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ACS Cent Sci. 2019 May 22;5(5):852-866. doi: 10.1021/acscentsci.9b00127. Epub 2019 May 10.

Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic.

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Robert F. Smith School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, New York 14853, United States.
Department of Chemical Engineering and Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.
Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853, United States.
Department of Chemistry, University of Southern California, Los Angeles, California 90089, United States.
Perlmutter Cancer Center, New York University Langone Medical Center, New York, New York 10016, United States.
Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, New York 10016, United States.


Manipulation of the ubiquitin-proteasome pathway to achieve targeted silencing of cellular proteins has emerged as a reliable and customizable strategy for remodeling the mammalian proteome. One such approach involves engineering bifunctional proteins called ubiquibodies that are comprised of a synthetic binding protein fused to an E3 ubiquitin ligase, thus enabling post-translational ubiquitination and degradation of a target protein independent of its function. Here, we have designed a panel of new ubiquibodies based on E3 ubiquitin ligase mimics from bacterial pathogens that are capable of effectively interfacing with the mammalian proteasomal degradation machinery for selective removal of proteins of interest. One of these, the Shigella flexneri effector protein IpaH9.8 fused to a fibronectin type III (FN3) monobody that specifically recognizes green fluorescent protein (GFP), was observed to potently eliminate GFP and its spectral derivatives as well as 15 different FP-tagged mammalian proteins that varied in size (27-179 kDa) and subcellular localization (cytoplasm, nucleus, membrane-associated, and transmembrane). To demonstrate therapeutically relevant delivery of ubiquibodies, we leveraged a bioinspired molecular assembly method whereby synthetic mRNA encoding the GFP-specific ubiquibody was coassembled with poly A binding proteins and packaged into nanosized complexes using biocompatible, structurally defined polypolypeptides bearing cationic amine side groups. The resulting nanoplexes delivered ubiquibody mRNA in a manner that caused efficient target depletion in cultured mammalian cells stably expressing GFP as well as in transgenic mice expressing GFP ubiquitously. Overall, our results suggest that IpaH9.8-based ubiquibodies are a highly modular proteome editing technology with the potential for pharmacologically modulating disease-causing proteins.

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