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Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA

The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has b...

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Autores principales: Liu, Yanli, Zhao, Zitong, Song, Yunyang, Yin, Yifeng, Wu, Fanghui, Jiang, Hui
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10455749/
https://www.ncbi.nlm.nih.gov/pubmed/37623702
http://dx.doi.org/10.3390/md21080421
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author Liu, Yanli
Zhao, Zitong
Song, Yunyang
Yin, Yifeng
Wu, Fanghui
Jiang, Hui
author_facet Liu, Yanli
Zhao, Zitong
Song, Yunyang
Yin, Yifeng
Wu, Fanghui
Jiang, Hui
author_sort Liu, Yanli
collection PubMed
description The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has been characterized in Conus literatus. However, the hydroxylation machinery of proline in conopeptides is still unclear. In order to address the hydroxylation mechanism of proline in μ-PIIIA, three recombinant plasmids encoding different hybrid precursors of μ-PIIIA were constructed and crossly combined with protein disulfide isomerase, prolyl 4 hydroxylase, and glutaminyl cyclase in a continuous exchange cell-free protein system. The findings showed that prolyl 4 hydroxylase might recognize the propeptide of μ-PIIIA to achieve the hydroxylation of proline, while the cyclization of glutamate was also formed. Additionally, in Escherichia coli, the co-expression plasmid encoding prolyl 4 hydroxylase and the precursor of μ-PIIIA containing pro and mature regions were used to validate the continuous exchange cell-free protein system. Surprisingly, in addition to the two hydroxyproline residues and one pyroglutamyl residue, three disulfide bridges were formed using Trx as a fusion tag, and the yield of the fusion peptide was approximately 20 mg/L. The results of electrophysiology analysis indicated that the recombinant μ-PIIIA without C-terminal amidate inhibited the current of hNa(V)1.4 with a 939 nM IC(50). Our work solved the issue that it was challenging to quickly generate post-translationally modified conopeptides in vitro. This is the first study to demonstrate that prolyl 4 hydroxylase catalyzes the proline hydroxylation through recognition in the propeptide of μ-PIIIA, and it will provide a new way for synthesizing multi-modified conopeptides with pharmacological activity.
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spelling pubmed-104557492023-08-26 Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA Liu, Yanli Zhao, Zitong Song, Yunyang Yin, Yifeng Wu, Fanghui Jiang, Hui Mar Drugs Article The post-translational modifications of conopeptides are the most complicated modifications to date and are well-known and closely related to the activity of conopeptides. The hydroxylation of proline in conopeptides affects folding, structure, and biological activity, and prolyl 4 hydroxylase has been characterized in Conus literatus. However, the hydroxylation machinery of proline in conopeptides is still unclear. In order to address the hydroxylation mechanism of proline in μ-PIIIA, three recombinant plasmids encoding different hybrid precursors of μ-PIIIA were constructed and crossly combined with protein disulfide isomerase, prolyl 4 hydroxylase, and glutaminyl cyclase in a continuous exchange cell-free protein system. The findings showed that prolyl 4 hydroxylase might recognize the propeptide of μ-PIIIA to achieve the hydroxylation of proline, while the cyclization of glutamate was also formed. Additionally, in Escherichia coli, the co-expression plasmid encoding prolyl 4 hydroxylase and the precursor of μ-PIIIA containing pro and mature regions were used to validate the continuous exchange cell-free protein system. Surprisingly, in addition to the two hydroxyproline residues and one pyroglutamyl residue, three disulfide bridges were formed using Trx as a fusion tag, and the yield of the fusion peptide was approximately 20 mg/L. The results of electrophysiology analysis indicated that the recombinant μ-PIIIA without C-terminal amidate inhibited the current of hNa(V)1.4 with a 939 nM IC(50). Our work solved the issue that it was challenging to quickly generate post-translationally modified conopeptides in vitro. This is the first study to demonstrate that prolyl 4 hydroxylase catalyzes the proline hydroxylation through recognition in the propeptide of μ-PIIIA, and it will provide a new way for synthesizing multi-modified conopeptides with pharmacological activity. MDPI 2023-07-25 /pmc/articles/PMC10455749/ /pubmed/37623702 http://dx.doi.org/10.3390/md21080421 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/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 (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Liu, Yanli
Zhao, Zitong
Song, Yunyang
Yin, Yifeng
Wu, Fanghui
Jiang, Hui
Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA
title Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA
title_full Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA
title_fullStr Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA
title_full_unstemmed Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA
title_short Usage of Cell-Free Protein Synthesis in Post-Translational Modification of μ-Conopeptide PIIIA
title_sort usage of cell-free protein synthesis in post-translational modification of μ-conopeptide piiia
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10455749/
https://www.ncbi.nlm.nih.gov/pubmed/37623702
http://dx.doi.org/10.3390/md21080421
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