Random mutagenesis of PDZ(Omi) domain and selection of mutants that specifically bind the Myc proto-oncogene and induce apoptosis.
Omi is a mammalian serine protease that is localized 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 X-chromosome 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 serine protease with a unique PDZ domain. PDZ domains recognize the extreme carboxyl terminus of target proteins. Internal peptides that are able to fold into a beta-finger are also reported to bind some PDZ domains. Using a modified yeast two-hybrid system, PDZ(Omi) 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 PDZ(m) 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.
Junqueira
Daniela
D
Biomolecular Science Center, University of Central Florida, 12722 Research Parkway, Orlando, FL 32826, USA.
Cilenti
Lucia
L
Musumeci
Lucia
L
Sedivy
John M
JM
Zervos
Antonis S
AS
D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D006367:HeLa Cells; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D007700:Kinetics; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D017433:Protein Structure, Secondary; D000090063:Proto-Oncogene Mas; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; C000715470:MAS1 protein, human; D024101:Mitochondrial Proteins; D000090063:Proto-Oncogene Mas; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2