This work was supported by a grant from the Russian Science Foundation (17-75-20102, the protocol development). Experiments described in the representative results section (analysis of phosphorylation) were supported by the Stockholm (181301) and Swedish (190345) Cancer Societies.

1. System preparation
NOTE: The molecular models of the native and mutant protein forms are built based on an appropriate crystal structure obtained from the Protein Data Bank15,16.
<ol>
	<li>To retrieve the selected PDB structure, use the Download Files drop-down list and click on PDB Format. Remove remarks and connectivity data, and insert a TER card between separate protein chains in the PDB file. Optionally, set the protonation state of histidine residues by replacing the residue name HIS with HIE, HID, or HIP (Supplementary File 1). 
	NOTE: It is reasonable not to remove crystallographically resolved water molecules (if present in the PDB file).</li>
	<li>To prepare the starting model, launch the tleap program (AmberTools package) and then enter commands that manipulate tleap objects.
	<ol>
		<li>Launch the program: tleap. Load the ff14SB force field to describe the protein with molecular mechanics: &#62; source leaprc.protein.ff14SB.</li>
		<li>Load force fields for water molecules and atomic ions (Na+, Cl-): &#62; source leaprc.water.tip3p.</li>
		<li>Load the PDB file and build coordinates for hydrogens, creating an object named mol: &#62; mol = loadpdb protein.pdb.</li>
		<li>Check for internal inconsistencies that could cause problems: &#62; check mol. 
		NOTE: Disulfide bridges, if present, must be specified manually. Replace the names of covalently bound cysteines CYS with CYX and type the following command in tleap to create a bond between SG atoms: bond mol.X.SG mol.Y.SG (X and Y are cysteine residue numbers).</li>
		<li>Create a solvent box around the protein (TIP3P water, 12 &#197; distance): &#62; solvatebox mol TIP3PBOX 12.0.</li>
		<li>Check the total charge: &#62; charge mol, and add counterions (Na+ or Cl-) to neutralize the system: 
		&#62; addions mol Na+ 0.</li>
		<li>Create the topology prmtop file and the coordinate inpcrd file (inputs for running a simulation): &#62; saveamberparm mol protein.prmtop protein.inpcrd. Quit the tleap program: &#62; quit. 
		&#8203;NOTE: Amber-format coordinate files can be easily converted to PDB format using the ambpdb tool (refer to the Users&#39; Manual, see Table of Materials).</li>
	</ol>
	</li>
</ol>
2. Energy minimization
NOTE: The energy minimization is necessary to remove any bad contacts and overlaps between atoms in the starting system that lead to instability when running MD.
<ol>
	<li>Carry out the first stage of the energy minimization (2,500 steps of the steepest descent algorithm + 2,500 steps of the conjugate gradient algorithm) to optimize the positions of added hydrogen atoms and water molecules while keeping the protein coordinates fixed by positional restraints on heavy atoms.
	<ol>
		<li>Run the pmemd program as follows: 
		pmemd -O -i min1.in -p protein.prmtop -c protein.inpcrd -o protein_min1.out 
		-r protein_min1.rst -ref protein.inpcrd.</li>
		<li>Follow the required arguments: -i FILE control data; -p FILE molecular topology, force field parameters, atom names; -c FILE initial coordinates; -o FILE user-readable log output; -r FILE final coordinates; -ref FILE reference coordinates for position restraints.</li>
		<li>Specify options in the input file min1.in: 
		&#38;cntrl 
		imin=1, maxcyc=5000, ncyc=2500, 
		cut=10.0, ntb=1, 
		ntc=1, ntf=1, 
		ntpr=10, 
		ntr=1, 
		restraintmask=&#39;:1-517 &#38; !@H=&#39;, 
		restraint_wt=2.0 
		/ 
		NOTE: Input options: imin=1 perform minimization; maxcyc=5000 maximum number of minimization cycles; ncyc=2500 minimization method is switched from steepest descent to conjugate gradient after ncyc cycles; cut=10.0 non-bonded cutoff (&#197;); ntb=1 periodic boundaries are imposed, constant volume; ntc=1 no constraints are applied to bond lengths (for better energy convergence); ntf=1 all interactions are calculated; ntpr=10 energy information is printed to the out file every ntpr steps; ntr=1 flag for restraining specified atoms using a harmonic potential; restraintmask=&#39;:1-517 &#38; !@H=&#39; specifies restrained atoms; restraint_wt=2.0 weight (kcal/mol&#8729;&#197;2) for positional restraints.</li>
	</ol>
	</li>
	<li>Carry out the second stage of the energy minimization (5,000 steepest descent steps + 5,000 conjugate gradient steps) without restraints to optimize the whole system.
	<ol>
		<li>Use min2.in and protein_min1.rst as inputs: pmemd -O -i min2.in -p protein.prmtop 
		-c protein_min1.rst -o protein_min2.out -r protein_min2.rst.</li>
		<li>Specify options in the input file min2.in: 
		&#38;cntrl 
		imin=1, maxcyc=10000, ncyc=5000, 
		cut=10.0, ntb=1, 
		ntc=1, ntf=1, 
		ntpr=10 
		/</li>
	</ol>
	</li>
</ol>
3. Heating
NOTE: This stage aims to heat the system from 0 K to 300 K. Initial velocities are assigned to atoms since the starting model based on the PDB file does not contain velocity information.
<ol>
	<li>Carry out the heating process with positional restraints on the protein atoms (50 ps, constant volume).
	<ol>
		<li>Use heat.in and protein_min2.rst as inputs: pmemd -O -i heat.in -p protein.prmtop 
		-c protein_min2.rst -o protein_heat.out 
		-r protein_heat.rst -x protein_heat.mdcrd -ref protein_min2.rst</li>
		<li>Follow the required arguments: -x FILE coordinate sets saved over MD trajectory.</li>
		<li>Specify options in the input file heat.in: 
		&#38;cntrl 
		imin=0, irest=0, ntx=1, 
		nstlim=25000, dt=0.002, 
		ntc=2, ntf=2, 
		cut=10.0, ntb=1, 
		ntpr=500, ntwx=500, 
		ntt=3, gamma_ln=2.0, 
		tempi=0.0, temp0=300.0, 
		ntr=1, restraintmask=&#39;:1-517&#39;, 
		restraint_wt=1.0, 
		nmropt=1 
		/ 
		&#38;wt TYPE=&#39;TEMP0&#39;, 
		istep1=0, istep2=25000, 
		value1=0.1, value2=300.0 / 
		&#38;wt TYPE=&#39;END&#39; / 
		NOTE: Input options: imin=0 run MD; irest=0 run a new simulation; ntx=1 no initial velocities are read from the rst file; nstlim=25000 number of MD steps; dt=0.002 time step (ps); ntc=2 bonds involving hydrogen are constrained using the SHAKE algorithm; ntf=2 forces for the constrained bonds are not calculated; ntwx=500 coordinates are written to the mdcrd file every ntwx steps; ntt=3 Langevin temperature regulation; gamma_ln=2.0 collision frequency for Langevin dynamics (ps&#8722;1); tempi=0.0 initial temperature; temp0=300.0 reference temperature; nmropt=1 parameters specified in an &#38;wt namelist are read; TYPE=&#39;TEMP0&#39; vary the target temperature.</li>
	</ol>
	</li>
</ol>
4. Equilibration
NOTE: This stage is necessary to adjust the density of water and obtain the equilibrium state of the protein.
<ol>
	<li>Perform the equilibration at 300 K without any restraints (500 ps, constant pressure).
	<ol>
		<li>Use equil.in and protein_heat.rst as inputs: pmemd -O -i equil.in -p protein.prmtop 
		-c protein_heat.rst -o protein_equil.out -r protein_equil.rst -x protein_equil.mdcrd.</li>
		<li>Specify options in the input file equil.in: 
		&#38;cntrl 
		imin=0, irest=1, ntx=5, 
		nstlim=250000, 
		dt=0.002, 
		ntc=2, ntf=2, 
		cut=10.0, ntb=2, ntp=1, taup=2.0, 
		ntpr=1000, ntwx=1000, ntwr=50000, 
		ntt=3, gamma_ln=2.0, 
		temp0=300.0 
		/ 
		NOTE: Input options: irest=1 restart the simulation; ntx=5 coordinates and velocities are read from a previously generated rst file; ntb=2 periodic boundaries&#160;are imposed, constant pressure; ntp=1 isotropic pressure scaling; taup=2.0 pressure relaxation time (ps); ntwr=50000 every ntwr steps, the rst file is written, ensuring recovery from a crash.</li>
	</ol>
	</li>
</ol>
5. Production dynamics
<ol>
	<li>After equilibrium has successfully been achieved, carry out a production MD simulation (10 ns or longer) at constant pressure, and generate the trajectory file for subsequent analysis of the protein structure.
	<ol>
		<li>Use prod.in and protein_equil.rst as inputs: pmemd -O -i prod.in -p protein.prmtop 
		-c protein_equil.rst -o protein_prod.out -r protein_prod.rst -x protein_prod.mdcrd.</li>
		<li>Specify options in the input file prod.in: 
		&#38;cntrl 
		imin=0, irest=1, ntx=5, 
		nstlim=5000000, 
		dt=0.002, 
		ntc=2, ntf=2, 
		cut=10.0, ntb=2, ntp=1, taup=2.0, 
		ntpr=1000, ntwx=1000, ntwr=50000, 
		ntt=3, gamma_ln=2.0, 
		temp0=300.0, ig=-1 
		/ 
		NOTE: The parallel version of pmemd (pmemd.MPI) or GPU-accelerated version (pmemd.cuda) can be used on computer clusters and supercomputers. A long MD simulation may be broken into several segments and performed sequentially. It is strongly recommended to set ig=-1 (random seed option) for every simulation restart. The ptraj or cppptraj programs can be used for the analysis and processing of coordinate trajectories. For more information, see the software Users&#39; Manual.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Olsson, M., Zhivotovsky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+and+cancer.">Caspases and cancer.</a> Cell Death and Differentiation. 18 (9), 1441-1449 (2011).</li><li>Lavrik, I. N., Golks, A., Krammer, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase:+Pharmacological+manipulation+of+cell+death.">Caspase: Pharmacological manipulation of cell death.</a> Journal of Clinical Investigation. 115 (10), 2665-2672 (2005).</li><li>Degterev, A., Boyce, M., Yuan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+decade+of+caspases.">A decade of caspases.</a> Oncogene. 22 (53), 8543-8567 (2003).</li><li>Pop, C., Salvesen, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+caspases:+Activation,+specificity,+and+regulation.">Human caspases: Activation, specificity, and regulation.</a> Journal of Biological Chemistry. 284 (33), 21777-21781 (2009).</li><li>Zamaraev, A. V., Kopeina, G. S., Prokhorova, E. A., Zhivotovsky, B., Lavrik, I. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-translational+modification+of+caspases:+The+other+side+of+apoptosis+regulation.">Post-translational modification of caspases: The other side of apoptosis regulation.</a> Trends in Cell Biology. 27 (5), 322-339 (2017).</li><li>Pearlman, S. M., Serber, Z., Ferrell, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+for+the+evolution+of+phosphorylation+sites.">A mechanism for the evolution of phosphorylation sites.</a> Cell. 147 (4), 934-946 (2011).</li><li>Zamaraev, A. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+Serine-384+in+Caspase-2+processing+and+activity.">Requirement for Serine-384 in Caspase-2 processing and activity.</a> Cell Death and Disease. 11 (10), 825 (2020).</li><li>Dagbay, K. B., Hill, M. E., Barrett, E., Hardy, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor-associated+mutations+in+caspase-6+negatively+impact+catalytic+efficiency.">Tumor-associated mutations in caspase-6 negatively impact catalytic efficiency.</a> Biochemistry. 56 (34), 4568-4577 (2017).</li><li>Ando, 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=Cancer-associated+missense+mutations+of+caspase-8+activate+nuclear+factor-&#954;B+signaling.">Cancer-associated missense mutations of caspase-8 activate nuclear factor-&#954;B signaling.</a> Cancer Science. 104 (8), 1002-1008 (2013).</li><li>Case, D. 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=.">.</a> AMBER 2020. , (2020).</li><li>Salomon-Ferrer, R., Case, D. A., Walker, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+the+Amber+biomolecular+simulation+package.">An overview of the Amber biomolecular simulation package.</a> WIREs Computational Molecular Science. 3 (2), 198-210 (2013).</li><li>Roe, D. R., Cheatham, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTRAJ+and+CPPTRAJ:+Software+for+processing+and+analysis+of+molecular+dynamics+trajectory+data.">PTRAJ and CPPTRAJ: Software for processing and analysis of molecular dynamics trajectory data.</a> Journal of Chemical Theory and Computation. 9 (7), 3084-3095 (2013).</li><li>Maier, J. 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=ff14SB:+Improving+the+accuracy+of+protein+side+chain+and+backbone+parameters+from+ff99SB.">ff14SB: Improving the accuracy of protein side chain and backbone parameters from ff99SB.</a> Journal of Chemical Theory and Computation. 11 (8), 3696-3713 (2015).</li><li>Salomon-Ferrer, R., G&#246;tz, A. W., Poole, D., Le Grand, S., Walker, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Routine+microsecond+molecular+dynamics+simulations+with+AMBER+on+GPUs.+2.+Explicit+solvent+particle+mesh+ewald.">Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. Explicit solvent particle mesh ewald.</a> Journal of Chemical Theory and Computation. 9 (9), 3878-3888 (2013).</li><li>Berman, H. 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=The+Protein+Data+Bank.">The Protein Data Bank.</a> Nucleic Acids Research. 28 (1), 235-242 (2000).</li><li>Burley, S. 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=RCSB+Protein+Data+Bank:+Powerful+new+tools+for+exploring+3D+structures+of+biological+macromolecules+for+basic+and+applied+research+and+education+in+fundamental+biology,+biomedicine,+biotechnology,+bioengineering+and+energy+sciences.">RCSB Protein Data Bank: Powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences.</a> Nucleic Acids Research. 49, 437-451 (2021).</li><li>Martinez, X., 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=Molecular+graphics:+Bridging+structural+biologists+and+computer+scientists.">Molecular graphics: Bridging structural biologists and computer scientists.</a> Structure. 27 (11), 1617-1623 (2019).</li><li>Lee, T. 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=GPU-accelerated+molecular+dynamics+and+free+energy+methods+in+Amber18:+Performance+enhancements+and+new+features.">GPU-accelerated molecular dynamics and free energy methods in Amber18: Performance enhancements and new features.</a> Journal of Chemical Information and Modeling. 58 (10), 2043-2050 (2018).</li><li>J&#228;ger, R., Zwacka, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+enigmatic+roles+of+caspases+in+tumor+development.">The enigmatic roles of caspases in tumor development.</a> Cancers. 2 (4), 1952-1979 (2010).</li></ol>

The authors have no conflicts of interest to disclose.

The described MD approach allows for modeling both the wild-type and mutant forms of caspase using the biomolecular simulation packages. Several important issues of the methodology are discussed here. First, a representative crystal structure of caspase needs to be selected from the Protein Data Bank. Importantly, both monomeric and dimeric forms of caspase are acceptable. Choosing high-resolution structures with a minimum number of missing residues is a good idea. The protonation state of some residues can be set manually in the input PDB file. For example, in Figure 1, a hydrogen atom was attached to the N&#949;2 atom (HIE form) of the catalytic residue His277. Second, a solvent box must be created around the protein for further explicit-solvent simulations. Energy minimization is required to optimize the coordinates of added hydrogen atoms and water molecules. Third, the heating and equilibration must be carried out prior to the production dynamics simulation. At the heating stage, one should ensure that the cavities and binding pockets on the protein surface fill up with water molecules (if necessary, one can increase the number of MD steps). At the equilibration stage, the acquisition of the equilibrium conformation of the protein needs to be confirmed by analyzing the root-mean-square deviation of its backbone atoms from the initial positions12. Fourth, monitoring the conformational changes of the protein structure during the production dynamics simulation is required. For this purpose, one should superimpose trajectory frames onto the starting structure by fitting the backbone atoms and then analyze the conformational changes using a molecular visualization tool (VMD, PyMOL, etc.)17. If necessary, one can increase the number of MD steps and/or break up the simulation into several segments performed sequentially. Fifth, it is recommended to use the GPU-accelerated version of the MD simulation program (pmemd.cuda), whose performance significantly exceeds that achievable by the traditional CPU implementation14,18.
A possible limitation of the described method is the availability of certain caspase structures in the Protein Data Bank. However, more than 900 PDB entries are found under the &#34;caspase&#34; request, indicating that caspase structures have been investigated in detail. Some difficulties may arise when a crystal structure is obtained at low resolution (i.e., at a coarse level of detail) or lacks some fragments important for the caspase function.
In summary, MD modeling approaches have proven effective in predicting structural changes of proteins after mutations of their genes, modifications, or intermolecular interactions. The application of MD simulation in the field of cell death opens great opportunities to study the molecular mechanisms of caspase regulation by post-translational modifications. In this regard, long-term MD simulation could assess the dynamic behavior of functional regions and the potential effect of amino acid substitutions to elucidate the molecular causes associated with mutated or modified caspase-2 as well as other caspases. For example, missense mutations of initiator caspase genes (caspase-2/caspase-8/caspase-9/caspase-10) were detected in gastrointestinal cancers and in tumors of the central and peripheral nervous systems19. Together, MD modeling of caspases is a powerful in silico approach for understanding the mechanisms regulating programmed cell death and associated disorders and for developing new effective therapies.

Apoptosis is one of the most widely-studied cellular processes that regulate the morphogenesis and tissue homeostasis of multicellular organisms. Apoptosis can be initiated by a wide range of external or internal stimuli, such as the activation of death receptors, disturbance in the cell cycle signals, DNA damage, endoplasmic reticulum (ER) stress, and various bacterial and viral infections1. The caspases - key apoptotic players - are conventionally classified into two groups: initiators (caspase-2, caspase-8, caspase-9, and caspase-10) and effectors (caspase-3, caspase-6, and caspase-7), depending on their domain structure and the place in the caspase cascade2,3. Upon cell death signals, the initiator caspases interact with adaptor molecules that facilitate proximity-induced dimerization and autoprocessing to form an active enzyme. The effector caspases are activated through cleavage by initiator caspases and perform downstream execution steps by cleaving multiple cellular substrates4.
The maturation and function of the initiator and effector caspases are regulated by a large number of different intracellular mechanisms, among which the post-translational modification plays an indispensable role in cell death modulation5. The addition of modifying groups (phosphorylation, nitrosylation, methylation, or acetylation) or proteins (ubiquitination or SUMOylation) changes the enzymatic activity of caspases or the protein conformation and stability that regulate apoptosis. Site-directed mutagenesis is widely applied to investigate the potential post-translational modification sites and discern their role. A putative modification site is usually replaced by another amino acid, which cannot be further modified. Thus, potentially phosphorylated serine and threonine are mutated to alanine, and lysine ubiquitination sites are replaced with arginine. Another strategy includes substituting an amino acid that particularly mimics post-translational modification (e.g., glutamate and aspartate have been used to mimic phosphorylated serine or threonine)6. However, some of these substitutions located in the high vicinity of an active site or in critical positions could change caspase structure, disturb catalytic activity, and suppress apoptotic cell death7. Similar effects could be observed in cases of tumor-associated missense mutations in caspase genes. For example, the tumor-associated mutation of caspase-6 - R259H - resulted in conformational changes of loops in the substrate-binding pocket, reducing the efficient catalytic turnover of substrates8. The G325A amino acid substitution in caspase-8 identified in head and neck squamous cell carcinoma could hamper caspase-8 activity, which led to the modulation of nuclear factor-kB (NF-kB) signaling and promoted tumorigenesis9.
To assess the potential effect of amino acid substitutions on caspase structure and function, molecular modeling can be applied. The molecular dynamics (MD) approach is described in this work for modeling the wild-type caspase and its mutant forms using the biomolecular simulation package (Amber). The MD method gives a view of the dynamic evolution of the protein structure following the introduction of mutations. Originally developed by Peter Kollman&#39;s group, the Amber package became one of the most popular software tools for biomolecular simulations10,11,12,13. This software is divided into two parts: (1) AmberTools, a collection of programs routinely used for system preparation (atom type assignment, adding hydrogens and explicit-water molecules, etc.) and trajectory analysis; and (2) Amber, which is centered around the pmemd simulation program. AmberTools is a free package (and a prerequisite for installing Amber itself), while Amber is distributed with a separate license and fee structure. Parallel simulations on a supercomputer and/or using graphics processing units (GPUs) can substantially improve the performance for the scientific research of protein structure dynamics14. The latest available software versions are AmberTools21 and Amber20, but the described protocols can also be used with the former versions.

<table><tbody><tr><td>Amber20</td><td>University of California, San Francisco</td><td></td><td>Software for molecular dynamics simulation&lt;br/&gt; http://ambermd.org</td></tr><tr><td>AmberTools21</td><td>University of California, San Francisco</td><td></td><td>Software for molecular modeling and analysis&lt;br/&gt; http://ambermd.org</td></tr></tbody></table>

exploring caspase mutations and post-translational modification by molecular modeling approaches

Apoptosis is a type of programmed cell death that eliminates damaged cells and controls the development and tissue homeostasis of multicellular organisms. Caspases, a family of cysteine proteases, play a key role in apoptosis initiation and execution. The maturation of caspases and their activity is fine-tuned by post-translational modifications in a highly dynamic fashion. To assess the effect of post-translational changes, potential sites are routinely mutated with residues persistent to any modifications. For example, the serine residue is replaced with alanine or aspartic acid. However, such substitutions could alter the caspase active site's conformation, leading to disturbances in catalytic activity and cellular functions. Moreover, mutations of other amino acid residues located in critical positions could also break the structure and functions of caspases and lead to apoptosis perturbation. To avoid the difficulties of employing mutated residues, molecular modeling approaches can be readily applied to estimate the potential effect of amino acid substitutions on caspase structure. The present protocol allows the modeling of both the wild-type caspase and its mutant forms with the biomolecular simulation package (Amber) and supercomputer facilities to test the effect of mutations on the protein structure and function.

The present protocol can be readily applied in studies of post-translational modification of caspases or pathogenic mutations. In this section, the MD modeling workflow is illustrated (Figure 1), which has been successfully used in the study of caspase-27. Using in vitro site-directed mutagenesis of potential phosphorylation sites (Ser/Thr to Ala) and biochemical approaches, it was demonstrated that the Ser384Ala mutation prevented caspase-2 processing and blocked enzymatic activity and the induction of apoptotic cell death (Supplementary Figure 1). However, neither mass-spectrometry analysis nor Phos-Tag technology confirmed that Ser384 undergoes phosphorylation7. To understand how Ser384 regulates caspase-2 activity, MD modeling of the three-dimensional protein structure was performed (Figure 1).
The molecular models of wild-type and mutant forms of caspase-2 were constructed using the 1pyo crystal structure (available caspase-2 structures are listed in Supplementary Table 1). The Ser384Ala mutant was created by removing the O&#947; atom in the Ser384 residue (in PDB, Ser and Ala residues are distinguished by having the O&#947; atom). Hydrogen coordinates are usually absent in crystal structures. Therefore, hydrogen atoms were added to the protein structure, and then it was solvated by 12 &#197; thick layer&#160;of TIP3P water to enable further explicit-solvent simulations. Sodium ions were added to neutralize the system. The obtained starting models of caspase-2 were subjected to energy minimization, equilibration, and subsequent 10 ns MD simulation according to the MD modeling protocol, using the min1.in, min2.in, heat.in, equil.in, and prod.in control data files.
In the crystal structure of caspase-2, Ser384, together with Arg219 and Arg378, are involved in forming the cavity&#39;s surface in the active site. Arg219 and Arg378 form hydrogen bonds with the substrate&#39;s carboxyl group, while Ser384 does not intervene in direct interactions with the substrate. MD simulations confirmed that the Ser384Ala substitution did not affect the catalytic residues Cys320 (nucleophile) and His277 (general base) but induced a major conformational change in Arg378. Its guanidinium group turned toward the bulk solvent so that the N&#949; atom could not form an essential hydrogen bond with the substrate&#39;s carboxyl group (Figure 1). In the native enzyme, an electrostatic interaction occurs between the O&#947; atom of Ser384 (partial negative charge) and the Arg378 guanidinium group (positive charge). However, the serine substitution apparently disrupts this interaction. Thus, it was shown that the Ser384Ala substitution affected the substrate recognition by the arginine residues in the caspase-2 active site, impairing enzymatic activity and the ability to trigger apoptotic cell death. The discovered mechanism seems evolutionarily conserved and common for other caspase family members. Thus, the implementation of MD simulations allowed for confirming the biochemical results&#160;and&#160;obtaining&#160;new insights into the molecular structure of the active center of caspases.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64206/64206fig1v2.jpg" /> 
Figure 1: MD modeling workflow used to study the caspase structure. The wild-type caspase-2 and its Ser384Ala mutant were investigated following the instructions in the protocol section. It was revealed that the Ser384Ala substitution induces an important conformational change in the active site residue Arg378. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64206/64206fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Function of caspase-2 in cell death. In response to extrinsic and intrinsic stimuli, wild-type caspase-2 can be activated by an autoproteolytic mechanism. Active caspase-2 cleaves Bid, which, in turn, promotes mitochondrial outer membrane permeabilization and apoptotic cell death. The Ser384Ala mutation prevents caspase-2 activation and the induction of cell death. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64206/Supplementary Figure 1 fin.jpg" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Caspase-2 structures available in the Protein Data Bank. The structures are sorted by release date. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64206/Supplementary Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary File 1: Different steps in editing a PDB file. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64206/Supplementary File 1.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches at JoVE.com

Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches

The present protocol uses a biomolecular simulation package and describes the molecular dynamics (MD) approach for modeling the wild-type caspase and its mutant forms. The MD method allows for assessing the dynamic evolution of the caspase structure and the potential effect of mutations or post-translational modifications.

Apoptosis is a type of programmed cell death that eliminates damaged cells and controls the development and tissue homeostasis of multicellular organisms. ...

exploring-caspase-mutations-post-translational-modification-molecular

Nanyang Technological University

Research

JoVE Journal

Biology

1.2K Views.  Lomonosov Moscow State University. The present protocol uses a biomolecular simulation package and describes the molecular dynamics (MD) approach for modeling the wild-type caspase and its mutant forms. The MD method allows for assessing the dynamic evolution of the caspase structure and the potential effect of mutations or post-translational modifications.

Video: Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches

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

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Neuroscience

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.

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

In Silico Structural Analysis of Protein Carbonylation by Lipid Derivatives

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations

Protein carbonylation by reactive aldehydes derived from lipid peroxidation leads to cross-linking, oligomerization, and aggregation of proteins, causing intracellular damage, impaired cell functions, and, ultimately, cell death. It has been described in aging and several age-related chronic conditions. However, the basis of structural changes related to the loss of function in protein targets is still not well understood. Hence, a route to the in silico construction of new parameters for amino acids carbonylated with reactive carbonyl species derived from fatty acid oxidation is described. The Michael adducts for Cys, His, and Lys with 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE), and a furan ring form for 4-Oxo-2-nonenal (ONE), were built, while malondialdehyde (MDA) was directly attached to each residue. The protocol describes details for the construction, geometry optimization, assignment of charges, missing bonds, angles, dihedral angles parameters, and its validation for each modified residue structure. As a result, structural effects induced by the carbonylation with these lipid derivatives have been measured by molecular dynamics simulations on different protein systems such as the thioredoxin enzyme, bovine serum albumin and the membrane Zu-5-ankyrin domain employing root-mean-square deviation (RMSD), root mean square fluctuation (RMSF), structural secondary prediction (DSSP) and the solvent-accessible surface area analysis (SASA), among others.

Author Spotlight: In Silico Creation and Impact of Carbonylated Amino Acids on Protein Structure and Function

Here, we describe a protocol for the optimization and parameterization of amino acid residues modified with reactive carbonyl species, adaptable to protein systems. The protocol steps include structure design and optimization, charge assignments, parameter construction, and preparation of protein systems.

Protein carbonylation by reactive aldehydes derived from lipid peroxidation leads to cross-linking, oligomerization, and aggregation of proteins, causing ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

Medicine

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 ...

Immunology and Infection

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.

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 ...

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 ...

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...

Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches

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Chapters in this video

Lighting Up the Pathways to Caspase Activation Using Bimolecular Fluorescence Complementation

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

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

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

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

Evaluation of Caspase Activation to Assess Innate Immune Cell Death

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila