The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities.

Chu-Chiao Wu,Sunhee Lee,Srinivas Malladi,Miao-Der Chen,Nicholas J. Mastrandrea,Shawn B. Bratton,Zhiwen Zhang

doi:10.1038/ncomms13565

Abstract

According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.

Highlights

According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the apoptosis protease-activating factor 1 (Apaf-1) apoptosome
As C9 transitions from a bound monomer, which associates with the apoptosome solely through prodomain-Apaf-1 caspase recruitment domains (CARDs) interactions, to a bound dimer that interacts via its small subunits, its affinity for the complex should increase markedly (Fig. 1a, step 2)
We developed and utilized a number of novel techniques to formally demonstrate, for the first time, that ProC9 is recruited to the apoptosome through a network of interactions, involving the formation of a ProC9 homodimer and two independent prodomain-Apaf-1 CARD interactions (Figs 1–4)

Summary

Introduction

Initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Monomeric ‘initiator’ caspases (cysteine proteases), possessing long prodomains, such as caspases-8, -9 and -10, are recruited to and activated within large caspase-activating complexes composed of specific adapter proteins[1]. These apical proteases in turn activate ‘effector’ caspases-3, -6 and -7, present in the cell as constitutive dimers, through cleavage of the intersubunit linkers connecting their large and small subunits. Once activated, these downstream caspases dismantle the cell through targeted cleavage of hundreds of substrates[1,2]. In the present study, using a combination of novel biochemical approaches, we demonstrate that both proximity-induced homodimerization and allosteric regulation of monomeric C9 play unique and essential roles in apoptosome function and are integrated well within the overall model of the apoptosome as a molecular timer

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Conclusion