A New Model for the Transition of APAF-1 from Inactive Monomer to Caspase-activating Apoptosome

Abstract

The cytosolic adaptor protein Apaf-1 is a key player in the intrinsic pathway of apoptosis. Binding of mitochondrially released cytochrome c and of dATP or ATP to Apaf-1 induces the formation of the heptameric apoptosome complex, which in turn activates procaspase-9. We have re-investigated the chain of events leading from monomeric autoinhibited Apaf-1 to the functional apoptosome in vitro. We demonstrate that Apaf-1 does not require energy from nucleotide hydrolysis to eventually form the apoptosome. Despite a low intrinsic hydrolytic activity of the autoinhibited Apaf-1 monomer, nucleotide hydrolysis does not occur at any stage of the process. Rather, mere binding of ATP in concert with the binding of cytochrome c primes Apaf-1 for assembly. Contradicting the current view, there is no strict requirement for an adenine base in the nucleotide. On the basis of our results, we present a new model for the mechanism of apoptosome assembly.Cells of most multicellular organisms can terminate themselves conducting the evolutionary conserved program of apoptosis. As the well regulated response to a death signal from outside or inside the cell, a cascade of cysteine proteases, socalled caspases, is activated that eventually leads to the orderly disintegration of cell organelles and chromatin (1-3). In contrast to necrotic cell death, which is accompanied by inflammation and damage of the surrounding tissue, apoptotic cells keep their degraded constituents contained in intact membrane. The cell corpses are then cleanly removed by phagocytes obviating inflammatory and autoimmune responses (4). Apoptosis is vital for development and homeostasis of metazoans (5-7), and malfunction of the human apoptosis machinery is implicated in autoimmune diseases, neurodegenerative disorders, and cancer (8 -11).The intrinsic pathway of apoptosis responds to cytotoxic stress, genomic damage, and developmental cues (12, 13). The pivotal point of this pathway is the release of cytochrome c from the mitochondria through permeabilization of the outer membrane (14, 15). In human cells, transduction of this decisive death signal is achieved via the 142-kDa cytosolic protein Apaf-1, the mammalian ortholog of Caenorhabditis elegans CED-4 (16). Apaf-1 contains an N-terminal caspase recruitment domain (CARD), 2 a central nucleotide binding and oligomerization domain (NOD), and a C-terminal regulatory domain of 13 WD40 repeats. It is thought that the WD40 repeat domain blocks the CARD and NOD until cytochrome c is released into the cytosol (17-20). Binding of cytochrome c to the WD40 repeat domain in the presence of ATP or dATP appears to induce a large conformational change in Apaf-1 that eventually bares the NOD and the effector-binding CARD. Mediated by the NOD, Apaf-1 oligomerizes into a wheel-like heteroheptameric protein complex (21) termed apoptosome. The apoptosome recruits the initiator caspase-9 via homophilic CARD-CARD interaction and the holoenzyme then activates downstream executioner caspases (22-24).So far, there is no consistent view...

The DNA repair complex Ku70/86 modulates Apaf1 expression upon DNA damage

“…Apaf1 normally being in an inactive conformation, its oligomerization is caused by cytochrome c release from mitochondria, after apoptotic stimuli, in the presence of dATP/ATP. 1 Seven Apaf1 molecules generate the apoptosome, 2,3 which is able to recruit the procaspase 9 and allow its activation. 4 In such a multimeric complex, the apoptosome activates executioner caspases, which orchestrate the 'safe' destruction of the cell.…”

Loss of Proapoptotic Gene Apaf-1 Expression in Pancreatic Cancer

“…Atsipalaidavęs citochromas C iš mitochondrijų ir dalyvaujant dATP/ ATP sukelia APAF-1 molekulių oligomerinio komplekso susidarymą. Septynios APAF-1 molekulės suformuoja apoptosomą, kuri jungiasi su prokaspaze-9 ir ją aktyvuoja, paleidžiant tolimesnę kaspazių aktyvavimo kaskadą, kuri pasibaigia ląstelės apoptoze [5]. Pažaidos apoptosomos susidarymo mechanizme siejamos su vėžio vystymusi ir atsparumu chemoterapiniam gydymui [6].…”

A Systems Biology Analysis of Apoptosome Formation and Apoptosis Execution Supports Allosteric Procaspase-9 Activation

“…Affinities and rate constants for the interaction of cytochrome c and Apaf-1 were published previously (30,31). k on and k off values for the interaction of dATP and Apaf-1 were calculated from the published K d (32) and the rate of dATP turnover by Apaf-1 (29). Furthermore, we allowed activated Apaf-1 monomers to fall back into their auto-inhibited state (29).…”

“…k on and k off values for the interaction of dATP and Apaf-1 were calculated from the published K d (32) and the rate of dATP turnover by Apaf-1 (29). Furthermore, we allowed activated Apaf-1 monomers to fall back into their auto-inhibited state (29). A list of all reactions and their parameterization is provided as supplemental Tables 1-4.…”

“…For simplicity, dATP and ATP were implemented as one species in the model. Because the binding order of cytochrome c and dATP to Apaf-1 is interchangeable (29), both reaction sequences were implemented in the core model. Affinities and rate constants for the interaction of cytochrome c and Apaf-1 were published previously (30,31).…”