For nearly 2 decades, investigators have debated whether cysteinyl-aspartate-specific protease 9 (caspase-9) is activated within the apoptotic protease-activating factor 1 (Apaf-1) apoptosome through proximityinduced homodimerization or through formation of a holoenzyme. Recently, we have demonstrated that caspase-9 forms (and likely transitions between) both caspase-9 homo-and Apaf-1:caspase-9 heterodimers, each of which plays unique roles in the recruitment and activation of caspase-9.
KEYWORDSApaf-1; apoptosome; caspase-3; caspase-9; molecular timer Mitochondrial outer membrane permeabilization, release of cytochrome c, and formation of the Apaf-1-caspase-9 apoptosome are critical events in stress-induced apoptosis.1 Nevertheless, due to difficulties associated with its reconstitution, how caspase-9 is activated within the apoptosome remains hotly contested.1 According to dogma, all initiator caspases are activated via proximity-induced homodimerization, and indeed, several studies suggest that the apoptosome serves merely as a platform to recruit and increase the local concentration of caspase-9, sufficient to overcome its relatively weak binding affinity.2,3 Other studies, however, suggest that the apoptosome is a holoenzyme in which oligomerized Apaf-1 acts as a positive allosteric regulator of caspase-9, promoting unique conformational changes in the protease that lead to its activation. [4][5][6][7] Importantly, unlike other initiator caspases that are released from their adaptor proteins as active proteases, caspase-9 must remain bound to the apoptosome to exhibit significant catalytic activity. Furthermore, cleavage of its intersubunit linker is not required for its activation, 8 and in fact, processing of procaspase-9 to its 2-chain (p35/p12) form reduces its affinity for the apoptosome, resulting in its dissociation from the complex. Continuous recruitment of procaspase-9 is therefore required to maintain apoptosome activity. 9 Based on these findings, we previously proposed that the Apaf-1 apoptosome functions as a proteolytic-based "molecular timer." In this model, the intracellular concentration of procaspase-9 sets the overall duration of the timer, procaspase-9 autoprocessing activates the timer, and the rate at which caspase-9-p35/p12 dissociates from the complex (and thus loses its activity) determines how fast the timer "ticks" over. Notably, since the prodomains of pro-and processed caspase-9 are identical and interact with the same caspase recruitment domain (CARD) in Apaf-1, it has remained a mystery why processed caspase-9, in particular, dissociates from the apoptosome. We recently hypothesized that caspase-9 cleavage might diminish its ability to homodimerize, resulting in a decrease in its overall avidity for the complex (Fig. 1A). To assess the abilities of pro-and processed caspase-9 to form active homodimers, we used a variety of biophysical and biochemical techniques, including size-exclusion chromatography coupled to multiangle light scattering (SEC-MALS), a surfaceplasmon...