- Expression, preparation, and high-throughput screening of caspase-8: discovery of redox-based and steroid diacid inhibition.
Expression, preparation, and high-throughput screening of caspase-8: discovery of redox-based and steroid diacid inhibition.
Because of the intimate role of caspase-8 in apoptosis signaling pathways from FAS, TNFR1, and other death receptors, the enzyme is a potentially important therapeutic target. We have generated an Escherichia coli expression construct for caspase-8 in which a His-tag sequence is inserted ahead of codon 217 of caspase-8. The strain produced a significant amount of soluble His-tagged 31-kDa inactive single-chain enzyme precursor. This 31-kDa protein could be purified to 98% purity. Hydroxyapatite resolved the enzyme into two species, one with the appropriate 31,090 relative mass and the other with 178 units additional mass. The latter proved to result from E. coli-based modification of the His-tag with one equivalent of glucono-1,5-lactone. The purified proteins could be activated by autoproteolysis to the appropriate 19- plus 11-kDa enzyme by the addition of dithiothreitol in appropriate buffer conditions. This yielded an enzyme with specific activity of 4-5 units/mg against 200 microM Ac-IETD-pNA at 25 degrees C. The fully active protein was used in a high-throughput screen for inhibitors of caspase-8. A preliminary robustness screen demonstrated that caspase-8 is susceptible to reactive oxygen-based inactivation in the presence of dithiothreitol (DTT) but not in the presence of cysteine. Investigation into the mechanism of this inactivation showed that quinone-like compounds were reduced by DTT establishing a reactive oxygen generating redox cycle the products of which (likely H(2)O(2)) inactivated the enzyme. A new class of caspase-8 inhibitors, steroid-derived diacids, with affinity in the low micromolar range were discovered in the refined screen. Structure--activity investigation of the inhibitors showed that both the steroid template and the acid moieties were required for activity.