Abstract 1917 Quantifying Enhanced Caspase Inhibition by Self-Associating an Inhibitor of Apoptosis Protein

One of the main regulatory pathways of apoptosis is the protein-protein interaction between inhibitor of apoptosis proteins (IAPs) and caspases, the enzymes that trigger intracellular cascades to initiate apoptosis. Caspases are multimers that contain two active sites, each of which can be bound and inhibited by an individual IAP. This project investigates if converting IAPs into dimers (pairs) can increase their potency for inhibiting caspases. An IAP dimer could have greater affinity for binding a caspase and it could potentially inhibit both caspase active sites in one binding event. To create a dimerizeable IAP, a caspase-inhibiting IAP fragment was fused to the DNA-binding domain of the transcription factor HAP1 (heme activation protein 1) which dimerizes when it binds cooperatively to its DNA site. Initial studies indicated that adding HAP1 binding site DNA did enhance the caspase inhibition by this fusion. Here we rigorously validate the HAP1 dimerization method and quantify the degree of enhanced caspase inhibition caused by dimerization. Validation was achieved using a maltose binding protein (MBP)-HAP1 fusion. In a fluorescence anisotropy assay, titrations of MBP-HAP1 binding to a labeled HAP1 site DNA were sigmoidal and fit by a model for cooperative binding of 2 proteins that yielded a KD of 45 nM. In gel filtration chromatography, mixing MBP-HAP1 with the HAP1 binding site DNA resulted in a complex eluting with a size consistent with being a dimer or larger with little to no monomer complex evident. Furthermore, the multimer complex did not dilute into monomer complexes when excess DNA was mixed with the protein which confirmed cooperativity in DNA binding. For testing caspase inhibition, three IAP-HAP1 fusions were created with variable tether lengths of 0 (no tether), 15 (short tether), or 30 (long tether) residues placed between the IAP and HAP1. In a caspase kinetics assay, all three fusions showed enhanced inhibition when HAP1 binding site DNA was added. Adding HAP1 site DNA to 10 nM of any of the fusions doubled the level of caspase inhibition compared with no DNA or with a DNA control containing no HAP1 binding site. When the IAP-HAP1 fusions were titrated to the caspase, 60% inhibition of caspase shifted from ∼30 nM IAP-HAP1 in the absence of HAP1 site DNA (or with control DNA) to ∼6 nM in the presence of HAP1 site DNA, a 5-fold enhancement in inhibition. No significant differences were observed among the different tether lengths suggesting no geometric constraints for the dimer inhibiting the caspase. The results suggest that self-association can be used to enhance the potency of inhibitors that target caspases or other dimeric enzymes. This work was funded by a Kutztown University Research Committee Grant to M. Junker.