USE OF THE YEAST 3-HYBRID SYSTEM AS A TOOL TO STUDY CASPASES

Caspases are a family of heteromeric (p20/p10) cysteine proteases with important functions in the regulation of apoptosis and inflammation. Up to now, tools to identify new substrates for caspases have mostly b een limited to the random screening of in vitro translated proteins th at are known, or assumed, to play a role in apoptosis. We describe the use of a yeast three-hybrid approach as a tool that adapts the classi cal two-hybrid system to the needs of heteromeric caspases for functio nal dissection of known interactions or screening for physiological su bstrates and inhibitors. Functional heteromeric caspase-1 was obtained by coexpression of p20(Cys285Ser) and p10 caspase-1 subunits that mer e each fused to the Ga14 DNA-binding domain. Upon coexpression of a th ird hybrid of the Ga14 activation domain and the viral caspase-1 pseud osubstrate inhibitors CrmA or p35, or the prototype physiological casp ase-1 substrate prointerleukin-1 beta, a functional Ga14 transcription factor could be reconstituted. In contrast, no interaction was found between CrmA or p35 and the immature p45 or p30 precursor forms of cas pase-1. Therefore, the three-hybrid system might allow screening for n ew physiological substrates and inhibitors of heteromeric caspases. (C ) 1998 Academic Press.