Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic
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 call...
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| Format: | Article |
| Language: | English |
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American Chemical Society (ACS)
2021
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| Online Access: | https://hdl.handle.net/1721.1/136198 |
| _version_ | 1811071814939443200 |
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| author | Ludwicki, Morgan B Li, Jiahe Stephens, Erin A Roberts, Richard W Koide, Shohei Hammond, Paula T DeLisa, Matthew P |
| author_facet | Ludwicki, Morgan B Li, Jiahe Stephens, Erin A Roberts, Richard W Koide, Shohei Hammond, Paula T DeLisa, Matthew P |
| author_sort | Ludwicki, Morgan B |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T08:56:36Z |
| format | Article |
| id | mit-1721.1/136198 |
| institution | Massachusetts Institute of Technology |
| language | English |
| last_indexed | 2024-09-23T08:56:36Z |
| publishDate | 2021 |
| publisher | American Chemical Society (ACS) |
| record_format | dspace |
| spelling | mit-1721.1/1361982022-04-01T14:44:57Z Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic Ludwicki, Morgan B Li, Jiahe Stephens, Erin A Roberts, Richard W Koide, Shohei Hammond, Paula T DeLisa, Matthew P 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. 2021-10-27T20:34:13Z 2021-10-27T20:34:13Z 2019 2019-08-20T18:14:48Z Article http://purl.org/eprint/type/JournalArticle https://hdl.handle.net/1721.1/136198 en 10.1021/acscentsci.9b00127 ACS Central Science Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf American Chemical Society (ACS) ACM |
| spellingShingle | Ludwicki, Morgan B Li, Jiahe Stephens, Erin A Roberts, Richard W Koide, Shohei Hammond, Paula T DeLisa, Matthew P Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic |
| title | Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic |
| title_full | Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic |
| title_fullStr | Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic |
| title_full_unstemmed | Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic |
| title_short | Broad-Spectrum Proteome Editing with an Engineered Bacterial Ubiquitin Ligase Mimic |
| title_sort | broad spectrum proteome editing with an engineered bacterial ubiquitin ligase mimic |
| url | https://hdl.handle.net/1721.1/136198 |
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