Monomerization of XIAP by Executioner Caspases

Abstract

A class of cysteine proteases known as caspases are responsible for a physiological process called Apoptosis. These enzymes are classified as initiator and executioner caspases based on the stage they enter the apoptosis process. The initiator caspases of −8 and −10 become activated by extracellular signals in the extrinsic apoptosis pathway and initiator caspase-9 is activated by intracellular signals during the intrinsic apoptosis pathway. These pathways intersect at the executioner caspase levels which consists of −3 and −7. Caspases involved in the intrinsic apoptotic pathway possess a tetrapeptide motif called IAP binding Motif, IBM, which plays crucial role in antagonizing and regulating their activities by Inhibitor of Apoptosis Proteins, IAPs. This evolutionary conserved motif is found at the N-termini of the small subunits of caspase-9, −7, and −3. We also identified an IBM motif at the large subunit of caspase-7 which showed higher affinity toward XIAP as compared to its small subunit. We therefore hypothesized that as far as XIAP interaction is concerned, caspase-3 and caspase-7 should behave differently. To decipher XIAP-caspase interactions size exclusion high pressured liquid chromatography was performed. The retention time analysis revealed that procaspase-3 is a dimeric protein as reported previously. However active caspase-3 as well as caspase-7 appeared as heterodimer of the large and the small subunits as opposed to a homodimer of a heterodimer. XIAP protein consisting of the BIR1-2-3 domains on the other hand appeared as dimer. Interestingly, interaction of either caspase-3 or caspase-7 caused XIAP dissociation into monomers while caspases maintaining their monomeric states. Given that dimerization is required for caspases activation it is intriguing to propose that one mechanism for caspase inhibition by XIAP is through locking the executioner caspases in the monomeric state.