Van Criekinge W, van Gurp M, Decoster E, Schotte P, Van de Craen M, Fiers W, Vandenabeele P, Beyaert R
Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Gent, Gent, B-9000, Belgium.
Anal Biochem. 1998 Oct 1;263(1):62-6. doi: 10.1006/abio.1998.2823.
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.
半胱天冬酶是一类异源二聚体（p20/p10）半胱氨酸蛋白酶，在细胞凋亡和炎症调节中具有重要功能。到目前为止，用于鉴定半胱天冬酶新底物的工具大多局限于对已知或假定在细胞凋亡中起作用的体外翻译蛋白质进行随机筛选。我们描述了一种酵母三杂交方法的应用，该方法将经典的双杂交系统进行了调整，以满足异源二聚体半胱天冬酶对已知相互作用进行功能剖析或筛选生理底物及抑制剂的需求。通过共表达分别与Gal4 DNA结合结构域融合的p20（Cys285Ser）和p10半胱天冬酶-1亚基，获得了功能性异源二聚体半胱天冬酶-1。当共表达Gal4激活结构域与病毒半胱天冬酶-1假底物抑制剂CrmA或p35，或原型生理半胱天冬酶-1底物白细胞介素-1β前体的第三个杂交体时，可重建功能性Gal4转录因子。相比之下，未发现CrmA或p35与半胱天冬酶-1的未成熟p45或p30前体形式之间存在相互作用。因此，三杂交系统可能有助于筛选异源二聚体半胱天冬酶的新生理底物和抑制剂。
