Caspase-7 Is Directly Activated by the ∼700-kDa Apoptosome Complex and Is Released as a Stable XIAP-Caspase-7 ∼200-kDa Complex

Abstract

MCF-7 cells lack caspase-3 but undergo mitochondrial-dependent apoptosis via caspase-7 activation. It is assumed that the Apaf-1-caspase-9 apoptosome processes caspase-7 in an analogous manner to that described for caspase-3. However, this has not been validated experimentally, and we have now characterized the caspase-7 activating apoptosome complex in MCF-7 cell lysates activated with dATP/cytochrome c. Apaf-1 oligomerizes to produce ϳ1.4-MDa and ϳ700-kDa apoptosome complexes, and the latter complex directly cleaves/activates procaspase-7. This ϳ700-kDa apoptosome complex, which is also formed in apoptotic MCF-7 cells, is assembled by rapid oligomerization of Apaf-1 and followed by a slower process of procaspase-9 recruitment and cleavage to form the p35/34 forms. However, procaspase-9 recruitment and processing are accelerated in lysates supplemented with caspase-3. In lysates containing very low levels of Smac and Omi/HtrA2, XIAP (X-linked inhibitor of apoptosis) binds tightly to caspase-9 in the apoptosome complex, and as a result caspase-7 processing is abrogated. In contrast, in MCF-7 lysates containing Smac and Omi/ HtrA2, active caspase-7 is released from the apoptosome and forms a stable ϳ200-kDa XIAP-caspase-7 complex, which apparently does not contain cIAP1 or cIAP2. Thus, in comparison to caspase-3-containing cells, XIAP appears to have a more significant antiapoptotic role in MCF-7 cells because it directly inhibits caspase-7 activation by the apoptosome and also forms a stable ϳ200-kDa complex with active caspase-7.The MCF-7 cell line was derived from a patient with metastatic breast cancer and is an often-used model system for studying estrogen receptor-positive breast cancer (for review, see Ref.

Caspase-9 cleavage, do you need it?

“…Taken together, the findings of Denault et al [11] agree with other studies, for example with caspase-3-null MCF-7 cell lysates and recombinant apoptosome complexes, which show that only caspase-3 and not caspase-7 produces the p37 form of caspase-9 [1]. However, the study by Denault et al [11] clearly consolidates the view that the most active form of apoptosome-bound caspase-9 contains either the p35 or p37 subunit in conjunction with the p10 subunit.…”

“…Apaf-1 oligomerizes to form the seven-spoked, wheel-like Apaf-1 apoptosome complex, which recruits caspase-9 to form the active holoenzyme caspase-activating apoptosome complex. This complex then efficiently recruits and directly cleaves procaspase-3 or -7 with high efficiency [1]. However, caspase-9 [like caspase-8 in the DISC (death-inducing signalling complex)] is an unusual caspase in that, in addition to its CARD (caspase-recruiting domain), it has a long interlinker peptide separating the small and large subunit domains.…”

MicroRNA-9 Inhibition of Cell Proliferation and Identification of Novel miR-9 Targets by Transcriptome Profiling in Breast Cancer Cells

“…Our results demonstrate that miR-9 overexpression decreases cell viability and increases apoptosis in MCF-7 breast cancer cells. As a caspase-3 null cell line (51), previous studies have shown that DEVDase activity can be increased in MCF-7 cells independent of caspase-3 whereby caspase-7 cleaves DEVD substituting caspase-3 (35,(52)(53)(54). In this study caspase-7 activity was increased, indicating pro-apoptotic activity of miR-9 in MCF-7 cells.…”

Pseudomonas aeruginosa Delays Kupffer Cell Death via Stabilization of the X-Chromosome-Linked Inhibitor of Apoptosis Protein

“…It directly binds to caspases-3, -7, and -9, rendering them inactive (7,34,35). IAPs are characterized by the presence of one or more baculovirus IAP repeat domains and a caspase activation recruitment domain.…”