Redirecting the specificity of ubiquitination by modifying ubiquitin-conjugating enzymes.
Depletion of specific cellular proteins is a powerful tool in biological research and has many medical and agricultural benefits. In contrast to genetic methods currently available to attenuate protein levels, we describe an alternative approach that redirects the ubiquitin-dependent proteolytic pathway to facilitate specific proteolytic removal. Degradation via the ubiquitin pathway requires the prior attachment of multiple ubiquitins to the target protein. This attachment is accomplished, in part, by a family of enzymes designated E2s (or ubiquitin-conjugating enzymes), some of which use domains near their C termini for target recognition. Here, we demonstrate that E2 target recognition can be redefined by engineering E2s to contain appropriate protein-binding peptides fused to their C termini. In five dissimilar examples, chimeric E2s were created that recognized and ubiquitinated their respective binding partners with high specificity. We also show that ubiquitination of one protein targeted by this method led to its ATP-dependent degradation in vitro. Thus, by exploiting interacting domains derived from natural and synthetic ligands, it may be possible to design E2s capable of directing the selective removal of many intracellular proteins.
Gosink
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MM
Department of Horticulture, University of Wisconsin, Madison 53706, USA.
Vierstra
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RD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002294:Carcinoma, Squamous Cell; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D017931:DNA Primers; D066246:ErbB Receptors / Q000096:biosynthesis; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012156:Reticulocytes / Q000378:metabolism; D016211:Transforming Growth Factor alpha / Q000096:biosynthesis; D014908:Triticum / Q000378:metabolism; D014407:Tumor Cells, Cultured; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*
D017931:DNA Primers; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D016211:Transforming Growth Factor alpha; D014452:Ubiquitins; D044763:Ubiquitin-Conjugating Enzymes; C063109:ubiquitin-conjugating enzyme UBC4; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D008025:Ligases