Use of the yeast three-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 been limited to the random screening of in vitro translated proteins that 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 classical two-hybrid system to the needs of heteromeric caspases for functional dissection of known interactions or screening for physiological substrates and inhibitors. Functional heteromeric caspase-1 was obtained by coexpression of p20(Cys285Ser) and p10 caspase-1 subunits that were each fused to the Gal4 DNA-binding domain. Upon coexpression of a third hybrid of the Gal4 activation domain and the viral caspase-1 pseudosubstrate inhibitors CrmA or p35, or the prototype physiological caspase-1 substrate prointerleukin-1beta, a functional Gal4 transcription factor could be reconstituted. In contrast, no interaction was found between CrmA or p35 and the immature p45 or p30 precursor forms of caspase-1. Therefore, the three-hybrid system might allow screening for new physiological substrates and inhibitors of heteromeric caspases.
Van Criekinge
W
W
Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Gent, Gent, B-9000, Belgium.
van Gurp
M
M
Decoster
E
E
Schotte
P
P
Van de Craen
M
M
Fiers
W
W
Vandenabeele
P
P
Beyaert
R
R
D001671:Biochemistry / Q000379:methods*; D020170:Caspase 1 / Q000235:genetics / Q000378:metabolism*; D061945:Caspase Inhibitors; D003594:Cytoplasmic Granules / Q000201:enzymology; D006822:Hybrid Cells; D007375:Interleukin-1 / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000378:metabolism*; D014764:Viral Proteins; D015003:Yeasts / Q000235:genetics*
D061945:Caspase Inhibitors; D007375:Interleukin-1; D011498:Protein Precursors; D011994:Recombinant Proteins; D015843:Serpins; D014764:Viral Proteins; C043830:interleukin 1 precursor; C074641:interleukin-1beta-converting enzyme inhibitor; D020170:Caspase 1