Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway

被引:43
作者
Matsuzawa, S [1 ]
Cuddy, M [1 ]
Fukushima, T [1 ]
Reed, JC [1 ]
机构
[1] Burnham Inst Med Res, La Jolla, CA 92037 USA
关键词
antizyme; ornithine decarboxylase; proteasome; protein degradation;
D O I
10.1073/pnas.0507512102
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
引用
收藏
页码:14982 / 14987
页数:6
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