Caspase-2: controversial killer or checkpoint controller?

The caspases are an evolutionarily conserved family of cysteine proteases, with essential roles in apoptosis or inflammation. Caspase-2 was the second caspase to be cloned and it resembles the prototypical nematode caspase CED-3 more closely than any other mammalian protein. An absence of caspase-2-specific reagents and the subtle phenotype of caspase-2-deficient mice have hampered definition of the physiological role of caspase-2 and identification of factors regulating its activity. Although some data implicate caspase-2 in apoptotic pathways, a link with apoptosis has been less firmly established for caspase-2 than for some other caspases. Emerging evidence suggests that caspase-2 regulates the cell cycle and may act as a tumour suppressor. This article critically reviews the current state of knowledge regarding the biochemistry and biology of this controversial caspase.

Modeling Metazoan Apoptotic Pathways in Yeast.

This chapter describes techniques for characterizing metazoan apoptotic pathways using Saccharomyces cerevisiae. Active forms of the major apoptotic effectors-caspases, Bax and Bak-are all lethal to yeast. Using this lethality as a readout of caspase/Bax/Bak activity, proteins and small molecules that directly or indirectly regulate the activity of these effectors can be investigated in yeast, and apoptotic inhibitors can be identified using functional yeast-based screens. Caspase activity can also be monitored in yeast by cleavage-dependent liberation of a transcription factor from the plasma membrane, enabling it to activate the lacZ reporter gene. This system can be used to define the sequences that can be efficiently cleaved by particular caspases.

Analysis of the minimal specificity of caspase-2 and identification of Ac-VDTTD-AFC as a caspase-2-selective peptide substrate.

Caspase-2 is an evolutionarily conserved but enigmatic protease whose biological role remains poorly understood. To date, research into the functions of caspase-2 has been hampered by an absence of reagents that can distinguish its activity from that of the downstream apoptotic caspase, caspase-3. Identification of protein substrates of caspase-2 that are efficiently cleaved within cells may also provide clues to the role of this protease. We used a yeast-based transcriptional reporter system to define the minimal substrate specificity of caspase-2. The resulting profile enabled the identification of candidate novel caspase-2 substrates. Caspase-2 cleaved one of these proteins, the cancer-associated transcription factor Runx1, although with relatively low efficiency. A fluorogenic peptide was derived from the sequence most efficiently cleaved in the context of the transcriptional reporter. This peptide, Ac-VDTTD-AFC, was efficiently cleaved by purified caspase-2 and auto-activating caspase-2 in mammalian cells, and exhibited better selectivity for caspase-2 relative to caspase-3 than reagents that are currently available. We suggest that this reagent, used in parallel with the traditional caspase-3 substrate Ac-DEVD-AFC, will enable researchers to monitor caspase-2 activity in cell lysates and may assist in the determination of stimuli that activate caspase-2 in vivo.