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Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants
Determining the function of proteins remains a key task of modern biology. Classical genetic approaches to knocking out protein function in plants still face limitations, such as the time-consuming nature of generating homozygous transgenic lines or the risk of non-viable loss-of-function phenotypes...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880479/ https://www.ncbi.nlm.nih.gov/pubmed/33577589 http://dx.doi.org/10.1371/journal.pone.0247015 |
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author | Sorge, Eberhard Demidov, Dmitri Lermontova, Inna Houben, Andreas Conrad, Udo |
author_facet | Sorge, Eberhard Demidov, Dmitri Lermontova, Inna Houben, Andreas Conrad, Udo |
author_sort | Sorge, Eberhard |
collection | PubMed |
description | Determining the function of proteins remains a key task of modern biology. Classical genetic approaches to knocking out protein function in plants still face limitations, such as the time-consuming nature of generating homozygous transgenic lines or the risk of non-viable loss-of-function phenotypes. We aimed to overcome these limitations by acting downstream of the protein level. Chimeric E3 ligases degrade proteins of interest in mammalian cell lines, Drosophila melanogaster embryos, and transgenic tobacco. We successfully recruited the 26S proteasome pathway to directly degrade a protein of interest located in plant nuclei. This success was achieved via replacement of the interaction domain of the E3 ligase adaptor protein SPOP (Speckle-type POZ adapter protein) with a specific anti-GFP nanobody (VHHGFP4). For proof of concept, the target protein CENH3 of A. thaliana fused to EYFP was subjected to nanobody-guided proteasomal degradation in planta. Our results show the potential of the modified E3-ligase adapter protein VHHGFP4-SPOP in this respect. We were able to point out its capability for nucleus-specific protein degradation in plants. |
format | Online Article Text |
id | pubmed-7880479 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-78804792021-02-19 Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants Sorge, Eberhard Demidov, Dmitri Lermontova, Inna Houben, Andreas Conrad, Udo PLoS One Research Article Determining the function of proteins remains a key task of modern biology. Classical genetic approaches to knocking out protein function in plants still face limitations, such as the time-consuming nature of generating homozygous transgenic lines or the risk of non-viable loss-of-function phenotypes. We aimed to overcome these limitations by acting downstream of the protein level. Chimeric E3 ligases degrade proteins of interest in mammalian cell lines, Drosophila melanogaster embryos, and transgenic tobacco. We successfully recruited the 26S proteasome pathway to directly degrade a protein of interest located in plant nuclei. This success was achieved via replacement of the interaction domain of the E3 ligase adaptor protein SPOP (Speckle-type POZ adapter protein) with a specific anti-GFP nanobody (VHHGFP4). For proof of concept, the target protein CENH3 of A. thaliana fused to EYFP was subjected to nanobody-guided proteasomal degradation in planta. Our results show the potential of the modified E3-ligase adapter protein VHHGFP4-SPOP in this respect. We were able to point out its capability for nucleus-specific protein degradation in plants. Public Library of Science 2021-02-12 /pmc/articles/PMC7880479/ /pubmed/33577589 http://dx.doi.org/10.1371/journal.pone.0247015 Text en © 2021 Sorge et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Sorge, Eberhard Demidov, Dmitri Lermontova, Inna Houben, Andreas Conrad, Udo Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants |
title | Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants |
title_full | Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants |
title_fullStr | Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants |
title_full_unstemmed | Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants |
title_short | Engineered degradation of EYFP-tagged CENH3 via the 26S proteasome pathway in plants |
title_sort | engineered degradation of eyfp-tagged cenh3 via the 26s proteasome pathway in plants |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880479/ https://www.ncbi.nlm.nih.gov/pubmed/33577589 http://dx.doi.org/10.1371/journal.pone.0247015 |
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