The Apoptosome Activates Caspase-9 by Dimerization

Abstract

The apical protease of the human intrinsic apoptotic pathway, caspase-9, is activated in a polymeric activation platform known as the apoptosome. The mechanism has been debated, and two contrasting hypotheses have been suggested. One of these postulates an allosteric activation of monomeric caspase-9; the other postulates a dimer-driven assembly at the surface of the apoptosome--the "induced proximity" model. We show that both Hofmeister salts and a reconstituted mini-apoptosome activate caspase-9 by a second-order process, compatible with a conserved dimer-driven process. Significantly, replacement of the recruitment domain of the apical caspase of the extrinsic apoptotic pathway, caspase-8, by that of caspase-9 allows activation of this hybrid caspase by the apoptosome. Consequently, apical caspases can be activated simply by directing their zymogens to the apoptosome, ruling out the requirement for allosteric activation and supporting an induced proximity dimerization model for apical caspase activation in vivo.

Inflammatory caspases and inflammasomes: master switches of inflammation

“…[48][49][50] These recruitment platforms integrate cellular signals, promote dimerization of initiator caspases and lead to the formation of an active enzyme proficient to initiate specific signaling cascades. 51,52 These platforms are multiprotein complexes consisting of various molecules assembled on a central scaffold protein that characteristically possesses three main domains: a region involved in ligand sensing, a domain driving oligomerization and a domain involved in recruiting the caspases. The prototypical example is the apoptosome scaffold protein Apaf-1.…”

Commonly used caspase inhibitors designed based on substrate specificity profiles lack selectivity

“…The most commonly used tools include caspase-specific anti-sera as well as fluorogenic substrates and inhibitors. Unfortunately, antibody reagents often do not provide an accurate measure of caspase activity since several caspase family members (caspases 8/10 and 9) do not require proteolytic processing for activation [2,3]. Furthermore, recent evidence suggests that caspase-7 (an executioner caspase) activation occurs via a catalytically active full-length intermediate that cannot be differentiated from the non-cleaved inactive zymogen using antibodies [4,5].…”

Apaf-1-deficient fog mouse cell apoptosis involves hypo-polarization of the mitochondrial inner membrane, ATP depletion and citrate accumulation

“…A faint band of 39 kDa caspase-9 was also detectable, reflecting procaspase-9 processing by caspase-3, which increased further as the apoptosis reactions progressed [26]. Since auto-processing of procaspase-9 occurs by its activation through homo-dimerization, the 37-kDa form was thought to signify induction of the caspase-9 pathway [7,19,20,22]. This initial experiment suggested that +/+ and fog/fog cells were equally capable of capase-9 activation in response to the oxidative stress.…”

“…In fact, procaspase-9 is able to homo-dimerize to gain its enzyme activity in the absence of Apaf-1 as evidenced by bacterial expression systems, in vitro translation and biochemical analyses [7,[19][20][21][22]. More importantly, procaspase-9 is dimerized by higher concentrations of kosmotropes, salts able to stabilize proteins, such as 1 M citrate [21,22].…”

“…More importantly, procaspase-9 is dimerized by higher concentrations of kosmotropes, salts able to stabilize proteins, such as 1 M citrate [21,22]. Citric acid is the first product in the Krebs (citric acid) cycle whose reactions are controlled by the electron transfer system maintaining ∆ψm for ATP synthesis.…”