Caspase-3/Sandbox

Caspase, as its name implies, is a protease that functions via a cysteine residue in its active site to cleave substrates after aspartic acid residues. Caspases are crucial for the initiation (e.g. caspase-8, -9, -10) and execution (e.g. caspase-3, -6, -7) of apoptosis, or programmed cell death either via the intrinsic or extrinsic pathway (Degterev, Boyce et al. 2003). In the intrinsic pathway, stimuli such as DNA damage or endoplasmic reticulum (ER) stress, activates B cell lymphoma 2 (BCL-2) homology 3 (BH3)-only proteins like Bim or Bad, leading to BCL-2-associated X protein (BAX) and BCL-2 antagonist or killer (BAK) activation and mitochondrial outer membrane permeabilization (MOMP). Anti-apoptotic BCL-2 proteins prevent MOMP by binding BH3-only proteins and activated BAX or BAK. Following MOMP, release of various proteins from the mitochondrial intermembrane space (IMS) promotes caspase activation and apoptosis. Cytochrome c binds apoptotic protease-activating factor 1 (APAF1), inducing its oligomerization and thereby forming a structure termed the apoptosome that recruits and activates an initiator caspase, caspase-9. Caspase-9 cleaves and activates effector caspases, caspase-3 and caspase-7, leading to apoptosis. Mitochondrial release of second mitochondria-derived activator of caspase (SMAC; also known as DIABLO) and OMI (also known as HTRA2) neutralizes the caspase inhibitory function of X-linked inhibitor of apoptosis protein (XIAP). The extrinsic apoptotic pathway is initiated by the ligation of death receptors with their cognate ligands, leading to the recruitment of adaptor molecules such as Fas-associated death domain protein (FADD) and then caspase-8. This results in the dimerization and activation of caspase-8, which can then directly cleave and activate caspase-3 and caspase-7, leading to apoptosis. Crosstalk between the extrinsic and intrinsic pathways occurs through caspase-8 cleavage and activation of the BH3-only protein BH3-interacting domain death agonist (BID), the product of which (truncated BID; tBID) is required in some cell types for death receptor-induced apoptosis (Li, Zhu et al. 1998). It has become appreciated more recently that caspase activity, specifically caspase-8, is also required for T cell growth (Alam, Cohen et al. 1999; Kennedy, Kataoka et al. 1999; Misra, Jelley-Gibbs et al. 2005), and that the location and level of active caspases within cells may be a key determinant of survival or death (Misra, Russell et al. 2007; Koenig, Russell et al. 2008). We have previously observed that murine αβ T cells bearing high levels of caspase activity manifest increased rates of both cell growth and cell death (Dohrman, Russell et al. 2005).

File:3kjf active site 1.png

Inhibitor bound Human Caspase-3, 3kjf

spinqualitylabelsall models PDB ID 1qx3 Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
Crystal Structure of Unliganded Human Caspase-3
Gene:	CASP3 OR CPP32 (Homo sapiens)
Structural annotation: Resources: CATH : 1Qx3A00 InterPro : Ipr001309, Ipr015917, Ipr011600, Ipr002138, Ipr015470, Ipr002398 Pfam : PF00656 SCOP : d1qx3a_ UniProt : P42574
Functional annotation: Cellular component: cytoplasm Biological process: apoptosis proteolysis induction of apoptosis nuclear fragmentation during apoptosis Molecular function: caspase activity peptidase activity protein binding hydrolase activity cysteine-type peptidase activity
Evolutionary conservation: Toggle Conservation Colors: Rows = identical sequences: Further Information: Caveats, Explanation, Complete Results at ConSurfDB
Resources:	FirstGlance, OCA, PDBsum, RCSB
Coordinates:	save as pdb, mmCIF, xml

Inhibitor DEVD-CHO bound to Caspase-3, 1PAU

Structure & FunctionStructure & Function

StructureStructure

(Structural Description) Caspase-3 chains are classified as alpha/beta, with one chain containing a 3-layer(aba) sandwich with Rossmann fold topology (This fold is made up of three or more parallel beta strands linked by two alpha helices in the topological order beta-alpha-beta-alpha-beta) and another chain containing a 2-layer sandwich with alpha-beta plaits.

The active form is made up of two chains, of which can be kept in its inactive form by inhibitors such as DEVD-CHO seen in 1pau.

Active Site/Ligand BindingActive Site/Ligand Binding

	spinqualitylabelsall models PDB ID 1qx3.3kjf.morph.pdb' Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image
	Morph showing conformational changes between unbound (1qx3) and inhibitor bound (3kjf).

and inhibitor of caspase-activated deoxyribonuclease (ICAD) by caspase-3 (Enari, Sakahira et al. 1998; Sakahira, Enari et al. 1998).

In all three adducts the “warhead” ketone oxygen hydrogen bonds with the backbone amide NH of both the catalytic cysteine and conserved glycine (caspase-3 Gly122). Similarly, the P1 carboxylate moiety forms hydrogen bonds with the side chains of three conserved residues (caspase-3 Arg64; caspase-3 Arg207; caspase-3 Gln161). The adjacent amide nitrogen hydrogen bonds with a main chain carbonyl (caspase-3 Ser205) as well as forming a close contact with the catalytic cysteine sulfur atom (3.0 Å). One of the carbonyls of the urazolopyridazine forms a hydrogen bond with a backbone main chain amide (caspase-3 Arg207). As can be surmised from this enumeration of interactions, the structures show that the core elements of the inhibitors bind to caspase-3 and caspase-8 in similar ways. These interactions closely mimic those of caspases with peptidic sub- strates (Fig. 8).

FunctionFunction

(Structural insights into its function) In its inactive form, caspases consist of a prodomain, large subunit, and small subunit, interjected with aspartic acid residues. These are cleavage sites of which active caspases can cleave leading to an active form in which the subunits come together to form a complex.

DiseaseDisease

Although the role of caspases in chronic neurodegenerative disease is controversial, their role in acute neurodegenerative disease, such as nerve crush injury and stroke, may be more evident. Inhibition of caspase activity increases survival of neurons. Studies have also shown that caspases are downregulated to allow for unchecked survival of neoplastic cancers and autoimmune diseases. In particular, caspase-8 has been found to be silenced in neuroblastoma and humans with mutations in caspase-8 and -10 develop ALPS (autoimmune lymphoproliferative syndrome). These raise the possibility that caspase-3 may play a role as well. Knocking out caspase-3 in 129x1/SvJ mice leads to hydrocephalus and subsequently perinatal lethality. Caspase-3 knockout in C57BL/6J however appear to have no brain abnormalities.

(Fuentes-Prior and Salvesen, 2004)

Evolutionarily Related ProteinsEvolutionarily Related Proteins

Proteins + (Links to evolutionarily related proteins) example Caspase 1 through 18

To date, eighteen caspases have been identified. Caspases can largely be grouped into three subfamilies in humans: inflammatory (caspase-1, -4, and -5), effector (caspase-3, -6, and -7), and initiator caspases (-2, -8, -9, and -10). Caspase-11 and -12 substitutes for caspase-4 and -5, respectively, in mice. (Fuentes-Prior and Salvesen, 2004)

Solved StructuresSolved Structures

(Links to available structures) 3PCX Caspase-3 E246A, K242A Double Mutant http://www.pdb.org/pdb/explore/explore.do?structureId=3PCX 3PD1 Caspase-3 K242A http://www.pdb.org/pdb/explore/explore.do?structureId=3PD1 3PD0 Caspase-3 E246A http://www.pdb.org/pdb/explore/explore.do?structureId=3PD03KJF 3KJF Caspase-3 bound to a covalent inhibitor http://www.pdb.org/pdb/explore/explore.do?structureId=3KJF

3ITN 3H0E 3GJQ, 3GJR, 3GJS, 3GJT 3EDQ 3DEH, 3DEI, 3DEJ, 3DEK 2CNK, 2CNL, 2CNN, 2CNO, 2CDR 2J30, 2J31, 2J32, 2J33 2C1E, 2C2K, 2C2M, 2C2O 2H51 2H5J 2H65 2DKO, 2CJX, 2CJY 1RE1 1RHJ, 1RHK, 1RHM, 1RHQ, 1RHR, 1RHU 1NME, 1NMQ, 1NMS 1CP3 1PAU 1I3O 1QX3

References & NotesReferences & Notes

Alam, A., L. Y. Cohen, et al. (1999). "Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells." J Exp Med 190(12): 1879-90.

Aouad, S. M., L. Y. Cohen, et al. (2004). "Caspase-3 is a component of Fas death-inducing signaling complex in lipid rafts and its activity is required for complete caspase-8 activation during Fas-mediated cell death." J Immunol 172(4): 2316-23.

Budd, R. C. (2001). "Activation-induced cell death." Curr Opin Immunol 13(3): 356-62.

Degterev, A., M. Boyce, et al. (2003). "A decade of caspases." Oncogene 22(53): 8543-67.

Dohrman, A., J. Q. Russell, et al. (2005). "Cellular FLIP long form augments caspase activity and death of T cells through heterodimerization with and activation of caspase-8." J Immunol 175(1): 311-8.

Enari, M., H. Sakahira, et al. (1998). "A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD." Nature 391(6662): 43-50.

Kennedy, N. J., T. Kataoka, et al. (1999). "Caspase activation is required for T cell proliferation." J Exp Med 190(12): 1891-6.

Koenig, A., J. Q. Russell, et al. (2008). "Spatial differences in active caspase-8 defines its role in T-cell activation versus cell death." Cell Death Differ 15(11): 1701-11.

Li, H., H. Zhu, et al. (1998). "Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis." Cell 94(4): 491-501.

Misra, R. S., D. M. Jelley-Gibbs, et al. (2005). "Effector CD4+ T cells generate intermediate caspase activity and cleavage of caspase-8 substrates." J Immunol 174(7): 3999-4009.

Misra, R. S., J. Q. Russell, et al. (2007). "Caspase-8 and c-FLIPL associate in lipid rafts with NF-kappaB adaptors during T cell activation." J Biol Chem 282(27): 19365-74.

Sakahira, H., M. Enari, et al. (1998). "Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis." Nature 391(6662): 96-9.

Vincent, M. S., K. Roessner, et al. (1996). "Apoptosis of Fashigh CD4+ synovial T cells by borrelia-reactive Fas-ligand(high) gamma delta T cells in Lyme arthritis." J Exp Med 184(6): 2109-17.

Wesselborg, S., O. Janssen, et al. (1993). "Induction of activation-driven death (apoptosis) in activated but not resting peripheral blood T cells." J Immunol 150(10): 4338-45.