Abstract:

XIAP, a member of the Inhibitor of Apoptosis Proteins, is an anti-apoptotic protein which inhibits controlled cell death, apoptosis, by non-covalently binding pro-apoptotic proteases called caspases. XIAP is inhibited during apoptosis by the Smac (Secondary Mitochondrial Activator of Caspases), which binds to XIAP's BIR3 domain through its four N-terminal residues (AVPI). However, in some cancers, XIAP is upregulated and can overwhelm Smac's regulatory response. Therefore, the development of small molecule XIAP inhibitors based on Smac's N-terminal sequence could be valuable in freeing caspases to facilitate apoptosis. Earlier work has shown that tetrapeptides based on Smac's sequence bind as well as or better than Smac. This thesis seeks to further characterize the interaction of peptides to the BIR3 domain.

A cell permeating probe was developed using the fluorophore badan to induce transport. This probe contained a Smac-like sequence for binding to XIAP and a lysine/DOTA conjugate. DOTA is a multidentate lanthanide chelator, capable of binding lanthanide ions with high affinity. Confocal microscopy was used to image the accumulation of this badan labeled peptide within cells. To date, this is believed to be the first example of lanthanide ion transport into living cells without the aid of a high molecular weight transport agent.

Probes were also developed to "map" the fluorescent aromatic residues within the BIR3 binding pocket. The AVPI sequence was modified to carry terbium ions at several different points within the sequence. Terbium ions are intrinsically fluorescent and can act as good fluorescent acceptors due to their large Stokes shift. By exciting tryptophan residues at 295 nm or both tryptophan and tyrosine residues at 280 nm, FRET (fluorescence resonance energy transfer) can be used to determine the distance from aromatic residues within BIR3 to the terbium ion on a binding peptide. By varying the location of the terbium ion, the relative distances to BIR3 aromatic residues can be mapped. Efforts are underway to apply this technique to other peptide binding proteins without known structures as a means to identify and locate tryptophan and tyrosine residues within unknown binding sites.