Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic
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acscentsci.9b00127.pdf
Description
Published version
Size
4.26 MB
Format
Unknown
Checksum (MD5)
001f259f0d51f7e882b1e6b1997f1d4d
Author(s)
Ludwicki, Morgan B
Li, Jiahe
Stephens, Erin A
Roberts, Richard W
Koide, Shohei
Hammond, Paula T
DeLisa, Matthew P
Date Issued
2019
Journal
ACS Central Science
Publisher
American Chemical Society (ACS)
Version
Final published version
Abstract
Copyright © 2019 American Chemical Society. 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.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Koch Institute for Integrative Cancer Research at MIT
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DOI of Published Version
https://doi.org/10.1021/acscentsci.9b00127
