Random mutagenesis of PDZOmi domain and selection of mutants that specifically bind the Myc proto-oncogene and induce apoptosis

Omi is a mammalian serine protease that is localised in the mitochondria and released to the cytoplasm in response to apoptotic stimuli. Omi induces cell death in a caspase-dependent manner by interacting with the Xchromosome linked inhibitor of apoptosis protein, as well as in a caspase-independent way that relies on its proteolytic activity. Omi is synthesized as a precursor polypeptide and is processed to an active serene protease with a unique PDZ domain. PDZ domains recognise the extreme carboxyl terminus of target proteins. Internal peptides that are able to fold into a b-finger are also reported to bind some PDZ domains. Using a modified yeast two-hybrid system, PDZOmi mutants were isolated by their ability to bind the carboxyl terminus of human Myc oncoprotein in yeast as well as in mammalian cells. One such PDZm domain (PDZ-M1), when transfected into mammalian cells, was able to bind to endogenous Myc protein and induce cell death. PDZ-M1-induced apoptosis was entirely dependent on the presence of Myc protein and was not observed when c-myc null fibroblasts were used. Our studies indicate that the PDZ domain of
Omi can provide a prototype that could easily be exploited to target specifically and inactivate oncogenes by binding to their unique carboxyl terminus.