Apoptosis is a process in all animals that safely removes unneeded or potentially harmful cells. Misregulation of apoptosis occurs in cancer (too little apoptosis) and many neurodegenerative diseases (too much apoptosis). Apoptosis requires the activity of caspase enzymes which are kept inhibited in living cells by IAP (inhibitor of apoptosis) proteins. Caspases are symmetric proteins with 2 catalytic sites. We investigated if self-associating 2 monomeric IAPs into a dimer (self-associated pair) enhances the IAP’s inhibition of caspases. Using recombinant DNA methods, a minimal caspase-inhibiting fragment of DIAP1 (<i>Drosophila</i> IAP1) was fused to the DNA-binding domain of a transcription factor known to dimerize cooperatively when it binds to its specific binding DNA site. Adding a duplex containing the DNA binding site was used to induce DIAP1 fragment dimerization. Three different lengths of a flexible tether were inserted between the DIAP1 fragment and DNA-binding domain to test for geometric constraints for the self-associated DIAP1 fragment to bind and inhibit a caspase. The addition of the DNA-binding site duplex to self-associate the DIAP1 fusion significantly enhanced caspase inhibition. At 0.2 μM, the fusion with the longest tether caused 32 ± 13% inhibition without the DNA and 82 ± 6% inhibition with the DNA. Similar patterns of enhanced inhibition by DNA were observed with the other fusions. A control DNA duplex containing no binding site sequence caused little to no enhanced inhibition, demonstrating that the enhanced inhibition was driven by sequence-dependent DNA binding. DNA-induced self-association of the DIAP1 fusions is being independently assessed by gel filtration chromatography. The results suggest that creating dimerization with appropriate tethers can be used to generate more potent caspase inhibitors.

Self-association enhances the potency of an inhibitor of apoptosis protein

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.