Cargando…

Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132

BACKGROUND: Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The characterization of ZFN protein stability and the development of simple methods to improve ZFN function would facilitate the application of this promising technology. However, the factors that a...

Descripción completa

Detalles Bibliográficos
Autores principales: Ramakrishna, Suresh, Kim, Young-Hoon, Kim, Hyongbum
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554775/
https://www.ncbi.nlm.nih.gov/pubmed/23365657
http://dx.doi.org/10.1371/journal.pone.0054282
_version_ 1782256973933182976
author Ramakrishna, Suresh
Kim, Young-Hoon
Kim, Hyongbum
author_facet Ramakrishna, Suresh
Kim, Young-Hoon
Kim, Hyongbum
author_sort Ramakrishna, Suresh
collection PubMed
description BACKGROUND: Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The characterization of ZFN protein stability and the development of simple methods to improve ZFN function would facilitate the application of this promising technology. However, the factors that affect ZFN protein stability and function are not yet clear. Here, we determined the stability and half-life of two ZFN proteins and examined the effect of MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-Hl), a proteasome inhibitor, on ZFN-mediated gene modifications. METHODOLOGY/PRINCIPAL FINDINGS: ZFN proteins were expressed in 293T cells after transfection of ZFN-encoding plasmids. We studied two ZFN pairs: Z-224, which targets the CCR5 gene, and K-230, which targets a region 230 kbp upstream of CCR5. Western blotting after treatment with cycloheximide showed that the half-life of these ZFN proteins was around two hours. An immunoprecipitation assay revealed that the ZFN interacts with ubiquitin molecules and undergoes polyubiquitination in vivo. Western blotting showed that the addition of MG132, a proteasomal inhibitor, increased ZFN protein levels. Finally, a surrogate reporter assay and a T7E1 assay revealed that MG132 treatment enhanced ZFN-directed gene editing. CONCLUSIONS: To our knowledge, this is the first study to investigate ZFN protein stability and to show that a small molecule can increase ZFN activity. Our protein stability study should lay the foundation for further improvement of ZFN technology; as a first step, the use of the small molecule MG132 can enhance the efficiency of ZFN-mediated gene editing.
format Online
Article
Text
id pubmed-3554775
institution National Center for Biotechnology Information
language English
publishDate 2013
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-35547752013-01-30 Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132 Ramakrishna, Suresh Kim, Young-Hoon Kim, Hyongbum PLoS One Research Article BACKGROUND: Zinc finger nucleases (ZFNs) are powerful tools for gene therapy and genetic engineering. The characterization of ZFN protein stability and the development of simple methods to improve ZFN function would facilitate the application of this promising technology. However, the factors that affect ZFN protein stability and function are not yet clear. Here, we determined the stability and half-life of two ZFN proteins and examined the effect of MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal-Hl), a proteasome inhibitor, on ZFN-mediated gene modifications. METHODOLOGY/PRINCIPAL FINDINGS: ZFN proteins were expressed in 293T cells after transfection of ZFN-encoding plasmids. We studied two ZFN pairs: Z-224, which targets the CCR5 gene, and K-230, which targets a region 230 kbp upstream of CCR5. Western blotting after treatment with cycloheximide showed that the half-life of these ZFN proteins was around two hours. An immunoprecipitation assay revealed that the ZFN interacts with ubiquitin molecules and undergoes polyubiquitination in vivo. Western blotting showed that the addition of MG132, a proteasomal inhibitor, increased ZFN protein levels. Finally, a surrogate reporter assay and a T7E1 assay revealed that MG132 treatment enhanced ZFN-directed gene editing. CONCLUSIONS: To our knowledge, this is the first study to investigate ZFN protein stability and to show that a small molecule can increase ZFN activity. Our protein stability study should lay the foundation for further improvement of ZFN technology; as a first step, the use of the small molecule MG132 can enhance the efficiency of ZFN-mediated gene editing. Public Library of Science 2013-01-24 /pmc/articles/PMC3554775/ /pubmed/23365657 http://dx.doi.org/10.1371/journal.pone.0054282 Text en © 2013 Ramakrishna 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Ramakrishna, Suresh
Kim, Young-Hoon
Kim, Hyongbum
Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132
title Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132
title_full Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132
title_fullStr Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132
title_full_unstemmed Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132
title_short Stability of Zinc Finger Nuclease Protein Is Enhanced by the Proteasome Inhibitor MG132
title_sort stability of zinc finger nuclease protein is enhanced by the proteasome inhibitor mg132
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3554775/
https://www.ncbi.nlm.nih.gov/pubmed/23365657
http://dx.doi.org/10.1371/journal.pone.0054282
work_keys_str_mv AT ramakrishnasuresh stabilityofzincfingernucleaseproteinisenhancedbytheproteasomeinhibitormg132
AT kimyounghoon stabilityofzincfingernucleaseproteinisenhancedbytheproteasomeinhibitormg132
AT kimhyongbum stabilityofzincfingernucleaseproteinisenhancedbytheproteasomeinhibitormg132