Pro-apoptotic Proteins Released from the Mitochondria Regulate the Protein Composition and Caspase-processing Activity of the Native Apaf-1/Caspase-9 Apoptosome Complex

Abstract

The apoptosome is a large caspase-activating (ϳ700 -1400 kDa) complex, which is assembled from Apaf-1 and caspase-9 when cytochrome c is released during mitochondrial-dependent apoptotic cell death. Apaf-1 the core scaffold protein is ϳ135 kDa and contains CARD (caspase recruitment domain), CED-4, and multiple (13) WD40 repeat domains, which can potentially interact with a variety of unknown regulatory proteins. To identify such proteins we activated THP.1 lysates with dATP/cytochrome c and used sucrose density centrifugation and affinity-based methods to purify the apoptosome for analysis by MALDI-TOF mass spectrometry. First, we used a glutathione S-transferase (GST) fusion protein (GST-casp9 1-130 ) containing the CARD domain of caspase-9-(1-130), which binds to the CARD domain of Apaf-1 when it is in the apoptosome and blocks recruitment/activation of caspase-9. This affinity-purified apoptosome complex contained only Apaf-1XL and GST-casp9 1-130 , demonstrating that the WD40 and CED-4 domains of Apaf-1 do not stably bind other cytosolic proteins. Next we used a monoclonal antibody to caspase-9 to immunopurify the native active apoptosome complex from cell lysates, containing negligible levels of cytochrome c, second mitochondria-derived activator of caspase (Smac), or Omi/HtrA2. This apoptosome complex exhibited low caspase-processing activity and contained four stably associated proteins, namely Apaf-1, pro-p35/34 forms of caspase-9, pro-p20 forms of caspase-3, X-linked inhibitor of apoptosis (XIAP), and cytochrome c, which was only bound transiently to the complex. However, in lysates containing Smac and Omi/HtrA2, the caspase-processing activity of the purified apoptosome complex increased 6 -8-fold and contained only Apaf-1 and the p35/p34-processed subunits of caspase-9. During apoptosis, Smac, Omi/HtrA2, and cytochrome c are released simultaneously from mitochondria, and thus it is likely that the functional apoptosome complex in apoptotic cells consists primarily of Apaf-1 and processed caspase-9.The morphological and biochemical changes of apoptotic cell death largely result from the activation of a group of cysteine aspartic acid-specific proteases known as caspases (for review see Refs. 1-3). The activation of caspases is a central feature of apoptosis, and key components of this mechanism are highly conserved throughout evolution from Caenorhabditis elegans to Drosophila melanogaster and ultimately to mammals. In C. elegans there are at least four genes, ced-3, ced-4, ced-9, and egl-1, which are critical for the execution of apoptotic cell death. In C. elegans, CED-4 is normally bound to mitochondria and cell membranes by CED-9 (4). Up-regulation of EGL-1 inactivates CED-9, releasing CED-4, which oligomerizes/recruits and induces autocatalytic processing and activation of CED-3 (5). ced-3 encodes for a cysteine protease, which is homologous to interleukin-1␤-converting enzyme or ICE, now known as caspase-1. Thirteen other caspases have now been identified in mammals, some of which are involved in...

Subcellular localization of Apaf-1 in apoptotic rat pituitary cells

“…1C and 4C). These data are in agreement with observations obtained from cell lysates (22,28). In cell lysates, the interacting proteins may bind less tightly once the apoptosome holoenzyme complex is formed and may be lost during purification (28).…”

Cellular Caspases: New Targets for the Action of Pharmacological Agents

“…Pore opening can be stimulated by inorganic phosphate, caspases, SH-reagents, cell exhaustion by reduced glutathione, formation of active forms of oxygen, uncoupling of oxidative phosphorylation, rise in Ca 2+ content in the cytoplasm, effect of ceramide, depletion of the mitochondrial ATP pool, etc. [24,27,28].…”

“…Procaspase-9 gets access to the CARD of Apaf-1. The thus activated caspase-9 recruits procaspase-3, which is, in turn, activated to form caspase-3 [24,25].…”

“…The release is promoted by Bcl-2 family proteins (Вах-protein). The flavoprotein AIF released from the mitochondrial intermembrane region is an apoptosis effector that would then act independently of caspases [24,29].…”

Cardiolipin drives cytochrome c proapoptotic and antiapoptotic actions

“…These events, tightly regulated by several heat shock proteins ( Fig. 1), allow for the catalytic maturation of caspase 3 and other caspases, which eventually mediate the biochemical and morphological features of apoptosis (9)(10)(11)(12)(13).…”