assessing caspase-mediated cleavage of viral and host proteins in virus-infected cells

Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells

To begin, seed MDCK or A549 cells in a 12-well cell culture plate, incubate the cells overnight at 37 degrees Celsius under a 5% carbon dioxide atmosphere. On the next day, remove the old culture medium from the cells and wash the cells in each well twice with 1 milliliter of serum-free MEM.
Then infect the cells by adding 400 microliters of influenza A virus inoculum and place the plate for incubation for one hour. After incubation, remove the virus inoculum and wash the cells with MEM. Add 1 milliliter of serum-free MEM to each well, and incubate the cells as shown in the previous step.
After 24 hours, harvest the cells by scraping them with a 1-milliliter syringe plunger, and transfer them to a 1.5-milliliter tube. Centrifuge the tube at 12,000 g for two minutes at room temperature, and collect the supernatant. Wash the cell pellet with 250 microliters of phosphate-buffered saline and centrifuge. Remove the supernatant and add 100 microliters of cell lysis buffer. Mix by vortex.
Heat the tube at 98 degrees Celsius for 10 minutes to completely lyse the cells. Then, resolve equal amounts of protein from uninfected and infected samples by standard SDS polyacrylamide gel electrophoresis. After electrophoresis, transfer the protein gel to a nitrocellulose or PVDF membrane and perform western blotting. Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes.

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1. Assessing the cleavage or degradation of proteins in Influenza A virus, IAV-infected cells by caspases
<ol>
	<li>Seed 3 x 105&#160;Madin-Darby Canine Kidney (MDCK) or A549 cells per well in a 12-well cell culture plate.&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: If using MDCK cells, ensure that the antibody against the protein of interest recognizes its canine species.</li>
	<li>Seed the wells in pairs, i.e., a control pair-one well for the uninfected-mock sample and one for the infected-mock sample, a test pair-one well for uninfected-inhibitor A sample and one for the infected-inhibitor A sample. Increase the number of pairs with each additional inhibitor.</li>
	<li>Incubate the cells at 37 &#176;C under a 5% CO2&#160;atmosphere overnight.</li>
	<li>The next day, infect the cells with IAV (an H1N1 subtype or another subtype).
	<ol>
		<li>For this, prepare the virus inoculum by diluting virus stock in 400 &#181;L of serum-free minimum essential medium (MEM) (see&#160;Table of Materials) at a multiplicity of infection (MOI) of 0.5-3.0 plaque-forming units (pfu)/cell&#160;(a factor of the doubling of cell number when calculating the MOI). 
		NOTE: For infecting MDCK cells, supplement the virus inoculum with tosyl phenylalanyl chloromethyl ketone (TPCK)-trypsin at a final concentration of 1 &#181;g/mL.</li>
	</ol>
	</li>
	<li>Remove the old culture medium from the cells (step 1.3) and wash the cells with 1 mL/well of serum-free MEM 2x.</li>
	<li>Add 400 &#181;L of virus inoculum (step 1.4.1) to the cells and incubate them for 1 h at 35 &#176;C under a 5% carbon dioxide (CO2)&#160;atmosphere.</li>
	<li>In the meantime, dilute a caspase inhibitor (e.g., Z-DEVD-FMK), a lysosome Inhibitor (e.g., ammonium chloride, NH4Cl), or a proteasome inhibitor (e.g., MG132) (see&#160;Table of Materials) at a final concentration of 40 &#181;M, 20 mM, or 10 &#181;M, respectively, in serum-free MEM&#160;(see&#160;Table of Materials). The latter two inhibitors serve as a control. Dilute an equal volume of the solvent (if other than water) used to reconstitute one of the inhibitors in serum-free medium as a mock.</li>
	<li>Remove the virus inoculum and wash the cells as in step 1.5 (1x).</li>
	<li>Add the serum-free MEM, 1 mL/well, mock or supplemented with an inhibitor, to the cells and incubate the cells as in step 1.6. This time point is considered as 0 h infection.</li>
	<li>After 24 h, harvest the cells by scraping them with a 1 mL syringe plunger (rubber side) and transfer them into a 1.5 mL polypropylene tube.</li>
	<li>Centrifuge the tube at 12,000 x&#160;g&#160;for 1-2 min at room temperature. Collect the supernatant using a pipette.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: This supernatant could be discarded or used for a plaque assay&#160;to measure the titer of the released virus progeny.</li>
	<li>Wash the cell pellet with 250 &#181;L of phosphate-buffered saline (PBS) by re-centrifugation as in step 1.11.</li>
	<li>Remove the supernatant and lyse the cells by adding 80-100 &#181;L of cell lysis buffer (50 mM Tris (hydroxymethyl) aminomethane (THAM) hydrochloride (Tris-HCl), pH 7.4, 150 mM sodium chloride (NaCl), 0.5% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, and 1x protease inhibitor cocktail, see&#160;Table of Materials) and vortexing.</li>
	<li>Heat the tube at 98 &#176;C for 10 min to completely lyse and prepare the total cell lysate. Store the lysates at 4 &#176;C for performing steps 1.15-1.17 the next day, but, for best results, finish these steps on the same day.</li>
	<li>Estimate the protein amount in each sample using a bicinchoninic acid (BCA) tableassay kit (see&#160;Table of Materials).</li>
	<li>Resolve equal amounts of protein from uninfected and infected samples by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE)&#160;along with molecular weight markers.</li>
	<li>Transfer the protein to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane (see&#160;Table of Materials). 
	NOTE: PVDF membrane may give a high background in some western blot imagers; check for compatibility.</li>
	<li>Perform western blotting to detect the protein of interest using the method described elsewhere.</li>
	<li>Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes.&#160;&#160;&#160; 
	NOTE: If the protein level has recovered in the caspase inhibitor-treated infected sample lane, then the protein is cleaved or degraded by caspases. Otherwise, it is degraded by either lysosome or proteasome.</li>
	<li>Quantify the protein recovery by measuring its band intensity in each lane from at least three replicates of the same experiment and normalizing it with the corresponding loading control band.</li>
	<li>Use any contemporary imager and associated software (see&#160;Table of Materials) to image the western blots and quantify the protein recovery.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A549 cells</td>
			<td>ATCC</td>
			<td>CRM-CCL-185</td>
			<td>Human, epithelial, lung</td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>A9434</td>
			<td></td>
		</tr>
		<tr>
			<td>Caspase 3 Inhibitor</td>
			<td>Sigma-Aldrich</td>
			<td>264156-M</td>
			<td>Also known as &#39;InSolution Caspase-3 Inhibitor II - Calbiochem&#39;</td>
		</tr>
		<tr>
			<td>Complete, Mini Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836153001</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-NP antibody</td>
			<td></td>
			<td></td>
			<td>Gift from Richard Webby (St Jude Children&#39;s Research Hospital, Memphis, USA) to MH</td>
		</tr>
		<tr>
			<td>MDCK cells</td>
			<td>ATCC</td>
			<td>CCL-34</td>
			<td>Dog, epithelial, kidney</td>
		</tr>
		<tr>
			<td>MG132</td>
			<td>Sigma-Aldrich</td>
			<td>M7449</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11095080</td>
			<td>Add L-glutamine, antibiotics or other supplements as required</td>
		</tr>
		<tr>
			<td>Odyssey Fc imager with Image Studio Lite software 5.2&#160;</td>
			<td>LI-COR</td>
			<td></td>
			<td>Odyssey Fc has been replaced with Odyssey XF and Image Studio Lite software has been replaced with Empiria Studio software.</td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Protran Premium nitrocellulose membrane</td>
			<td>Cytiva (Fomerly GE Healthcare)</td>
			<td>10600003</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-actin antibody</td>
			<td>Abcam</td>
			<td>ab8227</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-cortactin antibody</td>
			<td>Cell Signaling</td>
			<td>3502</td>
			<td></td>
		</tr>
		<tr>
			<td>SeeBlue Pre-stained Protein Standard</td>
			<td>ThermoFisher Scientific</td>
			<td>LC5625</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl, NaCl, SDS, Sodium Deoxycholate, Triton X-100</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, TPCK-Treated</td>
			<td>Sigma-Aldrich</td>
			<td>4370285</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Husain, M. <a href="https://app.jove.com/v/64189">Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection.</a>&#160;J. Vis. Exp. (2022)
The video demonstrates a method to assess caspase-mediated protein cleavage in virus-infected cells. Post-viral infection, caspases get activated and degrade various host cellular proteins and viral nucleoproteins while specific caspase inhibitors prevent this degradation, as confirmed by western blotting.

1. Assessing the cleavage or degradation of proteins in Influenza A virus, IAV-infected cells by caspases

	Seed 3 x 105&#160;Madin-Darby Canine Kidney ...

Begin with virus-infected mammalian cells that produce multiple caspases, including caspase-3.
Add a growth medium to the control well and a caspase-3 inhibitor-supplemented medium to the test well.
In the control well, caspase-3 recognizes and degrades the viral nucleoproteins and host proteins essential for viral propagation.
In the test well, the inhibitor enters the cell, inhibiting caspase-3 activity and preventing protein degradation.
Dislodge cells from the plate. Add a lysing reagent and heat to induce cell lysis, releasing intracellular proteins.
Load both samples on an SDS-polyacrylamide gel and electrophorese to separate proteins based on size.
After electrophoresis, transfer the gel to a blotting membrane.
Treat the membrane with primary antibodies against viral nucleoproteins and host proteins.
Wash and overlay with fluorophore-conjugated secondary antibodies binding with primary antibodies, generating fluorescent bands.
The higher fluorescence in the test sample indicates reduced caspase-3 mediated viral and host protein cleavage compared to the control.

21 조회수. 바이러스에 감염된 세포에서 바이러스 및 숙주 단백질의 카스파제 매개 절단 평가

비디오: 바이러스에 감염된 세포에서 바이러스 및 숙주 단백질의 카스파제 매개 절단 평가 - 실험

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

형광 분석 또는 유세포분석을 사용한 카스파제 활성 측정

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

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JoVE Journal

생화학

Caspase-3 Activity in the Rat Amygdala Measured by Spectrofluorometry After Myocardial Infarction

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala &#8211; peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 &#176;C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

Myocardial infarction (MI) is not only followed by impaired cardiac function but also by apoptosis in the amygdala, a brain region involved in the behavioral consequences of MI. This protocol describes how to induce MI, collect amygdala tissue and measure caspase-3 activity, a marker of apoptosis, therein.

심근 경색 후 Spectrofluorometry에 의해 측정 된 쥐 편도체에서 카스파 제 3 활동

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still ...

신경과학

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is the initiation of innate immune programmed cell death (PCD) to eliminate infected or damaged cells and propagate immune responses. However, excess PCD is associated with inflammation and pathology. Therefore, understanding the activation and regulation of PCD is a central aspect of characterizing innate immune responses and identifying new therapeutic targets across the disease spectrum.
This protocol provides methods for characterizing innate immune PCD activation by monitoring caspases, a family of cysteine-dependent proteases that are often associated with diverse PCD pathways, including&#160;apoptosis,&#160;pyroptosis, necroptosis, and PANoptosis. Initial reports characterized caspase-2, caspase-8, caspase-9, and caspase-10 as initiator caspases and caspase-3, caspase-6, and caspase-7 as effector caspases in apoptosis, while later studies found the inflammatory caspases, caspase-1, caspase-4, caspase-5, and caspase-11, drive pyroptosis. It is now known that there is extensive crosstalk between the caspases and other innate immune and&#160;cell death molecules across the previously defined PCD pathways, identifying a key knowledge gap in the mechanistic understanding of innate immunity and PCD and&#160;leading to the characterization of PANoptosis. PANoptosis is a unique innate immune inflammatory PCD pathway regulated by PANoptosome complexes, which integrate components, including caspases,&#160;from other cell death pathways.
Here, methods for assessing the activation of caspases in response to various stimuli are provided. These methods allow for the characterization of PCD pathways both in vitro and in vivo, as activated caspases undergo proteolytic cleavage that can be visualized by western blotting using optimal antibodies and blotting conditions. A protocol and western blotting workflow have been established that allow for the assessment of the activation of multiple caspases from the same cellular population, providing a comprehensive characterization of the PCD processes. This method can be applied across research areas in development, homeostasis, infection, inflammation, and cancer to evaluate PCD pathways throughout cellular processes in health and disease.

This protocol describes a comprehensive method for assessing caspase activation (caspase-1, caspase-3, caspase-7, caspase-8, caspase-9, and caspase-11) in response to both in vitro and in vivo (in mice) models of infection, sterile insults, and cancer to determine the initiation of cell death pathways, such as pyroptosis, apoptosis, necroptosis, and PANoptosis.

선천면역세포사멸을 평가하기 위한 카스파아제 활성화 평가

Innate immunity provides the critical first line of defense in response to pathogens and sterile insults. A key mechanistic component of this response is ...

Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells

내레이션 대본

Assessing Caspase-Mediated Cleavage of Viral and Host Proteins in Virus-Infected Cells