Apoptotic pathways seem to be full of adaptor proteins, a recently identified example of which is the apoptosis-associated speck-like protein (ASC). But not only does ASC have both a pyrin domain and a carboxy-terminal caspase-recruitment domain (CARD) — which is quite unusual — it's now reported to associate with the pro-apoptotic protein Bax and to regulate a p53–Bax-intrinsic mitochondrial pathway of apoptosis. This work, by Ohtuka and co-workers, is presented in Nature Cell Biology.

On comparing gene expression plus or minus the tumour suppressor p53, the authors saw an induction of ASC messenger RNA and protein expression in the presence of p53. The ASC promoter contains a potential p53 recognition sequence, and further assays indeed confirmed this specific protein–DNA interaction. Several p53 target genes can affect cell-cycle arrest or death, so the authors overexpressed ASC — which caused significant apoptosis. This apoptotic effect, however, was markedly reduced in cells lacking Bax. ASC also exacerbated cell death when overexpressed in the presence of genotoxic agents.

The authors then used small interfering RNA (siRNA) to assess a direct role for ASC induction in p53- or genotoxic-stress-mediated apoptosis. A reduction in ASC expression inhibited apoptosis in both cases, which indicates that ASC is a downstream effector of p53 in DNA-damage-mediated apoptosis. As the absence of Bax inhibited ASC-induced apoptosis, and as Bax causes mitochondrial dysfunction followed by release of apoptotic stimulators, Ohtuka and co-workers investigated the effect of ASC overexpression on mitochondrial membrane potential and cytochrome c release. Bax+/+, but not Bax−/−, cells that expressed ASC had a significantly reduced membrane potential and released cytochrome c into the cytoplasm.

Most of the ASC protein localized to the mitochondrial fraction, and by analysis of deletion mutants, the authors found that the amino-terminal pyrin region was necessary for this localization. The presence of the CARD prompted Ohtuka and co-workers to assess the effect of ASC on caspases. Procaspase-2, -3 and -9 were cleaved to produce their active forms, but only when Bax was present. ASC, like Bax, therefore seems to localize to mitochondria and induce apoptosis. As both ASC and Bax have several properties in common, such as their regulation by p53, their mitochondrial and cytoplasmic localizations and their pro-apoptotic function, the authors investigated the possibility of a physical link between the two proteins, which was required in the p53–Bax-intrinsic mitochondrial apoptotic pathway. Endogenous Bax, but not other Bcl2-family proteins, interacted with endogenous ASC, again through ASC's pyrin domain, and both proteins colocalized in the cytoplasm and at mitochondria.

The all-important question, though, was whether ASC affected Bax localization. Overexpressing ASC increased the amount of Bax in the mitochondrial fraction coincident with a decrease in Bax levels in the cytoplasm. Further investigation showed that ASC expression induced a conformational change in Bax and resulted in its translocation to mitochondria. Finally, siRNA treatment against ASC inhibited the Bax conformational change and reduced the translocation of Bax to mitochondria after genotoxic stress.

So ASC functions downstream of p53 in DNA-damage-mediated apoptosis to regulate the Bax-intrinsic mitochondrial pathway. There is also emerging evidence that ASC might mediate caspase activation independently of Bax, showing that further investigation of this unusual adaptor is obviously required.