Molecular determinants of caspase-9 activation by the Apaf-1 apoptosome.
ABSTRACT: Autocatalytic activation of an initiator caspase triggers the onset of apoptosis. In dying cells, caspase-9 activation is mediated by a multimeric adaptor complex known as the Apaf-1 apoptosome. The molecular mechanism by which caspase-9 is activated by the Apaf-1 apoptosome remains largely unknown. Here we demonstrate that the previously reported 1:1 interaction between Apaf-1 caspase recruitment domain (CARD) and caspase-9 CARD is insufficient for the activation of caspase-9. Rather, formation of a multimeric CARD:CARD assembly between Apaf-1 and caspase-9, which requires three types of distinct interfaces, underlies caspase-9 activation. Importantly, an additional surface area on the multimeric CARD assembly is essential for caspase-9 activation. Together, these findings reveal mechanistic insights into the activation of caspase-9 by the Apaf-1 apoptosome and support the induced conformation model for initiator caspase activation by adaptor complexes.
Project description:Apoptosis is executed by a cascade of caspase activation. The autocatalytic activation of an initiator caspase, exemplified by caspase-9 in mammals or its ortholog, Dronc, in fruit flies, is facilitated by a multimeric adaptor complex known as the apoptosome. The underlying mechanism by which caspase-9 or Dronc is activated by the apoptosome remains unknown. Here we report the electron cryomicroscopic (cryo-EM) structure of the intact apoptosome from Drosophila melanogaster at 4.0 Å resolution. Analysis of the Drosophila apoptosome, which comprises 16 molecules of the Dark protein (Apaf-1 ortholog), reveals molecular determinants that support the assembly of the 2.5-MDa complex. In the absence of dATP or ATP, Dronc zymogen potently induces formation of the Dark apoptosome, within which Dronc is efficiently activated. At 4.1 Å resolution, the cryo-EM structure of the Dark apoptosome bound to the caspase recruitment domain (CARD) of Dronc (Dronc-CARD) reveals two stacked rings of Dronc-CARD that are sandwiched between two octameric rings of the Dark protein. The specific interactions between Dronc-CARD and both the CARD and the WD40 repeats of a nearby Dark protomer are indispensable for Dronc activation. These findings reveal important mechanistic insights into the activation of initiator caspase by the apoptosome.
Project description:During stress-induced apoptosis, the initiator caspase-9 is activated by the Apaf-1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase-9 is paradoxically observed outside the complex. We show herein that apoptosome-mediated cleavage of procaspase-9 occurs exclusively through a CARD-displacement mechanism, so that unlike the effector procaspase-3, procaspase-9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase-9 possessed higher affinity for the apoptosome and could displace the processed caspase-9 from the complex, thereby facilitating a continuous cycle of procaspase-9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase-9 per se contributed little to the activation of procaspase-3. Thus, the Apaf-1 apoptosome functions as a proteolytic-based 'molecular timer', wherein the intracellular concentration of procaspase-9 sets the overall duration of the timer, procaspase-9 autoprocessing activates the timer, and the rate at which the processed caspase-9 dissociates from the complex (and thus loses its capacity to activate procaspase-3) dictates how fast the timer 'ticks' over.
Project description:The apoptosome is a platform that activates apical procaspases in response to intrinsic cell death signals. Biochemical and structural studies in the past two decades have extended our understanding of apoptosome composition and structure, while illuminating the requirements for initiator procaspase activation. A number of studies have now provided high-resolution structures for apoptosomes from C. elegans (CED-4), D. melanogaster (Dark), and H. sapiens (Apaf-1), which define critical protein interfaces, including intra and interdomain interactions. This work also reveals interactions of apoptosomes with their respective initiator caspases, CED-3, Dronc and procaspase-9. Structures of the human apoptosome have defined the requirements for cytochrome c binding, which triggers the conversion of inactive Apaf-1 molecules to an extended, assembly competent state. While recent data have provided a detailed understanding of apoptosome formation and procaspase activation, they also highlight important evolutionary differences with functional implications for caspase activation. Comparison of the CARD/CARD disks and apoptosomes formed by CED-4, Dark and Apaf-1. Cartoons of the active states of the CARD-CARD disks, illustrating the two CED-4 CARD tetrameric ring layers (CED4a and CED4b; top row) and the binding of 8 Dronc CARDs and between 3-4 pc-9 CARDs, to the Dark and Apaf-1 CARD disk respectively (middle and lower rows). Ribbon diagrams of the active CED-4, Dark and Apaf-1 apoptosomes are shown (right column).
Project description:Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full length Apaf-1 and a single particle, electron density map at ~9.5 Å resolution. The apoptosome model includes N-terminal domains of Apaf-1, cognate ?-propellers, and cytochrome c. A direct comparison of Apaf-1 in the apoptosome and as a monomer reveals conformational changes that occur during the first two steps of assembly. This includes an induced-fit mechanism for cytochrome c binding to regulatory ?-propellers, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange. These linked conformational changes create an extended Apaf-1 monomer and drive apoptosome assembly. Moreover, the N-terminal CARD in the inactive Apaf-1 monomer is not shielded from other proteins by ?-propellers. Hence, the Apaf-1 CARD may be free to interact with a procaspase-9 CARD either before or during apoptosome assembly. Irrespective of the timing, the end product of assembly is a holo-apoptosome with an acentric CARD-CARD disk and tethered pc-9 catalytic domains. Subsequent activation of pc-9 leads to a proteolytic cascade and cell death.
Project description:Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is indispensable for caspase-9 activation by together forming a holoenzyme. The molecular mechanism of caspase-9 activation remains largely enigmatic. Here, we report the cryoelectron microscopy (cryo-EM) structure of an apoptotic holoenzyme and structure-guided biochemical analyses. The caspase recruitment domains (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: a 4:4 complex docks onto the central hub of the apoptosome, and a 2:1 complex binds the periphery of the central hub. The interface between the CARD complex and the central hub is required for caspase-9 activation within the holoenzyme. Unexpectedly, the CARD of free caspase-9 strongly inhibits its proteolytic activity. These structural and biochemical findings demonstrate that the apoptosome activates caspase-9 at least in part through sequestration of the inhibitory CARD domain.
Project description:Apoptosis is induced by caspases, which are members of the cysteine protease family. Caspases are synthesized as inactive zymogens and initiator caspases first gain activity by associating with an oligomeric complex of their adaptor proteins, such as the apoptosome. Activated initiator caspases subsequently cleave and activate effector caspases. Although such a proteolytic cascade would predict that a small number of active caspases could irreversibly amplify caspase activity and trigger apoptosis, many cells can maintain moderate levels of caspase activity to perform non-apoptotic roles in cellular differentiation, shape change and migration. Here we show that the Drosophila melanogaster apoptosome engages in a feedback inhibitory loop, which moderates its activation level in vivo. Specifically, the adaptor protein Apaf-1 lowers the level of its associated initiator caspase Dronc, without triggering apoptosis. Conversely, Dronc lowers Apaf-1 protein levels. This mutual suppression depends on the catalytic site of Dronc and a caspase cleavage site within Apaf-1. Moreover, the Drosophila inhibitor of apoptosis protein 1 (Diap1) is required for this process. We speculate that this feedback inhibition allows cells to regulate the degree of caspase activation for apoptotic and non-apoptotic purposes.
Project description:Caspases are critical for the initiation and execution of apoptosis. Nitric oxide (NO) or derived species can prevent programmed cell death in several cell types, reportedly through S-nitrosation and inactivation of active caspases. Although we find that S-nitrosation of caspases can occur in vitro, our study questions whether this post-translational modification is solely responsible for NO-mediated inhibition of apoptosis. Indeed, using Jurkat cells as a model system, we demonstrate that NO donors block Fas- and etoposide-induced caspase activation and apoptosis (downstream of mitochondrial membrane depolarization) and cytochrome c release. However, caspase activity was not restored by the strong reducing agent dithiothreitol, as predicted for S-nitrosation reactions, thereby excluding active-site-thiol modification of caspases as the only anti-apoptotic mechanism of NO donors in cells. Rather, we observed that processing of procaspases-9, -3 and -8 was decreased due to ineffective formation of the Apaf-1/caspase-9 apoptosome. Gel-filtration and in vitro binding assays indicated that NO donors inhibit correct assembly of Apaf-1 into an active approx. 700 kDa apoptosome complex, and markedly attenuate caspase-recruitment domain (CARD)-CARD interactions between Apaf-1 and procaspase-9. Therefore we suggest that NO or a metabolite acts directly at the level of the apoptosome and inhibits the sequential activation of caspases-9, -3 and -8, which are required for both stress- and receptor-induced death in cells that use the mitochondrial subroute of cell demise.
Project description:Apaf-1-like molecules assemble into a ring-like platform known as the apoptosome. This cell death platform then activates procaspases in the intrinsic cell death pathway. In this review, crystal structures of Apaf-1 monomers and CED-4 dimers have been combined with apoptosome structures to provide insights into the assembly of cell death platforms in humans, nematodes, and flies. In humans, the caspase recognition domains (CARDs) of procaspase-9 and Apaf-1 interact with each other to form a CARD-CARD disk, which interacts with the platform to create an asymmetric proteolysis machine. The disk tethers multiple pc-9 catalytic domains to the platform to raise their local concentration, and this leads to zymogen activation. These findings have now set the stage for further studies of this critical activation process on the apoptosome.
Project description: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.
Project description:The Drosophila Apaf-1 related killer forms an apoptosome in the intrinsic cell death pathway. In this study we show that Dark forms a single ring when initiator procaspases are bound. This Dark-Dronc complex cleaves DrICE efficiently; hence, a single ring represents the Drosophila apoptosome. We then determined the 3D structure of a double ring at ?6.9 Å resolution and created a model of the apoptosome. Subunit interactions in the Dark complex are similar to those in Apaf-1 and CED-4 apoptosomes, but there are significant differences. In particular, Dark has "lost" a loop in the nucleotide-binding pocket, which opens a path for possible dATP exchange in the apoptosome. In addition, caspase recruitment domains (CARDs) form a crown on the central hub of the Dark apoptosome. This CARD geometry suggests that conformational changes will be required to form active Dark-Dronc complexes. When taken together, these data provide insights into apoptosome structure, function, and evolution.