TheCASP3protein is a member of thecysteine-aspartic acid protease (caspase) family.[5] Sequential activation of caspases plays a central role in the execution-phase ofcell apoptosis. Caspases exist as inactiveproenzymes that undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the activeenzyme. This protein cleaves and activatescaspases 6 and7; and the protein itself is processed and activated by caspases 8, 9, and10. It is the predominant caspase involved in the cleavage ofamyloid-beta 4A precursor protein, which is associated with neuronal death inAlzheimer's disease.[6] Alternative splicing of this gene results in two transcript variants that encode the same protein.[7]
Caspase-3 shares many of the typical characteristics common to all currently-known caspases. For example, its active site contains acysteine residue (Cys-163) andhistidine residue (His-121) that stabilize thepeptide bond cleavage of a protein sequence to the carboxy-terminal side of anaspartic acid when it is part of a particular 4-amino acid sequence.[9][10] This specificity allows caspases to be incredibly selective, with a 20,000-fold preference for aspartic acid overglutamic acid.[11] A key feature of caspases in the cell is that they are present aszymogens, termed procaspases, which are inactive until a biochemical change causes their activation. Each procaspase has an N-terminal large subunit of about 20 kDa followed by a smaller subunit of about 10 kDa, called p20 and p10, respectively.[12]
Under normal circumstances, caspases recognize tetra-peptide sequences on theirsubstrates andhydrolyze peptide bonds afteraspartic acid residues. Caspase 3 andcaspase 7 share similar substrate specificity by recognizing tetra-peptide motif Asp-x-x-Asp.[13] The C-terminal Asp is absolutely required while variations at other three positions can be tolerated.[14] Caspase substrate specificity has been widely used in caspase basedinhibitor and drug design.[15]
Caspase-3, in particular, (also known as CPP32/Yama/apopain)[16][17][18] is formed from a 32 kDa zymogen that is cleaved into 17 kDa and 12 kDa subunits. When the procaspase is cleaved at a particular residue, the active heterotetramer can then be formed by hydrophobic interactions, causing four anti-parallel beta-sheets from p17 and two from p12 to come together to make a heterodimer, which in turn interacts with another heterodimer to form the full 12-strandedbeta-sheet structure surrounded byalpha-helices that is unique to caspases.[12][19] When the heterodimers align head-to-tail with each other, an active site is positioned at each end of the molecule formed by residues from both participating subunits, though the necessary Cys-163 and His-121 residues are found on the p17 (larger) subunit.[19]
The p12 (pink) and p17 (light blue) subunits of caspase-3 with the beta-sheet structures of each in red and blue, respectively; image generated in Pymol from 1rhm.pdb
The catalytic site of caspase-3 involves the thiol group of Cys-163 and theimidazole ring of His-121. His-121 stabilizes thecarbonyl group of the key aspartate residue, while Cys-163 attacks to ultimately cleave the peptide bond. Cys-163 and Gly-238 also function to stabilize the tetrahedraltransition state of the substrate-enzyme complex throughhydrogen bonding.[19]In vitro, caspase-3 has been found to prefer the peptide sequence DEVDG (Asp-Glu-Val-Asp-Gly) with cleavage occurring on the carboxy side of the second aspartic acid residue (between D and G).[11][19][20] Caspase-3 is active over a broadpH range that is slightly higher (more basic) than many of the other executioner caspases. This broad range indicates that caspase-3 will be fully active under normal and apoptotic cell conditions.[21]
Cys-285 (yellow) and His-237 (green and dark blue) in the active site of caspase-3, p12 subunit in pink and p17 subunit in light blue; image generated in Pymol from 1rhr.pdb
Caspase-3 is activated in the apoptotic cell both by extrinsic (death ligand) and intrinsic (mitochondrial) pathways.[12][22] The zymogen feature of caspase-3 is necessary because if unregulated, caspase activity would kill cells indiscriminately.[23] As an executioner caspase, the caspase-3 zymogen has virtually no activity until it is cleaved by an initiator caspase after apoptotic signaling events have occurred.[24] One such signaling event is the introduction ofgranzyme B, which can activate initiator caspases, into cells targeted for apoptosis by killerT cells.[25][26] This extrinsic activation then triggers the hallmark caspase cascade characteristic of the apoptotic pathway, in which caspase-3 plays a dominant role.[10] In intrinsic activation,cytochrome c from themitochondria works in combination withcaspase-9, apoptosis-activating factor 1 (Apaf-1), andATP to process procaspase-3.[20][26][27] These molecules are sufficient to activate caspase-3 in vitro, but other regulatory proteins are necessaryin vivo.[27]Mangosteen (Garcinia mangostana) extract has been shown to inhibit the activation of caspase 3 in B-amyloid treated human neuronal cells.[28]
One means of caspase inhibition is through the IAP (inhibitor of apoptosis) protein family, which includes c-IAP1, c-IAP2,XIAP, and ML-IAP.[19] XIAP binds and inhibits initiator caspase-9, which is directly involved in the activation of executioner caspase-3.[27] During the caspase cascade, however, caspase-3 functions to inhibit XIAP activity by cleaving caspase-9 at a specific site, preventing XIAP from being able to bind to inhibit caspase-9 activity.[29]
Caspase-3 has been found to be necessary for normalbrain development as well as its typical role in apoptosis, where it is responsible forchromatin condensation andDNA fragmentation.[20] Elevated levels of a fragment of Caspase-3, p17, in the bloodstream is a sign of a recentmyocardial infarction.[51] It is now being shown that caspase-3 may play a role in embryonic and hematopoieticstem cell differentiation.[52]
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