Abstract

Caspase-2 is an initiating caspase required for stress-induced apoptosis in various human cancer cells. However, it is not clear how caspase-2 exerts its apoptotic function in cells. By using both in vitro mitochondrial cytochrome c release assays and cell culture apoptosis analyses, we showed that active caspase-2, and not a catalytically mutated caspase-2, can cause cytochrome c release. Caspase-2 failed to induce cytochrome c release from mitochondria with Bid^-/- background, and the release could be restored by addition of the wild-type Bid protein, but not by a mutant Bid with the caspase-2 cleavage site mutated. Caspase-2 was not able to induce cytochrome c release from Bax^-/-Bak^-/- mitochondria either. In cultured cells, gene deletion of Bax/Bak or Bid abrogated apoptosis induced by overexpression of caspase-2. Collectively, these results indicate that proteolytic activation of Bid and the subsequent induction of the mitochondrial apoptotic pathway through Bax/Bak are essential for apoptosis triggered by caspase-2.

Caspase activation, the executing event of apoptosis, is under deliberate regulation. IAP (inhibitor of apoptosis) proteins inhibit caspase activity, whereas Smac/Diablo antagonizes IAP. XIAP, an X chromosome-linked IAP, can inhibit both caspase-9, the initiator caspase of the mitochondrial apoptotic pathway, and the downstream effector caspases, caspase-3 and caspase-7. Smac neutralizes XIAP inhibition of caspase-9 by competing for the binding of the BIR3 domain of XIAP with caspase-9. However, it is not clear how Smac liberates effector caspases from XIAP inhibition. It is generally believed that the binding of Smac with IAP generates a steric hindrance that prevents XIAP from inhibiting effector caspases, and therefore small molecule mimics of Smac are not able to reverse the inhibition of the effector caspases. Surprisingly, we showed that binding of a dimeric Smac N-terminal peptide with the BIR2 domain of XIAP effectively antagonizes inhibition of caspase-3 by XIAP. Further, we defined a dynamic and cooperative interaction of Smac with XIAP: binding of Smac with the BIR3 domain anchors the subsequent binding of Smac with the BIR2 domain, which in turn attenuates the caspase-3 inhibitory function of XIAP.

In our endeavor to investigate the regulation of autophagy and its role in cell death, we showed that histone deacetylase (HDAC) inhibitors, such as SAHA and butyrate, can induce programmed cell death in cancer cells, via two distinct pathways: mitochondria/cytochrome c-mediated apoptotic pathway and caspase-independent pathway with autophagic properties. Apaf-1 knockout, overexpression of Bcl-XL, or pharmacological inhibition of caspase activity did not prevent SAHA and butyrate-induced cell death. The cells undergoing such caspase-independent death had unambiguous morphological features of autophagic cell death.

Finally, through biochemical purification using HeLa cell extract, we have also identified the 20S proteasome as a novel activity for LC3B cleavage in vitro. This activity is highly specific to LC3B. LC3A, another isoform of LC3, as well as ATG3 and ATG10, two other autophagy-related proteins, can not be processed by the purified active 20S proteasome.