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...

<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 manual...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amber20</td>
			<td>University of California, San Francisco</td>
			<td></td>
			<td>Software for molecular dynamics simulation 
			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 
			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 bl...

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. ...

play a key role in initiation and execution using molecular modeling. We study the effect of post translation modifications and mutations on the Caspase structure and function. We describe the molecular dynamics approach that gives a view of the evolution of the protein, following the introduction of structural modifications at the atomic level.Such an approach is a particular significance of understanding the mechanisms regulating program cell death and associated disorders and developing new effective therapies. We demonstrate molecular modeling capabilities with one of the most popular tools, Amber 20. But the presented protocol can also be used with the formal versions of this software.The following video describes step by step instructions on preparing the Caspase structure for simulation and performing an ascilica investigation of this proteins wild type and modified forms. To retrieve the selected protein data bank or P D B structure, use the download files dropdown list and click on P D B format. Remove remarks and connectivity data and insert a T E R card between separate protein chains in the P D B file.To prepare the starting model launch the T-LEAP program from Amber Tools package. Then load the F F 14 S B force field to describe the protein with molecular mechanics and the parameters for water molecules and atomic ions such as sodium and chloride by entering the indicated commands in the command line. Next, load the P D B file and build coordinates for hydrogens creating an object named mol.Check for internal inconsistencies that could cause problems using check mol command. Create a solvent box around the protein. Then check the total charge by entering charge mol and add counter ions to neutralize the system.Create the topology P R M top file and the coordinate I N P C R D file by entering the indicated command. Once done, quit the T-lEAP program. Carry out the first stage of the energy minimization to optimize the positions of added hydrogen atoms and water molecules while keeping the protein coordinates fixed by positional restraints on heavy atoms.Run the P M E M D program by entering the indicated command. Follow the required arguments as I control data. P molecular topology, force field parameters and atom names.C initial coordinates. O user readable log output R final coordinates. REF, reference coordinates for position restraints.Next, carry out the second stage of the energy minimization without restraints to optimize the whole system using the inputs of indicated command. This stage aims to heat the system from zero to 300 kelvins. Carry out the heating process with positional restraints on the protein atoms with 50 picoseconds at constant volume by using the given command as inputs.Follow the required argument as X coordinate sets saved over molecular dynamics trajectory. The next stage is necessary to adjust the density of water and obtain the equilibrium state of the protein. Perform the equilibration at 300 kelvins for 500 picoseconds at constant pressure without any restraints using the indicated command after equilibrium has successfully been achieved.Carry out a production molecular dynamics simulation for 10 nanoseconds or longer at constant pressure and generate the trajectory file for subsequent analysis of the protein structure using the indicated command. The parallel version of the program, P M E M D M P I or G P U accelerated version P M E M D cuda can be used on computer clusters and supercomputers. A long molecular dynamics simulation may be broken into several segments and performed sequentially.In this analysis, wild type Caspase two and its Serine-384 alanine mutant were investigated following the molecular dynamics modeling workflow. The Serine-384 alanine substitution induced important conformational change in the active site residue Arginine-378. Further, it was shown that the Serine-384 alanine substitution affected the substrate recognition by the arginine residues in the active site impairing cast base activity.The site directed mutagenesis and biochemical tests demonstrated that the Serine-384 alanine mutation blocked the enzymatic activity in processing of Caspase two and suppressed the apoptosis of cancer cells. The described approach can be used to assess the effect of amino acid mutations and post-translational modifications in caspase two as well as other caspase involved in various cancer diseases.

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

Research

JoVE Journal

Biology

1.2K visualizações.  Lomonosov Moscow State University. desempenham um papel fundamental na iniciação e execução usando modelagem molecular. Estudamos o efeito de modificações e mutações pós-tradução na estrutura e função da caspase. Descrevemos a abordagem da dinâmica molecular que dá uma visão da evolução da proteína, após a introdução de modificações estruturais no nível atômico.Tal abordagem é um significado particular para a compreensão dos mecanismos que regulam a morte celular do programa e distúrbios ass...