Exploring the functional complexity of cellular proteins by protein knockout.
Comprehensive dissection of protein functions entails more complicated manipulations than simply eliminating the protein of interest. Established knockdown technologies, such as RNA interference, antisense oligodeoxynucleotides, or ribozymes, are limited for specific applications such as modulating protein levels or specific targeting of a posttranslationally modified subpopulation. Here we show that the engineered Skp1, Cullin 1, and F-box-containing betaTrCP substrate receptor ubiquitin-proteolytic system, designated protein knockout, could achieve not only total elimination but also rapid and systematic reduction of a given cellular protein. Stable expression of a single engineered betaTrCP demonstrated simultaneous and sustained degradation of the entire retinoblastoma family proteins. Furthermore, the engineered betaTrCP was capable of selecting hypo- but not hyperphosphorylated forms of retinoblastoma for degradation. The engineered betaTrCP has been extensively modified to increase its specificity in substrate selection. This optimized protein-knockout system offers a powerful and versatile proteomic tool to dissect diverse functional properties of cellular proteins in somatic cells.
Department of Pathology and Laboratory Medicine, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
Jianxuan
J
Zhang
Ning
N
Zheng
Pengbo
P
Zhou
D020134:Catalytic Domain; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008958:Models, Molecular; D016296:Mutagenesis; D015202:Protein Engineering; D011506:Proteins / Q000235:genetics* / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D044843:SKP Cullin F-Box Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D046911:Macromolecular Substances; D011506:Proteins; D011994:Recombinant Proteins; D044843:SKP Cullin F-Box Protein Ligases; D044767:Ubiquitin-Protein Ligases