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Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1

Nucleic acid aptamers hold promise as therapeutic tools for specific, tailored inhibition of protein targets with several advantages when compared to small molecules or antibodies. Nuclear WW domain containing E3 ubiquitin ligase 1 (WWP1) ubiquitin ligase poly-ubiquitinates Runt-related transcriptio...

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Autores principales: Tucker, Wesley O., Kinghorn, Andrew B., Fraser, Lewis A., Cheung, Yee-Wai, Tanner, Julian A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877624/
https://www.ncbi.nlm.nih.gov/pubmed/29518962
http://dx.doi.org/10.3390/ijms19030763
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author Tucker, Wesley O.
Kinghorn, Andrew B.
Fraser, Lewis A.
Cheung, Yee-Wai
Tanner, Julian A.
author_facet Tucker, Wesley O.
Kinghorn, Andrew B.
Fraser, Lewis A.
Cheung, Yee-Wai
Tanner, Julian A.
author_sort Tucker, Wesley O.
collection PubMed
description Nucleic acid aptamers hold promise as therapeutic tools for specific, tailored inhibition of protein targets with several advantages when compared to small molecules or antibodies. Nuclear WW domain containing E3 ubiquitin ligase 1 (WWP1) ubiquitin ligase poly-ubiquitinates Runt-related transcription factor 2 (Runx2), a key transcription factor associated with osteoblast differentiation. Since WWP1 and an adapter known as Schnurri-3 are negative regulators of osteoblast function, the disruption of this complex has the potential to increase bone deposition for osteoporosis therapy. Here, we develop new DNA aptamers that bind and inhibit WWP1 then investigate efficacy in an osteoblastic cell culture. DNA aptamers were selected against three different truncations of the HECT domain of WWP1. Aptamers which bind specifically to a C-lobe HECT domain truncation were observed to enrich during the selection procedure. One particular DNA aptamer termed C3A was further evaluated for its ability to bind WWP1 and inhibit its ubiquitination activity. C3A showed a low µM binding affinity to WWP1 and was observed to be a non-competitive inhibitor of WWP1 HECT ubiquitin ligase activity. When SaOS-2 osteoblastic cells were treated with C3A, partial localization to the nucleus was observed. The C3A aptamer was also demonstrated to specifically promote extracellular mineralization in cell culture experiments. The C3A aptamer has potential for further development as a novel osteoporosis therapeutic strategy. Our results demonstrate that aptamer-mediated inhibition of protein ubiquitination can be a novel therapeutic strategy.
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spelling pubmed-58776242018-04-09 Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1 Tucker, Wesley O. Kinghorn, Andrew B. Fraser, Lewis A. Cheung, Yee-Wai Tanner, Julian A. Int J Mol Sci Article Nucleic acid aptamers hold promise as therapeutic tools for specific, tailored inhibition of protein targets with several advantages when compared to small molecules or antibodies. Nuclear WW domain containing E3 ubiquitin ligase 1 (WWP1) ubiquitin ligase poly-ubiquitinates Runt-related transcription factor 2 (Runx2), a key transcription factor associated with osteoblast differentiation. Since WWP1 and an adapter known as Schnurri-3 are negative regulators of osteoblast function, the disruption of this complex has the potential to increase bone deposition for osteoporosis therapy. Here, we develop new DNA aptamers that bind and inhibit WWP1 then investigate efficacy in an osteoblastic cell culture. DNA aptamers were selected against three different truncations of the HECT domain of WWP1. Aptamers which bind specifically to a C-lobe HECT domain truncation were observed to enrich during the selection procedure. One particular DNA aptamer termed C3A was further evaluated for its ability to bind WWP1 and inhibit its ubiquitination activity. C3A showed a low µM binding affinity to WWP1 and was observed to be a non-competitive inhibitor of WWP1 HECT ubiquitin ligase activity. When SaOS-2 osteoblastic cells were treated with C3A, partial localization to the nucleus was observed. The C3A aptamer was also demonstrated to specifically promote extracellular mineralization in cell culture experiments. The C3A aptamer has potential for further development as a novel osteoporosis therapeutic strategy. Our results demonstrate that aptamer-mediated inhibition of protein ubiquitination can be a novel therapeutic strategy. MDPI 2018-03-07 /pmc/articles/PMC5877624/ /pubmed/29518962 http://dx.doi.org/10.3390/ijms19030763 Text en © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Tucker, Wesley O.
Kinghorn, Andrew B.
Fraser, Lewis A.
Cheung, Yee-Wai
Tanner, Julian A.
Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1
title Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1
title_full Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1
title_fullStr Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1
title_full_unstemmed Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1
title_short Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1
title_sort selection and characterization of a dna aptamer specifically targeting human hect ubiquitin ligase wwp1
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877624/
https://www.ncbi.nlm.nih.gov/pubmed/29518962
http://dx.doi.org/10.3390/ijms19030763
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