Caspase-3/Sandbox: Difference between revisions

{{STRUCTURE_1qx3 |  PDB=1qx3  |  SCENE=Caspase-3/Sandbox/Unliganded_human_caspase-3/4 | CAPTION= Crystal Structure of Unliganded Human Caspase-3}}

==Structure & Function==

===Structure===  

Procaspase-3, unlike initiator procaspases, is a stable dimer with little enzymatic activity. Procaspase-3 contains a tri-aspartate ‘safety-catch’ (Asp179–Asp181) in the intersubunit linker to remain nonfunctional. During the process of activation, this linker undergoes a pH-dependent conformational change, exposing Asp175 for cleavage. This leads to the formation of a heterotetramer (Walters, 2011). The heterotetramer consists of two anti-parallel arranged heterodimers, each one formed by a 17 kDa (p17) and a 12 kDa (p12) subunit (http://www.uniprot.org/uniprot/P42574).

====Salt Bridge====

Lys242 is located at the C-terminus of helix 5 and Glu246 is located at the base of loop L4 of caspase-3. It has been suggested that the salt bridge between Lys242-Glu246 (NH3+ of Lys with COO- of Glu) is not found in the zymogen form of caspase-3 but forms only upon caspase-3 maturation. Rather, these residues are involved in other interactions in procaspase-3. Lys242 maintains the native procaspase dimer, whereas glutamate is involved in stabilizing loop L3, the substrate binding loop. In procaspase-3, Glu246 participates in hydrogen bonding with Trp214. In the mature, active caspase-3, the stability of the active site of caspase-3 depends on the salt bridge between Lys242 and Glu246 to maintain the correct conformation of loop L4. In caspase-3, Glu246 neutralizes the positive charge of Lys242 by forming a salt bridge within the same hydrophobic cluster of residues between helices 4 and 5 that buries the aliphatic portion of Lys242. Mutations of either K242A or E246A resulted in the complete loss of procaspase-3 activity as measured by fluorescence emission following addition of substrate (Ac-DEVD-AFC) and excitation at 400nm. These mutants were active in the mature, fully cleaved caspase-3, although to a much lower extent than wild type mature caspase-3, perhaps due to altered accessibility of its active site. The measurements of kcat/Km for these mutants at the optimal pH (7.5) for caspase-3 activity are 14 to 60-fold lower than that of wild type, with an overall increase in Km and a decrease in kcat. This may be due to alterations caused by the mutation(s) in the loop bundle interaction between loops L2, L4 and L2’. Loss of the Lys242-Glu246 salt bridge may alter the position of helix 4, which consequently alter the position of loop L3, leading to altered hydrogen-bonding in the P4 site. This may explain the decrease in activity seen in the mutants. Furthermore, because Phe250 in loop L4 participates in hydrogen bonding to P4, mutations in loop L4 could result in disruptions of these interactions (Feeney, 2004; Walters, 2011).  

Thioredoxin, the main intracellular oxidoreductase, does not inhibit caspase-3 activity. However, Trx-Cys73-SNO inhibits apoptosis by decreasing caspase-3 activity via transnitrosylation at Cys163 catalytic residue. This reaction is reversible, although this is not as likely since Cys163 on caspase-3 is more nucleophilic than Cys73 on Trx. GSNO is capable of transferring its nitroso-group to Trx-Cys73, which in turn can S-nitrosylate caspase-3, leading to inactive caspase-3 (Mitchell, 2005).

In human B- and T-cell lines, nitrosylation of caspase-3 depends on localization: mitochondrial but not cytoplasmic caspase-3 zymogens are S-nitrosylated. S-nitrosylation may prevent improper autoactivation in the mitochondria due to close proximity in the intermembrane space (Mannick, 2001).  Mitochondrial caspase-3 is released into the cytoplasm and becomes denitrosylated when FasL binds Fas receptor (Mannick, 2001). Fas receptor activation by FasL activates the extrinsic pathway of apoptosis through activation of caspase-8, which can either directly cleave procaspase-3 to caspase-3 or lead to Bid cleavage to tBid. tBID then permeates into the mitochondria, leading to cytochrome c release, apoptosome formation with caspase-9, and finally caspase-3 cleavage to its activated form. Smac/DIABLO is also released from the mitochondria and leads to inhibition of XIAP (X-linked inhibitor of apoptosis), abrogating its inhibitory effect on caspase-3 activity within the cytosol. XIAP also functions as an E3 ubiquitin ligase, targeting caspase-3 for proteasomal degradation (Nakamura, 2010). Aside from this, Fas-induced apoptosis also leads to denitrosylation of caspase-3 and therefore activation by freeing its catalytic Cys residue (Mannick, 1999).  Perhaps, this denitrosylation occurs via transnitrosylation of XIAP RING domain at Cys450, which in effect inhibits its E3 ligase activity. This further prolongs caspase-3 activity by blocking its degradation, and thus promotes increased cell death (Nakamura, 2010). Thus, S-nitrosylation of caspases inhibits caspase activation and apoptosis, whereas denitrosylation activates caspases and promotes apoptosis (Mannick, 2007).  

==Solved Structures==

==References & Notes==