Caspase-3 (H. sapiens)
Created by Krystal Sing
Caspase-3 (PDB ID: 3KJF) belongs in the aspirate-specific cysteinyl proteases in the C1A family. Activation by caspases-8, 9, and 10 causes protein caspase-3 to become the primary mediator in the apoptosis pathway in mammlian cells. Caspase-3 continues the pathway by activating caspases-6 and 7. Along with caspases-6 and 7, caspase-3 belongs in the group III of the capase family called the apoptosis effector caspases. Caspase 3, a downstream caspase, cleaves other cellular proteins which includes critical cellular substrates like poly (ADP-ribose) polymerase and lamins1.
The protein sequence of caspase-3 consists of 277 amino acids. Expasy software computationally derived caspase-3’s theoretical isoelectric point and molecular weight. Carried out on the complete sequence of 277 amino acids, caspase 3’s molecular weight was 31607.88 Da and the theoretical pI lies at the pH of 6.09. Chain A’s sequence length is 147 residues. Of the chain, 25% are helical and 29% are beta sheets. 5 helices consisting of 38 residues make up helical helices and 10 strands of 43 residues are beta sheets. Chain B’s sequence length is 109 residues. Of the chain, 28% are helical and 14% are beta sheets. 3 helices consisting 31 residues make up helical helices and 7 strands of 16 residues are beta sheets.
The protein consists of 2 chains and is heterotetramer with sequences from Homo sapiens.The two heterodimers are aligned anti-parallel in regards to one another with each chain consisting of a 17 kDa (p17) subunit and a 12 kDa (p12) subunit. The subunits dimerize in order to form an active enzyme. In its inactive form, the protein is inhibited by isatin sulfonamides. The heterodeimer forms from hydrophobic interactions resulting from parallel beta sheets which comprise of six antiparallel beta strands. The structure caspase-3 comprise of 12 stranded beta sheets surrounded by alpha helices2. This unique quaternary structural fold is a characteristic for caspases. This antiparallel configuration results in two active sites located at opposite ends of the molecule. The active site consists of amino acid residues from both subunits. Specifically, the catalytic component comprises of the aspartic acid in the binding pocket from which cysteine (Cys 163) residue will aid the formation of the oxyanion hole in order for the catalytic reaction to proceed3.
Using BLAST on Caspase-3 subunit p12, a homolog of this subunit is the crystal structure of the chain B or D in caspase-7 complexed with XIAP. Chain B and D contain 105 residues each and are identical. The PDB ID is 1I51 and the sequence was found be 81% in comparison to Caspase-3 subunit p12. Caspase-7 complexed with XIAP also contains two unique subunit, subunits p20 and p11. The length of Caspase-7 is 303 amino acids, and the B and D chain stretch from amino acids 212 to 303. Selecting this fragment range on ExPasy software computation derivation, chain B or D’s molecular weight was calculated to be 10097.49 and theoretical pI was calculated to be 6.06. Using ClustalW’s aligning of the subunit p12 of 109 amino acids against chain B or D in caspase-7 of 105 amino acids showed 66 conserved amino acid residues. The function of caspase-7 is highly associated with caspase-3. This smaller subunit of caspase-7 will form an active heterodimer with the larger subunit of caspase-3, and vice versa. Caspase-7 also belong in the same family as Caspase-3 and its primary role is in apoptosis. Like caspase-3, caspase-7 exists as an inactive enzyme precursor. The protein is activated through cleaving by caspase-3, 10, and 9. XIAP, the inhibitor of apoptosis proteins, prohibit cell death by stopping the catalytic function of the caspase. XIAP is in contact with those residues essential for the catalytic function. The regions conserved are a lot of residues present in their active sites, which contains a Cys-285 and His-237 the carboxyl-terminal end of aspartic acid. The catalytic site for both caspase-3 and caspase-7 may be similar because the stability of the active site formed and how both functions similarly. The A and B subunits of caspase-7 and caspase-3 are superimposed for comparison.
In the process of procaspase activation, the cleavage of procaspase from the specific Asp-X bonds results in the formation of the mature caspase. The mature caspase consists of heterotetramer p202 and p102. The formation of this heterotetramer releases the prodomain activation center with the residues involved SHG and QACXG2. Caspases recognizes four amino acids, S4-S3-S2-S1. The cleavage occurs after the C-terminal residue (S1), which is the amino acid asparagine. The S3 location however is preferred is a glutamine and holds true for all mammalian caspases. Asp-Glu-X-Asp becomes the specificity of caspace cleavage. Caspase-3 prefers substrate DEXD4. In order for catalysis to occur, Asp residue must be present at position S1 and S4. The cleavage sequence accepts a hydrophobic amino acid residue at S2 and hydrophilic amino acid residue at S3. However, at S3, Val or Ala are acceptable. The most effective interaction is with inhibitor acetyl-Asp-Glu-Val-Asp-CHO. The reason for the Asp residue at S1 pocket is because of the strictly conserved S1 pocket of residues Arg-179, Arg-341 and Gln-283, which fits an aspartate side chain. With a glutamate residue at S1, four magnitude of lower catalytic efficiency would be observed5. The aromatic S2 pockets consisting of Tyr-338, Trp-340, and Ph3-381 prefers small residues like Ala and Val. During substrate binding, S2 pocket will alter. When caspase-3 is unoccupied, the pocket is occupied by the side chain of Tyr-338. When binded with a tetrapeptide inhibitor, Tyr-338 will rotate 90 degrees to move out of the pocket.
The free ligand molecule binding to caspase-3 is a covalent inhibitor. The name identifier is(3S)-3-({[(5S,10aS)-2-{(2S)-4-carboxy-2-[(phenylacetyl)amino]butyl}-1,3-dioxo-2,3,5,7,8,9,10,10a-octahydro-1H-[1,2,4]triazolo[1,2-a]cinnolin-5-yl]carbonyl}amino)-4-oxopentanoic acid and its formula is C29H35N5O9 with a molecular weight of 592.62 g/mol. The ligand is comprised of LTyrosine, L-a-aspartyl-L-prolyglycyl-L-phenylalanyl and has 4 hydrogen bond donors and 9 hydrogen bond acceptors from the total bond count of 81. 6 Bonds are aromatic.
This inhibitor of apoptosis (IAP) is specific for caspase-3. It binds and inhibits the substrate caspase 3 to prevent the casace of proteolysis and protect Fas or caspase-8 induced apoptosis6. This results in the direct inhibition of caspase-9. In the mitochondrial pathway for caspase activation, XIAP, c-IAP1 and cIAP2 directly bind to the caspase-3, caspase-3 interferes with procaspase-9, activating additional pro-caspase-9 molecules7. For XIAP, the residues involved in the domain include Leu and Asp, seaparated by six amino acids. Another inhibitor is the baculoviral p35 protein. The binding involves an inhibitory complex of the thioester link between caspase-3 and p35. Another inhibitor is the serpin CrmA and involves modifying the covalent character of the active center8.
IAP is characterized by the BIR domain. The inhibitory function of XIAP of caspase-3 involves residues 163-240, which covers the N-terminal extension at residues 124-162. The BIR domain consist of 70 residues that comprise of three beta strand and four alpha helices, coordinated by Cys and His residues, as shown in the crystal structure of BIR domain of XIAP in complex with caspase-39. The two caspase catalytic domains each consist of a large and small subunit and are structurally identical. The two active sites form a functional dimer. Contacts made between the inhibitor XIAP and caspase-3 includes both the large caspase-3 and small caspase-3 subunit of the inhibited catalytic domain. A short stretch of caspase-3 domain just adjacent of the catalytic domain is also made in contact. The majority of the interactions are hydrophobic with two isoleucine side chains (I149 and I153). Meanwhile, the N-terminal hook is a primary binding unit that helps align the alpha helix. The first interaction involves the hook of XIAP at residues 138-146. XIAP residues L140, V146, and L141 make contact with caspase-3 residues L290, Y338, W340, and F381, forming a hydrophobic cluster. Hydrogen bonds strengthen this interaction, from residues T143, G144, and V1469. A hydrophobic interaction mutation such as L141A interrupts the inhibitory action of XIAP10. The other major interaction site involves the two peptide bonds on the sides of V147 and the site consists of residues 148-156 which is responsible for the hydrophobic and hydrogen bond interactions. Again, a hydrophobic cluster is formed. A particular important residue is D148, which is responsible for hydrogen bonding as well as forming a buried salt bridge with R233 at the C terminus of BIR domain. This salt bridge strengthens the position of the BIR domain against caspase-3. A mutant of D148 would result in a total loss of inhibitory control.
In diseases such as Parkinson's and Alzheimer's, at the sites of cellular damage, an increased level of caspase activity is detected. Caspase-3 is the primary caspase that cleaves the amyloid-beta 4A precursor protein which in the case of inappropriate apoptosis leads to neuronal death in neurodegenerative diseases such as Parkinson's and Alzheimer's. Aside from its role in apoptosis, caspase-3 is also necessary for the development of the brain. In fact, in caspase-3-deficient animals, there is massive imparied development apoptosis in the brain and in these brains, normal development do not occur11. These caspase-3 deficient mice are also smaller in size and die at one to three weeks of age1. Also in the event of myocardial infection in humans, there is an elevatd level of caspase-3 p17 subunit fragments. Other highly expressed levels are also present in the lungs, spleen, liver and kidneys. On a more moderate level of expression, caspase-3 is expressed in the brain and skeletal muscle. A low level of expression is also found in the testis. The highest expression remains in the immune system. The role of caspase-3 in apoptotic processes in MCF-7 breast cancer cells showed that caspase-3 was essential for procaspase-9 processing. Caspase-9 contains a caspase-3 cleave site at reside 330 and procaspase-9 acts as a substrate for caspase-3 during apoptosis as well11. Pathways exist for caspase-3 activation that are both independent and depedent of caspase-9 function. Recent research has also demonstrated that caspase-3 may be involved in directing of embryonic and hematopoetic stem cell differentiation. Caspase-3 promotes differentiation by limiting self-renewal aspect of the stem cell pool and also engages factors that push for gene expression and phenotype of a differentiated cell type12
The caspases are target in drug development with inhibition of caspase activity targeted. In cancer cells, research has focused on selective activation of caspases in cancer cells which would lead to apoptosis. The strategy involves the approach “forward chemical genetics13.” This research has been heavily studied on caspase-3 as it is a key mediator of apoptosis pathway. Another strategy involves preventing molecules from binding to caspase-3. Peptides had been made that bind to the BIR3 pocket of XIAP, inhibiting XIAP from binding to caspase-32.
Caspase-3, at the onset of apoptosis, cleaves poly(ADP-ribose) polymerase (PARP) at ‘216-Asp-|-Gly-217’ bond, activating and cleaving between the helix-loop-helix leucine zipper domain and membrane attachment domain14.
The fas apoptosis activates the S-nitrosylated on the catalytic site cysteine in human cell lines in caspase 3. The increased intracellular caspace activity results from denitrosylation from this activation. The activation of caspase-3 is not only the result of cleavage but also the denitrosylation of the active site thiol as well.