Name: identifying caspases and their motifs that cleave proteins during influenza a virus infection
Uploaded: 2022-07-21T15:00:00.000+00:00
Duration: 8 min 16 s
Description: Watch this Scientific Journal Video about Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection at JoVE.com

The author acknowledges Jennifer Tipper, Bilan Li, Jesse vanWestrienen, Kevin Harrod, Da-Yuan Chen, Farjana Ahmed, Sonya Mros, Kenneth Yamada, Richard Webby, the BEI Resources (NIAID), the Health Research Council of New Zealand, the Maurice and Phyllis Paykel Trust (New Zealand), the H.S. and J.C. Anderson Trust (Dunedin), and the Department of Microbiology and Immunology and School of Biomedical Sciences (University of Otago).

Regulatory approvals were obtained from the University of Otago Institutional Biological Safety Committee to work with the IAV and mammalian cells. Madin-Darby Canine Kidney (MDCK) or human lung alveolar epithelial A549 cells and IAV H1N1 subtypes were used for the present study. IAV was grown in chicken eggs, as described elsewhere17. Sterile and aseptic conditions were used to work with mammalian cells, and a Biosafety Level 2 (or Physical Containment 2) facility and Class II biosafety cabinet were used to work with IAV subtypes.
1. Assessing the cleavage or degradation of proteins in IAV-infected cells by caspases
<ol>
	<li>Seed 3 x 105 MDCK or A549 cells per well in a 12-well cell culture plate. 
	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-and 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 (see references7,8).</li>
	<li>Incubate the cells at 37 &#176;C under a 5% CO2 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 Table of Materials) at a multiplicity of infection (MOI) of 0.5-3.0 plaque-forming units (pfu)/cell7,11 (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% CO2 atmosphere.</li>
	<li>In the meantime, dilute a caspase inhibitor (e.g., Z-DEVD-FMK), a lysosome Inhibitor (e.g., NH4Cl), or a proteasome inhibitor (e.g., MG132) (see Table of Materials) at a final concentration of 40 &#181;M, 20 mM, or 10 &#181;M, respectively, in serum-free MEM7 (see 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 g for 1-2 min at room temperature. Collect the supernatant using a pipette. 
	NOTE: This supernatant could be discarded or used for a plaque assay8 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-HCl, pH 7.4, 150 mM NaCl, 0.5% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1% Triton X-100, and 1x protease inhibitor cocktail, see 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 BCA assay kit (see Table of Materials).</li>
	<li>Resolve equal amounts of protein from uninfected and infected samples by standard SDS-polyacrylamide gel electrophoresis (SDS-PAGE)11 along with molecular weight markers11.</li>
	<li>Transfer the protein to a nitrocellulose or polyvinylidene difluoride (PVDF) membrane (see 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 elsewhere11.</li>
	<li>Compare the protein levels in the mock-treated and inhibitor-treated infected sample lanes. 
	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 Table of Materials) to image the western blots and quantify the protein recovery.</li>
</ol>
2. Confirmation of caspase-mediated cleavage or degradation of proteins in IAV-infected cells by RNA interference
<ol>
	<li>Design or obtain a pre-design small-interfering RNA (siRNA) targeting either canine or human caspase 3, caspase 6, and caspase 7 (executioner caspases1) and a non-targeting control siRNA (see Table of Materials).</li>
	<li>Deliver each siRNA to MDCK or A549 cells by reverse transfection8,11.</li>
	<li>For this, dilute in separate tubes 100 nM of control siRNA or caspase siRNA or a recommended volume of transfection reagent (see Table of Materials) in 100 &#181;L of appropriate medium (like OptiMEM, see Table of Materials), and incubate for 5 min at room temperature.</li>
	<li>Prepare each dilution in duplicate. 
	NOTE: This duplicate includes one replicate for analyzing the knockdown of caspase expression by RT-qPCR or western blotting (if antibodies to caspases are available) and another for assessing the recovery of the protein of interest by western blotting.</li>
	<li>Mix 100 &#181;L of each siRNA solution with 100 &#181;L of transfection reagent solution (step 2.3) and incubate for 20-45 min at room temperature to form the siRNA-transfection reagent complex.</li>
	<li>In the meantime, split and add 1 x 105 MDCK or A549 cells in 800 &#181;L of the complete growth medium, mix with 200 &#181;L of siRNA-transfection reagent complex (step 2.5), and add 1 mL of the suspension to a well of a 12-well culture plate. This dilution brings the final concentration of control siRNA or caspase siRNA to 10 nM.</li>
	<li>Incubate the cells at 37 &#176;C under a 5% CO2 atmosphere for 72 h.</li>
	<li>Harvest the cells from one replicate as in step 1.10, and process them to validate or confirm the knockdown of the expression of caspase 3, caspase 6, and caspase 7 either by RT-qPCR or western blotting, respectively, using standard methods and protocols8,11.</li>
	<li>Infect the cells in the other replicate with IAV as described in steps 1.4-1.9 (without inhibitors).</li>
	<li>After 24 h, harvest, process, and analyze the cells by western blotting and measure and quantify the protein recovery as described in steps 1.10-1.20.</li>
</ol>
3. Site-directed mutagenesis for locating the caspase cleavage site(s) in the polypeptide
<ol>
	<li>Clone the gene-encoding protein of interest in a mammalian expression plasmid11 or obtain it from a research lab or commercial source (see Table of Materials). 
	NOTE: It is preferred if the gene is cloned with an epitope tag or as GFP fusion to distinguish the ectopically expressed polypeptide from the endogenously expressed one during western blotting.</li>
	<li>Using caspase substrate databases4 or protein alignment tools, locate the consensus caspase cleavage motifs, XXXD and DXXD, on the polypeptide. Almost all caspase cleavage motifs possess the aspartic acid at the cleavage site (P1)3,4.</li>
	<li>Mutate the putative P1 aspartic acid to glutamic acid or a non-polar amino acid (alanine, valine) by site-directed mutagenesis followed by DNA sequencing11 using standard methods and protocols.</li>
	<li>Deliver the wild-type (WT) and mutant plasmid DNA to MDCK or A549 cells by reverse transfection11.
	<ol>
		<li>For this, dilute 2 &#181;g of plasmid DNA or a recommended volume of the transfection reagent in 100 &#181;L of an appropriate medium (see step 2.3 and Table of Materials) in separate tubes, and incubate for 5 min at room temperature.</li>
		<li>Mix 100 &#181;L of each plasmid DNA solution with 100 &#181;L of the transfection reagent solution and incubate for 20-45 min at room temperature to form the DNA-transfection reagent complex.</li>
	</ol>
	</li>
	<li>In the meantime, split and add 3 x 105 MDCK or A549 cells to 800 &#181;L of the complete growth medium, mix with 200 &#181;L of DNA-transfection reagent complex, and add the 1 mL suspension to a well of a 12-well culture plate.</li>
	<li>Incubate the cells at 37 &#176;C under a 5% CO2 atmosphere.</li>
	<li>After 48 h, infect the cells with IAV as described in steps 1.4-1.9 (without adding the inhibitors).</li>
	<li>After 24 h, harvest, process, and analyze the cells by western blotting (if applicable, using the antibody to an epitope tag or GFP). Then, measure and quantify the protein recovery as described in steps 1.10-1.20.</li>
</ol>

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The author has no conflicts of interest to disclose.

It is established that viruses tailor the host factors and pathways to their benefit. In turn, the host cells resist that by employing various strategies. One of those strategies is PANoptosis, which host cells use as an antiviral strategy against virus infections. However, viruses like IAV have evolved their own strategies to counter PANoptosis and exploit it to their advantage1,3,6. This interplay involves the cleavage of vari...

Influenza A virus (IAV) is the prototypic member of the Orthomyxoviridae family and is known to cause global epidemics and unpredictable pandemics. IAV causes human respiratory disease, influenza, commonly known as &#34;flu&#34;. The flu is an acute disease that results in the induction of host pro- and anti-inflammatory innate immune responses and the death of epithelial cells in the human respiratory tract. Both processes are governed by a phenomenon called programmed cell death1. The signaling for programmed cell death is induced as soon as various pathogen recognition receptors sense the incoming virus particles in host cells. This leads to the programming of the death of infected cells and signaling to the neighboring healthy cells by three interconnected pathways called pyroptosis, apoptosis, and necroptosis-recently coined as one process, PANoptosis1.
PANoptosis involves the proteolytic processing of many host and viral proteins from induction to execution. Such processing of proteins is primarily spearheaded by a family of cysteine proteases called caspases1,2. Up to 18 caspases (from caspase 1 to caspase 18) are known3. Most caspases are expressed as pro-caspases and activated by undergoing their own proteolytic processing either by autocatalysis or other caspases4 in response to a stimulus like a virus infection. The PANoptosis of IAV-infected cells was thought to be a host defense mechanism, but IAV has evolved ways to evade and exploit it to facilitate its replication1,2,5,6. One of them is to antagonize the host factors via caspase-mediated cleavage or degradation that are either inherently antiviral or interfere with one of the steps of the IAV life cycle. To this end, host factors, cortactin, HDAC4, and HDAC6 have been discovered to undergo caspase-mediated cleavage or degradation in IAV-infected epithelial cells7,8,9. The HDAC4 and HDAC6 are anti-IAV factors8,10, and cortactin interferes with IAV replication at a later stage of infection, potentially during viral assembly and budding11.
In addition, various caspases are also activated, which, in turn, cleave multiple proteins to activate the host inflammatory response during IAV infection1,2. Furthermore, nucleoprotein (NP), ion-channel M2 protein of IAV12,13,14, and various proteins of other viruses3,15,16 also undergo caspase-mediated cleavage during infection, which influences viral pathogenesis. Therefore, there is a continuous need to study caspase-mediated cleavage or degradation of host and viral proteins during IAV and other virus infections to understand the molecular basis of viral pathogenesis. Herein, the methods are presented to (1) assess the cleavage or degradation of such proteins by caspases, (2) identify those caspases, and (3) locate the cleavage sites.

<table><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 &#039;InSolution Caspase-3 Inhibitor II - Calbiochem&#039;</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&amp;rsquo;s Research Hospital, Memphis, USA) to MH</td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>ThermoFisher Scientific</td><td>31985062</td><td></td></tr><tr><td>Lipofectamine RNAiMAX Transfection Reagent</td><td>ThermoFisher Scientific</td><td>13778150</td><td></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>MISSION siRNA Universal Negative Control #1</td><td>Sigma-Aldrich</td><td>SIC001</td><td></td></tr><tr><td>Odyssey Fc imager with Image Studio Lite software 5.2&amp;nbsp;</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>Plasmid expressing human cortactin-GFP fusion&amp;nbsp;</td><td>Addgene</td><td>50728</td><td>Gift from Kenneth Yamada to Addgene</td></tr><tr><td>Pre-designed small interferring RNA (siRNA) to caspase 3</td><td>Sigma-Aldrich</td><td>NM_004346</td><td>siRNA ID: SASI_Hs01_00139105</td></tr><tr><td>Pre-designed small interferring RNA to caspase 6</td><td>Sigma-Aldrich</td><td>NM_001226</td><td>siRNA ID: SASI_Hs01_00019062</td></tr><tr><td>Pre-designed small interferring RNA to caspase 7</td><td>Sigma-Aldrich</td><td>NM_001227</td><td>siRNA ID: SASI_Hs01_00128361</td></tr><tr><td>Pre-designed SYBR Green RT-qPCR Primer pairs</td><td>Sigma-Aldrich</td><td>KSPQ12012</td><td>Primer Pair IDs: H_CASP3_1; H_CASP6_1; H_CASP7_1</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>Rabbit anti-GFP antibody</td><td>Takara</td><td>632592</td><td></td></tr><tr><td>SeeBlue Pre-stained Protein Standard</td><td>ThermoFisher Scientific</td><td>LC5625</td><td></td></tr><tr><td>Transfection medium, Opti-MEM</td><td>ThermoFisher Scientific</td><td>11668019</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>

identifying caspases and their motifs that cleave proteins during influenza a virus infection

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially described to occur by apoptosis, programmed cell death is now known to encompass three interconnected pathways: pyroptosis, apoptosis, and necroptosis, together coined as one process, PANoptosis. Influence A virus (IAV) infection induces PANoptosis in mammalian cells by inducing the activation of different caspases, which, in turn, cleave various host as well as viral proteins, leading to processes like the activation of the host innate antiviral response or the degradation of antagonistic host proteins. In this regard, caspase 3-mediated cleavage of host cortactin, histone deacetylase 4 (HDAC4), and histone deacetylase 6 (HDAC6) has been discovered in both animal and human epithelial cells in response to the IAV infection. To demonstrate this, inhibitors, RNA interference, and site-directed mutagenesis were employed, and, subsequently, the cleavage or resistance to cleavage and the recovery of cortactin, HDAC4, and HDAC6 polypeptides were measured by western blotting. These methods, in conjunction with RT-qPCR, form a simple yet effective strategy to identify the host as well as viral proteins undergoing caspase-mediated cleavage during an infection of IAV or other human and animal viruses. The present protocol elaborates the representative results of this strategy, and the ways to make it more effective are also discussed.

Treatment with caspase 3 inhibitor 
It has been discovered that host cortactin, HDAC4, and HDAC6 polypeptides undergo degradation in response to IAV infection in both canine (MDCK) and human (A549, NHBE) cells7,8,9. By using the above approaches, it was uncovered that IAV-induced host caspases, particularly caspase 3, cause their degradation7,8...

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Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Influenza A virus (IAV) infection activates the caspases that cleave host and viral proteins, which, in turn, have pro- and antiviral functions. By employing inhibitors, RNA interference, site-directed mutagenesis, and western blotting and RT-qPCR techniques, caspases in infected mammalian cells that cleave host cortactin and histone deacetylases were identified.

Caspases, a family of cysteine proteases, orchestrate programmed cell death in response to various stimuli, including microbial infections. Initially ...

identifying-caspases-their-motifs-that-cleave-proteins-during

Research

JoVE Journal

Immunology and Infection

1.4K Views.  University of Otago. Deze methode zal helpen om te bepalen en te bevestigen of een eiwit in met influenzavirus geïnfecteerde cellen afbraak of splitsing door caspasen ondergaat en de caspase-splitsingsplaatsen te identificeren. Deze methode vereist alleen elementaire moleculaire en cellulaire biologie laboratoriuminstellingen en onderzoeksvaardigheden en vereist geen gespecialiseerde apparatuur of softwaretraining. Deze methode kan worden aangepast om vergelijkbare doelstellingen te bereiken met betrekking tot i...

Video: Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first to be activated in these pathways. This group includes caspase-2, -8, and -9, as well as the inflammatory caspases, caspase-1, -4, and -5. The initiator caspases are all activated by dimerization following recruitment to specific multiprotein complexes called activation platforms. Caspase Bimolecular Fluorescence Complementation (BiFC) is an imaging-based approach where split fluorescent proteins fused to initiator caspases are used to visualize the recruitment of initiator caspases to their activation platforms and the resulting induced proximity. This fluorescence provides a readout of one of the earliest steps required for initiator caspase activation. Using a number of different microscopy-based approaches, this technique can provide quantitative data on the efficiency of caspase activation on a population level as well as the kinetics of caspase activation and the size and number of caspase activating complexes on a per cell basis.

This protocol describes caspase Bimolecular Fluorescence Complementation (BiFC); an imaging-based method that can be used to visualize induced proximity of initiator caspases, which is the first step in their activation.

The caspase family of proteases play essential roles in apoptosis and innate immunity. Among these, a subgroup known as initiator caspases are the first ...

Biochemistry

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Analysis of Group IV Viral SSHHPS Using In Vitro and In Silico Methods

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce active enzymes essential for viral replication. Viral proteases are highly specific and recognize conserved cleavage site motif sequences (~6-8 amino acids). In several Group IV viruses, the nsP protease(s) cleavage site motif sequences can be found in specific host proteins involved in generating the innate immune responses and, in some cases, the targeted proteins appear to be linked to the virus-induced phenotype. These viruses utilize short stretches of homologous host-pathogen protein sequences (SSHHPS) for targeted destruction of host proteins. To identify SSHHPS the viral protease cleavage site motif sequences can be inputted into BLAST and the host genome(s) can be searched. Cleavage initially can be tested using the purified nsP viral protease and fluorescence resonance energy transfer (FRET) substrates made in E. coli. The FRET substrates contain cyan and yellow fluorescent protein and the cleavage site sequence (CFP-sequence-YFP). This protease assay can be used continuously in a plate reader or discontinuously in SDS-PAGE gels. Models of the bound peptide substrates can be generated in silico to guide substrate selection and mutagenesis studies. CFP/YFP substrates have also been utilized to identify protease inhibitors. These in vitro and in silico methods can be used in combination with cell-based assays to determine if the targeted host protein affects viral replication.

We present a general protocol for identifying short stretches of homologous host-pathogen protein sequences (SSHHPS) embedded in the viral polyprotein. SSHHPS are recognized by viral proteases and direct the targeted destruction of specific host proteins by several Group IV viruses.

Alphaviral enzymes are synthesized in a single polypeptide. The nonstructural polyprotein (nsP) is processed by its nsP2 cysteine protease to produce ...

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening

Here we present a protocol to express and purify recombinant Drosophila caspases Dronc and Drice, and their use in in vitro cleavage assays.

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis ...

Bioengineering

Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct regulation of inflammatory signaling and is activated by proximity-induced dimerization following recruitment to inflammasomes. Caspase-1 is abundant in the monocytic cell lineage and induces maturation of the pro-inflammatory cytokines interleukin (IL)-1&#946; and IL-18 to active secreted molecules. The other inflammatory caspases, caspase-4 and -5 (and their murine homolog caspase-11) promote IL-1&#946; release by inducing pyroptosis. Caspase Bimolecular Fluorescence Complementation (BiFC) is a tool used to measure inflammatory caspase induced proximity as a readout of caspase activation. The caspase-1, -4, or -5 prodomain, which contains the region that binds to the inflammasome, is fused to non-fluorescent fragments of the yellow fluorescent protein Venus (Venus-N [VN] or Venus-C [VC]) that associate to reform the fluorescent Venus complex when the caspases undergo induced proximity. This protocol describes how to introduce these reporters into primary human monocyte-derived macrophages (MDM) using nucleofection, treat the cells to induce inflammatory caspase activation, and measure caspase activation using fluorescence and confocal microscopy. The advantage of this approach is that it can be used to identify the components, requirements, and localization of the inflammatory caspase activation complex in living cells. However, careful controls need to be considered to avoid compromising cell viability and behavior. This technique is a powerful tool for the analysis of dynamic caspase interactions at the inflammasome level as well as for the interrogation of the inflammatory signaling cascades in living MDM and monocytes derived from human blood samples.

This protocol describes the workflow to obtain monocytes-derived macrophages (MDM) from human blood samples, a simple method to efficiently introduce inflammatory caspase Bimolecular Fluorescence Complementation (BiFC) reporters into human MDM without compromising cell viability and behavior, and an imaging-based approach to measure inflammatory caspase activation in living cells.

Inflammatory caspases include caspase-1, -4, -5, -11, and -12 and belong to the subgroup of initiator caspases. Caspase-1 is required to ensure correct ...

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.

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

Assessment of the Activation of Multiple Caspases in Macrophages

Source: Han, J. et al., <a href="https://app.jove.com/v/64308">Evaluation of Caspase Activation to Assess Innate Immune Cell Death.</a>&#160;J. Vis. Exp.&#160;(2023)
This video demonstrates the assessment of multiple caspase activations in bone marrow-derived macrophages through stimulation with the influenza virus. Following macrophage lysis, heat-inactivation of lysate, and centrifugation, intracellular caspases are collected for subsequent immunological analysis.

1. Preparing the solutions

	Prepare L929-conditioned media.
	
		Plate 1 &#215; 106&#160;L929 cells (see&#160;Table of Materials) in an 182 ...

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Cellular Immunodiagnostics

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

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 ...

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

Caspases

Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside cells.
Caspase Activation
Caspases are produced as inactive zymogens, called procaspases. The procaspases contain four loops, L1 to L4. During activation, L2 is cleaved, and the monomeric subunits dimerize. The initiator procaspases also have long pro-domains such as the death effector domain (DED) or caspase recruitment domain (CARD). These pro-domains interact with the adaptor molecules to cleave and activate the initiator caspases. Conversely, executioner caspases have short pro-domains and are cleaved by the active initiator caspases.
Caspases and Homeostasis
Cell proliferation and cell death are both essential to maintain homeostasis in multicellular organisms. The over- and under-activation of caspases, thus, can lead to many diseases. For example, decreased caspase activity is a common feature of cancerous cells. Many infectious diseases also decrease caspase activity. For example, the p35 protein released by certain viruses binds the caspases, thus preventing caspase activation and blocking apoptosis. In contrast, the over-activation of inflammatory caspases causes sepsis due to increased inflammatory response. Excessive activation of caspases can also cause diseases such as Alzheimer&#39;s, Parkinson&#8217;s, and Huntington&#39;s.

Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. ...

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

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