Enhancing Caspase 3 To Promote Apoptosis Biology Essay


Apoptosis is a selective process for deletion of cells. It is generally involved in embryogenesis, tissue remodeling and also to maintain organ size and function. Sequential activation of caspases plays an important role in programmed cell death. In the caspase family, caspase-3 is an important effector protease that cleaves many downstream proteins. Damage in apoptotic pathways promotes uncontrolled growth of cancer cell. Analyses on clinical researches indicate that anticancer drugs activate cascade of apoptotic pathways either by positively or negatively regulating cell death induction. PAC-1 is such a compound, synthesized chemically that selectively induces apoptosis in cancer cells. This article reviews the effect of PAC-1 computationally, as a promoter for caspase-3. Docking of caspase-3 proteins with the specific ligand i.e., compound PAC-1, showed high interaction. The result shows that the PAC-1 facilitates the activation of caspase-3 and thus promotes the activation of apoptotic pathways and prevents the uncontrolled proliferation of cancer cells.

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Cell deaths are of two types: apoptosis and necrosis. Apoptosis or programmed cell death is a physiological process in which cells respond to a variety of stimuli and undergo a controlled and regulated manner of cell death [1, 2, 3]. Activation of apoptosis is maintained by diverse cell-signals, which originate extra-cellularly (toxins, hormones, nitric oxides, growth factors) or intra-cellularly [4]. Both pathways finally end up in the level of activation of caspases for cell death. Caspases are a family of cysteine proteases, with specific cysteine residue that cleaves proteins after the aspartic acid residue, a specificity which is not normal among proteases to produce the active mature caspases [5]. These cleavages remove an NH2 peptide terminal and separate the small and large domains of the pro-enzyme so that it produces the mature hetero tetrameric caspases containing two large and two small domains. There are 14 caspases found in human [6].

The formation of active caspases forms a cascade in which initiator caspases (8, 9) interact with the downstream effector molecules (3, 7) to facilitate their own activation. Finally, this leads to cell apoptosis. This sequence of activation in caspases is broadly classified into two pathways; the extrinsic pathway characterized by involvement of cell surface death receptors and the intrinsic pathway involved in key mitochondrial events. Caspases 2,3,6,7,8,9,10 mainly play a role in initiation of apoptosis and caspse-1, 4, 5, are important in proteolytic activation (interleukin 1 connecting enzyme) [7].

In this paper, we describe the extrinsic pathway which are activated by death receptors like Fas receptor (APO-1 or CD95) that belongs to TNF receptor super family [8,9]. Its natural ligand is FasL that amounts to 40-kDa and binds to receptor. This induces Fas, having an 85-amino acid region called death domain (DD). DISC (death initiating signaling complex) is formed which contains an adaptor protein called FADD (Fas associated death domain) and procaspases-8. By the homophilic interactions of DD and the N-terminal domain of procaspase-8 via an interacting site named DED (death effector domain), FADD binds to the cytoplasmic region of Fas.

Oligomerization of procaspase-8 in the DISC leads to self activation with release of active caspase-8 in to the cytosol [10]. This caspase-8 initiates other downstream proteins including procaspase-3 in two ways: the first is a complex pathway, wherein caspase8 cleaves Bcl-2 interacting protein named Bid and also releases cytochrome-c. The released cyt c binds to Apaf-1 together with dATP and procaspase-9. This procaspase-9 comes and activates caspase-9 and simultaneously this activates procaspase-3 and then activates caspase-3. In the second simple pathway, caspase-8 cleaves procaspase-3 directly and activates it. Out of the complex DFF40 and DFF45 (DNA fragmentation factor 45), the caspase-3 cleaves DFF45 and as a result of that, DFF45 dissociates from DFF40, causing oligomerization of DFF40 which has DNase activity. The active DFF40 oligomer induces the internucleosomal DNA fragmentation, which is an apoptotic feature indicative of chromatin condensation.

In the family, Caspase-3 in its inactive zymogens, pro-caspase-3 is a remarkable protein as the enzyme shows a large substrate diversity, as a variety of proteins have been cleaved in cell maintenance. Their disruption in regulation is involved as a key role in the progress of a variety of diseases, including Cancer, Alzheimer's, and Parkinson's disease [11].

PAC-1 plays a role to activate procaspase-3 indiscriminately. When administered to cancer cells, it signals the cells to auto-destruct by initiating the effector markable protein procaspase-3.It is followed by the activation of sequential proteins that are involved in apoptosis pathway [12]. Procaspase-3 has a safety-catch made of three aspartate amino-acids. Cell releases the safety catch and procaspase-3 is activated to caspase-3. PAC-1 cleaves three amino acids and initiates procaspase-3 to caspase-3 and further activates other molecules in the pathway and causes a gradual increase in the concentration of caspse-3.

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The proteins for Human caspase-3 were collected from PDB (Protein Data Bank) [13]. Then these molecules were subjected to sequence analysis, structural analysis and computational modeling to characterize them. Initially multiple sequence alignment for the protein caspase-3 was obtained through ClustalW tool [14]. It calculates the best match for the selected sequences and helps in identifying the similarities and differences can be observed. The secondary structures were found out using the tools 'Secondary Classification of Proteins (SCOP)' [15] and 'Self-Optimized Prediction Method with 2 Alignment tools (SOPMA)' [16].

Using ProtParam [17], the physio-chemical properties of the proteins was found. The instability index, aliphatic index, grand average hydropathy (GRAVY), molecular weight and the half life of these proteins were calculated. Active sites of capsase-3 proteins were determined using PROSITE [18]. For finding the repeated regions in the proteins, RADAR tool [19] was used. Subcellular location of the proteins were identified from Uniprot database [20].

The structural Analysis of the proteins were done using Accelry's Discovery Studio [21] with 'smart minimiser' and CHARMm force field. The input for the Discovery studio modeling was the PDB file collected from the repository - Protein Data Bank. The collected proteins were subjected to geometry optimization and the corresponding minimum interactional potential energy values were computed.

Docking analysis of the receptor caspase-3 proteins with ligand PAC-1 was done using CDOCKER tool of Discovery Studio.


The results obtained by multiple sequence alignment showed the presence of conserved regions in these proteins. From the SCOP database it was found that all the caspase-3 proteins belongs to alpha and beta proteins and they are in the caspase catalytic domain family.

The instability index values of these Caspase-3 proteins have been computed and it was found that these proteins were stable in nature (figure 1). Aliphatic index of these proteins was calculated. It can be considered as a positive factor for the enhancement of thermo stability of globular proteins. The half life of these proteins was found to be 1 hour. Grand hydropathy of these proteins was found out. All the caspase-3 proteins were found to be hydrophilic in nature (figure 2). The common functional sites of these proteins have been identified and can be extended as the targets for ligand binding. The Subcellular location of the proteins was found to be cytoplasm.

The Interaction potential energy of each of these proteins has been computed by the molecular modeling technique (figure 3). The interaction potential energy was found to be negative for all the proteins. The Vander Waals energy was also found to be negative. The RMS gradient value was very close to zero indicating them to be in a stable state. A comparison of interactional potential energy of other protein molecules has been made. Structural and sequence studies, interactional analysis and thermodynamic characterization can be effectively used in the designing of biomolecular systems. With this background a novel approach has been developed for designing a promoter drug targeting the cancerous cells to initiate the apoptosis.

CDOCKER result analysis showed that the three proteins in caspase-3 class showed interactions with the PAC-1 ligand. The interactional energies of the three proteins with the ligand have been tabulated (Table 1).


Death receptors activate initiator procaspases-8 when the ligand is bound to them. Auto-catalytically cleaved procaspase-8 then initiates executioner caspses-3, 6, 7 and leads to the apoptosis pathway. Over expression or mutation of any proteins involved in the pathway leads to the inactivation of caspases. Inactivation of upstream pathway disrupts the activation of caspase-3 and leads to uncontrolled growth of cells. For activation of these inactivated caspase-3 proteins, PAC-1 plays an important role. PAC-1 compound has active ligand interaction with the receptor and from computational analysis it has been found out that it can be used an active effector for the activation of caspase-3 in order to promote apoptosis in the cancer cells.

Figure 1

Figure 2

Figure 3

Table 1

Table 3: Docking Results

Serial no

Protein ID

Minimum Interactional Energy