Changes in Apaf-1 Conformation That Drive Apoptosome Assembly
ABSTRACT 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 β-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 β-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 β-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.
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ABSTRACT: Cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (c-FLIP) is a major antiapoptotic protein and an important cytokine and chemotherapy resistance factor that suppresses cytokine- and chemotherapy-induced apoptosis. c-FLIP is expressed as long (c-FLIPL), short (c-FLIPS), and c-FLIPR splice variants in human cells. c-FLIP binds to FADD and/or caspase-8 or -10 and TRAIL receptor 5 (DR5). This interaction in turn prevents Death-Inducing Signaling Complex (DISC) formation and subsequent activation of the caspase cascade. c-FLIPL and c-FLIPS are also known to have multifunctional roles in various signaling pathways, as well as activating and/or upregulating several cytoprotective and pro-survival signaling proteins including Akt, ERK, and NF-κB. In addition to its role in apoptosis, c-FLIP is involved in programmed necroptosis (necrosis) and autophagy. Necroptosis is regulated by the Ripoptosome, which is a signaling intracellular cell death platform complex. The Ripoptosome contains receptor-interacting protein-1/Receptor-Interacting Protein-3 (RIP1), caspase-8, caspase-10, FADD, and c-FLIP isoforms involved in switching apoptotic and necroptotic cell death. c-FLIP regulates the Ripoptosome; in addition to its role in apoptosis, it is therefore also involved in necrosis. c-FLIPL attenuates autophagy by direct acting on the autophagy machinery by competing with Atg3 binding to LC3, thereby decreasing LC3 processing and inhibiting autophagosome formation. Upregulation of c-FLIP has been found in various tumor types, and its silencing has been shown to restore apoptosis triggered by cytokines and various chemotherapeutic agents. Hence, c-FLIP is an important target for cancer therapy. This review focuses on (1) the anti-apoptotic role of c-FLIP splice variants in preventing apoptosis and inducing cytokine and chemotherapy drug resistance, as well as its roles in necrosis and autophagy, and (2) modulation of c-FLIP expression as a means to enhance apoptosis and modulate necrosis and autophagy in cancer cells.01/2013; Suppl 6. DOI:10.4172/2157-2518.S6-003
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ABSTRACT: 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.Structure 04/2013; 21(4):501-15. DOI:10.1016/j.str.2013.02.024 · 5.62 Impact Factor
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ABSTRACT: NOD-like receptor (NLR) proteins oligomerize into multiprotein complexes termed "inflammasomes" when activated. Their autoinhibition mechanism remains poorly defined. Here, we report the crystal structure of the mouse NLRC4 in a closed form. The ADP-mediated interaction between the central nucleotide-binding domain (NBD) and the winged-helix domain (WHD) was critical for stabilizing the closed conformation of NLRC4. The helical domain (HD2) repressively contacted a conserved and functionally important α-helix of the NBD. The C-terminal leucine-rich repeat (LRR) domain is positioned to sterically occlude one side of the NBD domain and consequently sequester NLRC4 in a monomeric state. Disruption of ADP-mediated NBD-WHD or NBD-HD2/NBD-LRR interactions resulted in constitutive activation of NLRC4. Together, our data reveal the NBD-organized cooperative autoinhibition mechanism of NLRC4 and provide insight into its activation.Science 06/2013; 341(6142). DOI:10.1126/science.1236381 · 33.61 Impact Factor