Name: identifying inhibitors of the hbx-ddb1 interaction using a split luciferase assay system
Uploaded: 2019-12-21T14:00:00.000+00:00
Duration: 5 min 55 s
Description: Watch this Scientific Journal Video about Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System at JoVE.com

This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (#19H03430 and #17K09405 to M.O., and #19J11829 to K.S.), by a Grant-in-Aid for Scientific Research on Innovative Areas (#18H05024 to M.O.), by the Research Program on Hepatitis from Japan Agency for Medical Research and Development, AMED (to M.O., #JP19fk021005), by program on the Innovative Development and the Application of New Drugs for Hepatitis B (#JP19fk0310102 to K.K.) from AMED, by grants from the Japan Foundation for Applied Enzymology and from the Kobayashi Foundation for Cancer Research (to M.O.), by GSK Japan Research Grant 2018 (to K.S.), and by a grant from the Miyakawa Memorial Research Foundation (to K.S.).

NOTE: A schematic representation of the split luciferase assay is shown in Figure 1A, and the assay process is outlined in Figure 1B. The interaction dynamics can be measured in real time without cell lysis.
1. Cell Preparation
<ol>
	<li>Maintain cultured HEK293T cells in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS), 1x penicillin/streptomycin at 37 &#176;C in 20% O2 and 5% CO2.</li>
	<li>Seed 5 x 106 cells into a 100 mm dish with 10 mL of DMEM and incubate at 37 &#176;C overnight.</li>
	<li>Transiently transfect 1 &#181;g of HBx and DDB1 with split luciferases into the cells according to the following method. 
	NOTE: The amount of the plasmid DNA transfected may depend on the transfection regent used. The optimal position of the split luciferase fused to the target protein must be determined beforehand. In this case, HBx fused to LgBit at the C-terminus of HBx (HBx-LgBit) and DDB1 fused to SmBit at the N-terminus of DDB1 (SmBit-DDB1) provided the best results (i.e., the brightest luciferase signals). This process has been reported previously in detail7.
	<ol>
		<li>Dilute 1 &#181;g of HBx-LgBit expressing DNA plasmid and 1 &#181;g of SmBit-DDB1 expressing DNA plasmid (Table of Materials) in DNA condensation buffer (Table of Materials) to a total volume of 300 &#181;L.</li>
		<li>Add 16 &#181;L of enhancer solution (Table of Materials) and mix by vortexing for 1 s.</li>
		<li>Incubate the sample at room temperature for 3 min.</li>
		<li>Add 60 &#181;L of transfection reagent (Table of Materials) to the sample and mix by vortexing for 10 s.</li>
		<li>Incubate the sample at room temperature for 8 min.</li>
		<li>During incubation, aspirate the culture medium from the dish (prepared in step 1.2), and wash cells with 5 mL of phosphate-buffered saline (PBS). Remove PBS by aspiration and add 7 mL of DMEM.</li>
		<li>Add 3 mL of DMEM to the tube containing the transfection complexes. Mix by pipetting and add the transfection complexes onto the cell in the 10 cm dish.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C in an incubator under 5% CO2 for 10 h.</li>
	<li>Reseed the cells into a white 96 well plate at 5 x 104 cells/well in 50 &#181;L of medium/well according to the following method.
	<ol>
		<li>Remove the spent cell culture medium and wash cells with 5 mL of PBS.</li>
		<li>Remove PBS by aspiration, add 1 mL of 0.25% trypsin-EDTA, and incubate at 37 &#176;C for 5 min to detach cells.</li>
		<li>Add 4 mL of DMEM and disperse the medium by pipetting over the surface of the cell layer several times. Transfer the cell suspension to a tube.</li>
		<li>Centrifuge cells at 500 x g for 5 min at room temperature.</li>
		<li>Discard supernatant and resuspend the cell pellet in 1 mL of PBS.</li>
		<li>Centrifuge the cell suspension at 500 x g for 5 min at room temperature and discard supernatant.</li>
		<li>Dilute the cell pellet with buffered cell culture medium (Table of Materials) supplemented with 10% FBS to a seeding density of 1.0 x 106 cells/mL.</li>
		<li>Pipette 50 &#181;L of cell suspension into each well of a 96 well plate and return the cells to the incubator.</li>
	</ol>
	</li>
	<li>Incubate cells at 37 &#176;C under 5% CO2 for 10 h.</li>
</ol>
2. Compound Screening
<ol>
	<li>During the incubation, dilute the screening compounds (Table of Materials) and solvent (dimethyl sulfoxide [DMSO]) at 13.5x concentration. For example, if the stock is 10 mM and the screening concentration is 10 &#181;M, add 1 &#181;L of stock solution to 73.1 &#181;L of buffered cell culture medium.</li>
	<li>Add 12.5 &#181;L of luminescent substrate (Table of Materials) to each well and incubate for 5 min at room temperature. 
	NOTE: As negative controls, the wells at both ends of the plate (i.e., columns 1 and 12) should contain no luminescent substrate.</li>
	<li>Measure the baseline luminescence using a luminometer (Table of Materials).</li>
	<li>Immediately after the initial measurement, add 5 &#181;L of compounds and control DMSO diluted in step 2.1 to each well. 
	NOTE: The final concentration will be 10 &#181;M.</li>
	<li>Measure luminescence values every 30 min for 2 h. 
	NOTE: The plate should be incubated in the dark at room temperature.</li>
	<li>Calculate the inhibitory effects by comparison with control DMSO treatment after normalization to the baseline signals. 
	NOTE: Screening each compound in duplicate or triplicate can reduce variation.</li>
</ol>

<ol>
	<li>Tang, L. S. Y., Covert, E., Wilson, E., Kottilil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+hepatitis+B+infection:+a+review.">Chronic hepatitis B infection: a review.</a> The Journal of the American Medical Association. 319, 1802-1813 (2018).</li><li>Sekiba, K., 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=Hepatitis+B+virus+pathogenesis:+Fresh+insights+into+hepatitis+B+virus+RNA.">Hepatitis B virus pathogenesis: Fresh insights into hepatitis B virus RNA.</a> World Journal of Gastroenterology. 24, 2261-2268 (2018).</li><li>Slagle, B. L., Bouchard, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatitis+B+virus+X+and+regulation+of+viral+gene+expression.">Hepatitis B virus X and regulation of viral gene expression.</a> Cold Spring Harbor Perspectives in Medicine. 6, a021402 (2016).</li><li>Murphy, C. 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=Hepatitis+B+Virus+X+Protein+Promotes+Degradation+of+SMC5/6+to+Enhance+HBV+Replication.">Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication.</a> Cell Reports. 16, 2846-2854 (2016).</li><li>Decorsi&#232;re, 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=Hepatitis+B+virus+X+protein+identifies+the+Smc5/6+complex+as+a+host+restriction+factor.">Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.</a> Nature. 531, 386-389 (2016).</li><li>Niu, C., 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+Smc5/6+complex+restricts+HBV+when+localized+to+ND10+without+inducing+an+innate+immune+response+and+is+counteracted+by+the+HBV+X+protein+shortly+after+infection.">The Smc5/6 complex restricts HBV when localized to ND10 without inducing an innate immune response and is counteracted by the HBV X protein shortly after infection.</a> PLoS One. 12, e0169648 (2017).</li><li>Sekiba, K., 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=Inhibition+of+HBV+Transcription+From+cccDNA+With+Nitazoxanide+by+Targeting+the+HBx-DDB1+Interaction.">Inhibition of HBV Transcription From cccDNA With Nitazoxanide by Targeting the HBx-DDB1 Interaction.</a> Cellular and Molecular Gastroenterology and Hepatology. 7, 297-312 (2019).</li><li>Eggers, C. T., 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=NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells.">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11, 400-408 (2015).</li><li>Zhang, J. H., Chung, T. D. Y., Oldenburg, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+statistical+parameter+for+use+in+evaluation+and+validation+of+high+throughput+screening+assays.">A simple statistical parameter for use in evaluation and validation of high throughput screening assays.</a> Journal of Biomolecular Screening. 4, 67-73 (1999).</li><li>Rao, V. S., Srinivas, K., Sujini, G. N., Kumar, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interaction+detection:+Methods+and+analysis.">Protein-protein interaction detection: Methods and analysis.</a> International Journal of Proteomics. 2014. 2014, 147648 (2014).</li><li>Michael, 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=A+Robotic+Platform+for+Quantitative+High-Throughput+Screening.">A Robotic Platform for Quantitative High-Throughput Screening.</a> ASSAY and Drug Development Technologies. 6, 637-657 (2008).</li><li>Skwarczynska, M., Ottmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-protein+interactions+as+drug+targets.">Protein-protein interactions as drug targets.</a> Future Medicinal Chemistry. 7, 2195-2219 (2015).</li><li>de Chassey, B., Meyniel-Schicklin, L., Vonderscher, J., Andr&#233;, P., Lotteau, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-host+interactomics:+New+insights+and+opportunities+for+antiviral+drug+discovery.">Virus-host interactomics: New insights and opportunities for antiviral drug discovery.</a> Genome Medicine. 6, 115 (2014).</li><li>Prasad, 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=Virus-Host+Interactions:+New+Insights+and+Advances+in+Drug+Development+Against+Viral+Pathogens.">Virus-Host Interactions: New Insights and Advances in Drug Development Against Viral Pathogens.</a> Current Drug Metabolism. 18, 942-970 (2017).</li></ol>

The authors have nothing to disclose.

We developed a convenient screening method using a split luciferase assay to find HBx-DDB1 binding inhibitors. The interaction dynamics can be detected in real time in living cells without the need for cell lysis. Inhibition of the HBx-DDB1 interaction leads to restoration of Smc5/6, which results in suppression of viral transcription, protein expression, and cccDNA production7. This novel mechanism of antiviral action may overcome the inadequacies of current HBV therapies.
Although a number of methods are available to investigate protein-protein interactions in living cells, examining these interactions remain difficult10. Our procedure is simple and requires only a short time to screen one 96 well plate. Moreover, the screening quality was satisfactory with a high Z&#39; score, the gold standard quality index for high-throughput screening9. Our assay may be suitable for robotic automation11 and is an efficient assay for drug discovery.
While the protocol described here used the HEK293T cell line because its high transfection efficacy and high proliferation ability are suitable for high-throughput screening, this screening method can be performed using other cell lines (e.g., HepG2) without modifications7. As a realistic strategy for screening compounds, HEK293T cells may be used in the first screening followed by HepG2 cells in a second validation screening. Some compounds may not show significant results in different cell lines when the effects are dependent on indirect mechanisms.
As our intention was to develop a high-throughput screening method, subsequent validation studies are necessary to confirm whether the identified compounds function as interaction inhibitors. Decreased levels of luminescent signals in this assay do not always correspond to inhibition of the HBx-DDB1 interaction. Cytotoxicity tests, co-immunoprecipitation studies, and further anti-HBV experiments are important to confirm the effects7.
Although we previously identified nitazoxanide as an inhibitor of the HBx-DDB1 interaction by screening a relatively small-scale compound library7, further studies involving screening of much larger compound libraries can be easily performed to identify novel compounds that are capable of inhibiting protein-protein interactions more efficiently. When performing such further screening, nitazoxanide can be used as a positive control for the assay. Furthermore, the system described here can be applied to other protein-protein interactions. Protein-protein interactions are an important class of drug targets12. Indeed, many other viruses interact with host factors to replicate or express their pathogenicity13,14. The split luciferase-based assay described here, which targets the interactions between viral and host proteins, may provide a new strategy to develop cures for HBV and other infectious diseases.

Hepatitis B virus (HBV) infection is a major public health concern worldwide, with annual estimates of 240 million people chronically infected with HBV and 90,000 deaths due to complications from the infection, including cirrhosis and hepatocellular carcinoma (HCC)1. Although the current anti-HBV therapeutic agents, nucleos(t)ide analogues, sufficiently inhibit viral reverse transcription, they rarely achieve elimination of viral proteins, which is the long-term clinical goal. Their poor effect on viral protein elimination is due to their lack of direct effect on viral transcription from episomal viral covalently closed circular DNA (cccDNA) minichromosomes in the hepatocyte nucleus2.
HBV transcription is activated by HBV regulatory X (HBx) protein3. Recent studies revealed that HBx degrades structural maintenance of chromosomes 5/6 (Smc5/6), a host restriction factor that blocks HBV transcription from cccDNA, via hijacking a DDB1-CUL4-ROC1 E3 ubiquitin ligase complex4,5,6. Therefore, a crucial step in promoting viral transcription from cccDNA is thought to be the HBx-DDB1 interaction. Compounds capable of inhibiting the binding between HBx and DDB1 may block viral transcription, and indeed nitazoxanide was identified as an inhibitor of the HBx-DDB1 interaction via a screening system developed in our laboratory7.
Here, we present our convenient screening system used to identify inhibitors of the HBx-DDB1 interaction, which utilizes a split luciferase complementary assay7,8. Split luciferase subunits are fused to HBx and DDB1, and the HBx-DDB1 interaction brings the subunits into close proximity to form a functional enzyme that generates a bright luminescent signal. As the interaction between the subunits is reversible, this system can detect rapidly dissociating HBx-DDB1 proteins (Figure 1). Using this system, a large compound library can be easily screened, which may result in the discovery of novel compounds capable of efficiently inhibiting the HBx-DDB1 interaction.

<table><tbody><tr><td>Cell culture microplate, 96 well, PS, F-BOTTOM</td><td>Greiner-Bio-One GmbH</td><td>655098</td><td></td></tr><tr><td>DMEM</td><td>Sigma Aldrich</td><td>D6046</td><td></td></tr><tr><td>DMSO</td><td>Tocris Bioscience</td><td>3176</td><td></td></tr><tr><td>Effectene transfection reagent</td><td>Qiagen</td><td>301425</td><td>Includes DNA-condensation buffer, enhancer solution and transfection reagent</td></tr><tr><td>FBS</td><td>Nichirei</td><td>175012</td><td></td></tr><tr><td>GloMax 96 microplate luminometer</td><td>Promega</td><td>E6521</td><td></td></tr><tr><td>HBx&amp;ndash;LgBit expressing DNA plasmid</td><td>Our laboratory</td><td></td><td>Available upon request</td></tr><tr><td>HEK293T cells</td><td>American Type Culture Collection</td><td>CRL-11268</td><td></td></tr><tr><td>NanoBiT PPI starter systems</td><td>Promega</td><td>N2015</td><td>Includes Nano-Glo Live Cell Reagent</td></tr><tr><td>Opti-MEM</td><td>Thermo Fisher Scientific</td><td>11058021</td><td>Described as &quot;buffered cell culture medium&quot; in the manuscript</td></tr><tr><td>PBS</td><td>Takara</td><td>T900</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma Aldrich</td><td>P0781</td><td></td></tr><tr><td>Screen-Well FDA-approved drug library V2 version 1.0</td><td>Enzo Life Sciences</td><td>BML-2841</td><td>Compounds used here were as follows: mequinol, mercaptopurine hydrate, mesna, mestranol, metaproterenol hemisulfate, metaraminol bitartrate, metaxalone, methacholine chloride, methazolamide, methenamine hippurate, methocarbamol, methotrexate, methoxsalen, methscopolamine bromide, methsuximide, methyclothiazide, methyl aminolevulinate&amp;middot;HCl, methylergonovine maleate, metolazone, metyrapone, mexiletine&amp;middot;HCl, micafungin, miconazole, midodrine&amp;middot;HCl, miglitol, milnacipran&amp;middot;HCl, mirtazapine, mitotane, moexipril&amp;middot;HCl, mometasone furoate, mupirocin, nadolol, nafcillin&amp;middot;Na, naftifine&amp;middot;HCl, naratriptan&amp;middot;HCl, natamycin, nebivolol&amp;middot;HCl, nelarabine, nepafenac, nevirapine, niacin, nicotine, nilotinib, nilutamide, nitazoxanide, nitisinone, nitrofurantoin, nizatidine, nortriptyline&amp;middot;HCl, olsalazine&amp;middot;Na, orlistat, oxaprozin, oxtriphylline, oxybutynin Chloride, oxytetracycline&amp;middot;HCl, paliperidone, palonosetron&amp;middot;HCl, paromomycin sulfate, pazopanib&amp;middot;HCl, pemetrexed disodium, pemirolast potassium, penicillamine, penicillin G potassium, pentamidine isethionate, pentostatin, perindopril erbumine, permethrin, perphenazine, phenelzine sulfate, phenylephrine, phytonadione, pimecrolimus, pitavastatin calcium, and podofilox</td></tr><tr><td>SmBit&amp;ndash;DDB1 expressing DNA plasmid</td><td>Our laboratory</td><td></td><td>Available upon request</td></tr><tr><td>Trypsin-EDTA</td><td>Sigma Aldrich</td><td>T4049</td><td></td></tr></tbody></table>

identifying inhibitors of the hbx-ddb1 interaction using a split luciferase assay system

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently inhibit viral replication, they have no direct effect on the expression of viral proteins transcribed from a viral covalently closed circular DNA (cccDNA). As high viral antigen load may play a role in this chronic and HBV-related carcinogenesis, the goal of HBV treatment is to eradicate viral proteins. HBV regulatory protein X (HBx) binds to the host DNA damage-binding protein 1 (DDB1) protein to degrade structural maintenance of chromosomes 5/6 (Smc5/6), resulting in activation of viral transcription from cccDNA. Here, using a split luciferase complementation assay system, we present a comprehensive compound screening system to identify inhibitors of the HBx-DDB1 interaction. Our protocol enables easy detection of interaction dynamics in real time within living cells. This technique may become a key assay to discover novel therapeutic agents for treatment of HBV infection.

Representative outcomes following the use of this protocol are shown in Figure 2A,B. The signal-to-background ratio was greater than 80 and the Z&#39; factor9 (the gold standard quality index for high-throughput screening) was greater than 0.5, indicating that this assay system was acceptable for high-throughput screening. With the threshold set to &#62;40% inhibition compared with the control (DMSO only), we identified nitazoxanide as a candidate drug7. Using this system, better candidate drugs can be found by screening other, larger compound libraries.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60652/60652fig1.jpg" /> 
Figure 1: Schematic representation of split luciferase analysis of the HBx-DDB1 interaction. (A) The principle underlying detection of HBx-DDB1 binding using the split luciferase complementary assay system. The separated luciferase subunits, LgBit and SmBit, are fused to HBx and DDB1, respectively. The HBx-DDB1 interaction brings the subunits into close proximity to form a functional enzyme that generates a luminescent signal. The interaction between the subunits is reversible. (B) Split luciferase assay. After co-transfection of plasmids for expression of HBx fused to LgBit and DDB1 fused to SmBit, cells were re-seeded into a 96 well plate. The addition of luminescent substrate enables measurement of luciferase activity without a cell lysis step. Luciferase activities can be measured after adding the screening compounds. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60652/60652fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60652/60652fig2.jpg" /> 
Figure 2: Successful results of the split luciferase assay. (A) Representative baseline luminescent signals from a 96 well plate. Luciferase intensity is represented by numbers and colors. Columns 1 and 12 are controls in which the luminescent substrate was not added. The Z&#39; factor was greater than 0.5. (B) Representative time-series result of relative luciferase activity levels after addition of screening compounds to a 96 well plate. The x-axis represents the inhibitory effects calculated compared to control (DMSO) after standardization to the baseline luciferase activity. The most effective compound was nitazoxanide. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60652/60652fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System at JoVE.com

Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

There is an urgent need for novel therapeutic agents for hepatitis B virus (HBV) infection. Although currently available nucleos(t)ide analogs potently ...

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6.6K Views.  The University of Tokyo. Here, we present a method for screening anti-hepatitis B viral agents that inhibit the HBx-DDB1 interaction using a split luciferase assay system. This system allows easy detection of protein-protein interactions and is suitable for identifying inhibitors of such interactions.

Video: Identifying Inhibitors of the HBx-DDB1 Interaction Using a Split Luciferase Assay System

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

Characterize Disease-related Mutants of RAF Family Kinases by Using a Set of Practical and Feasible Methods

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental disorders. A characterization of disease-related RAF mutants will help us select appropriate therapeutic strategies for treating these diseases. Recent studies have shown that RAF family kinases have both catalytic and allosteric activities, which are tightly regulated by dimerization. Here, we constructed a set of practical and feasible methods to determine the catalytic and allosteric activities and the relative dimer affinity/stability of RAF family kinases and their mutants. Firstly, we amended the classical in vitro kinase assay by reducing the detergent concentration in buffers, utilizing a gentle quick wash procedure, and employing a glutathione S-transferase (GST) fusion to prevent RAF dimers from dissociating during purification. This enables us to measure the catalytic activity of constitutively active RAF mutants appropriately. Secondly, we developed a novel RAF co-activation assay to evaluate the allosteric activity of kinase-dead RAF mutants by using N-terminal truncated RAF proteins, eliminating the requirement of active Ras in current protocols and thereby achieving a higher sensitivity. Lastly, we generated a unique complementary split luciferase assay to quantitatively measure the relative dimer affinity/stability of various RAF mutants, which is more reliable and sensitive compared to the traditional co-immunoprecipitation assay. In summary, these methods have the following advantages: (1) user-friendly; (2) able to carry out effectively without advanced equipment; (3) cost-effective; (4) highly sensitive and reproducible.

In this article, we presented a set of practical and feasible methods for characterizing disease-related mutants of RAF family kinases, which include in vitro kinase assay, RAF co-activation assay, and complementary split luciferase assay.

The rapidly accelerated fibrosarcoma (RAF) family kinases play a central role in cell biology and their dysfunction leads to cancers and developmental ...

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  ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Immunology and Infection

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 ...

Biochemistry

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Biology

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 ...

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has been used to study protein turnover in many cellular contexts, including the cell cycle and signal transduction pathways1-3. Herein, a method is described for isolating Xenopus egg extract that has been optimized to promote the degradation of the critical Wnt pathway component, &beta;-catenin. Two different methods are described to assess &beta;-catenin protein degradation in Xenopus egg extract. One method is visually informative ([35S]-radiolabeled proteins), while the other is more readily scaled for high-throughput assays (firefly luciferase-tagged fusion proteins). The techniques described can be used to, but are not limited to, assess &beta;-catenin protein turnover and identify molecular components contributing to its turnover. Additionally, the ability to purify large volumes of homogenous Xenopus egg extract combined with the quantitative and facile readout of luciferase-tagged proteins allows this system to be easily adapted for high-throughput screening for modulators of &beta;-catenin degradation.

Reconstitution Of &#946;-catenin Degradation In Xenopus Egg Extract

A method is described for analyzing protein degradation using radiolabeled and luciferase-fusion proteins in Xenopus egg extract and its adaptation for high-throughput screening for small molecule modulators of protein degradation.

Xenopus laevis egg extract is a well-characterized, robust system for studying the biochemistry of diverse cellular processes. Xenopus egg extract has ...

A Rapid In Vivo Bioassay for Developmentally Active Enhancers

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin immunoprecipitation followed by next generation sequencing (TF ChIP-seq). TF ChIP-seq generates a large data set (TF-regulatome) that includes PRSs and background interactions. These approaches require secondary low throughput validation in model systems that replicate the temporal and spatial specificity of the PRS. 
In this report, a protocol is described to rapidly validate activity of PRSs in their developmental context. Following selection of candidate PRSs, the associated sequences are isolated, cloned into a GFP reporter construct, and then transfected into chick embryos. For PRSs that are anticipated to be functional during early development, from gastrulation through early organogenesis, whole embryo electroporation (EP) is recommended. With this technique transfection, incubation and monitoring occurs ex ovo during early gastrulation. The entire embryo is transfected and as structures form, they can be monitored live for PRS activity. This ex ovo approach supports development up to early limb bud outgrowth. 
If the functional activity of a PRS is expected to be later in development or if the investigation is to ascertain its activity during the development of a specific organ, targeted regional electroporation (TREP) is used. This approach requires injection of construct DNA directly into the developing organ, precursor or adjacent cavity prior to electroporation. An approach to transfect the presumptive limb bud using TREP is also outlined and illustrated. TREP supports a rapid organ-specific validation of a PRS' activity. Both techniques can also be used to investigate regulatory domains by site-directed mutagenesis of the PRS-reporter construct. 
The chick as a bioassay offers a rapid effective tool for PRS validation, functional domain determination, and further characterization of the regulatory events responsible for development.

With the increased use of transcription factor-specific ChIP-seq technology, techniques to validate potential regulatory regions are critical. The authors describe a rapid chick bioassay to validate the activity of regulatory sequences functioning during development.

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin ...

Suppressing Hepatitis B Virus Replication Using Viral Antigen-Specific T Cells

Source: Xiong, X., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/60043">Stem Cell-Derived Viral Ag-Specific T Lymphocytes Suppress HBV Replication in Mice.</a> J. Vis. Exp. (2019).
The video demonstrates an assay using human induced pluripotent stem cell (iPSC)-derived CD8+ T cells to inhibit hepatitis B virus (HBV) replication in mice. These T cells, engineered with HBV-specific receptors, are transferred into transgenic mice. Replication-competent HBV genome plasmids are then injected directly into the liver, enabling viral DNA uptake. Once viral antigens are expressed and presented by hybrid MHC-I molecules, the engineered T cells recognize and bind to these complexes. This triggers the release of cytotoxic molecules, resulting in the destruction of HBV-infected liver cells and suppression of viral replication.

Hamowanie replikacji wirusa zapalenia wątroby typu B za pomocą limfocytów T specyficznych dla antygenu wirusowego

All procedures involving animal models have been reviewed by the local
institutional animal care committee and the JoVE veterinary review board. All ...