title: Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway.
abstract: With the euchromatic portion of several mammalian genomes now sequenced, emphasis has turned to ascertaining the functions of gene products. A method for targeting destruction of selected proteins in mammalian cells is described, based on the ubiquitin-independent mechanism by which ornithine decarboxylase (ODC) is degraded by the 26S proteasome in collaboration with antizyme (AZ). We show that expressing whole proteins, protein domains, or peptide ligands fused to the N terminus of ODC promotes proteasome-dependent degradation of these chimeric fusion proteins and their interacting cellular target proteins. Moreover, the degradation of the interacting (targeted) protein depends on coexpression of AZ in about half of cases, providing an inducible switch for triggering the degradation process. By using 12 pairs of interacting proteins for testing, direct comparisons with several alternative strategies for achieving targeted protein destruction based on the concept of induced ubiquitination revealed advantages of the ODC/AZ system, which does not require posttranslational attachment of ubiquitin to target proteins. As proof of concept, the ODC/AZ system was used to ablate expression of specific endogenous proteins (e.g., TRAF6; Rb), and was shown to create the expected lesions in cellular pathways that require these proteins. Altogether, these findings reveal a strategy for achieving targeted destruction of cellular proteins, thus providing an additional tool for revealing the cellular phenotypes of gene products.
authors.affiliation: Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
authors.forename: Shu-ichi
authors.initials: S
authors.lastname: Matsuzawa
authors.forename: Michael
authors.initials: M
authors.lastname: Cuddy
authors.forename: Toru
authors.initials: T
authors.lastname: Fukushima
authors.forename: John C
authors.initials: JC
authors.lastname: Reed
mesh_terms: D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D009955:Ornithine Decarboxylase / Q000235:genetics / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D047992:TNF Receptor-Associated Factor 2; D048029:TNF Receptor-Associated Factor 6; D025801:Ubiquitin; C031017:ornithine decarboxylase antizyme; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D009955:Ornithine Decarboxylase