBR pathways in cells. Upon transient transfection into cells of interest, repair of pathway-specific reporter substrates can be measured in under 24 h by the detection of Nanoluciferase (NanoLuc) luminescence.
These robust assays are quantitative, sensitive, titratable, and amenable to a high-throughput screening format. These properties provide broad applications in DNA repair research and drug discovery, complementing the currently available toolkit of cellular DSBR assays.

Author Spotlight: Developing Novel Anticancer Therapeutics Targeting the DNA Damage Response

We present protocols for the assessment of double strand break repair pathway proficiency in cells using a suite of luminescence-based extrachromosomal reporter substrates.

The repair of DNA double strand breaks (DSBs) is crucial for the maintenance of genome stability and cell viability. DSB repair (DSBR) in cells is ...

Cancer Research

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

Immunology and Infection

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

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse formation and maintenance. Many of these fundamental interactions occur in trans and are typically induced by heterophilic or homophilic interactions between cells expressing membrane anchored binding pairs. Elucidating how disease relevant mutations disrupt these fundamental protein interactions can provide insight into a myriad of cell biology fields. Many protein-protein interaction assays do not typically disambiguate between cis and trans interactions, which potentially leads to an overestimation of the extent of binding that is occurring in vivo and involve labor intensive purification of protein and/or specialized monitoring equipment. Here, we present an optimized simple protocol that allows for the observation and quantification of only trans interactions without the need for lengthy protein purifications or specialized equipment. The HEK cell aggregation assay involves the mixing of two independent populations of HEK cells, each expressing membrane-bound cognate ligands. After a short incubation period, samples are imaged and the resulting aggregates are quantified.

Here, we present an optimized protocol to rapidly and semiquantitatively measure ligand-receptor interactions in trans in a heterologous cell system using fluorescence microscopy.

Protein interactions at cellular interfaces dictate a multitude of biological outcomes ranging from tissue development and cancer progression to synapse ...

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes associated with neurodevelopmental disorders, but the functional significance of the vast majority of variants remains unknown. The present protocol describes the study of variants for Ube3a, a gene that encodes an E3 ubiquitin ligase linked to both autism and Angelman syndrome. Duplication or triplication of Ube3a is strongly linked to autism, whereas its deletion causes Angelman syndrome. Thus, understanding the valence of changes in UBE3A protein activity is important for clinical outcomes. Here, a rapid, cell-based method that pairs Ube3a variants with a Wnt pathway reporter is described. This simple assay is scalable and can be used to determine the valence and magnitude of activity changes in any Ube3a variant. Moreover, the facility of this method allows the generation of a wealth of structure-function information, which can be used to gain deep insights into the enzymatic mechanisms of UBE3A.

A Scalable, Cell-Based Method for the Functional Assessment of Ube3a Variants

A simple and scalable method was developed to assess the functional significance of missense variants in Ube3a, a gene whose loss and gain of function are linked to both Angelman syndrome and autism spectrum disorder.

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes ...

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, ...

Quantitative Measurement of γ-Secretase-mediated Amyloid Precursor Protein and Notch Cleavage in Cell-based Luciferase Reporter Assay Platforms

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