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Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model

Zip family proteins are involved in the control of zinc (Zn) ion homeostasis. The present study cloned the promoters and investigated the transcription responses and protein subcellular localizations of three LIV-1 subfamily members (zip10, zip13, and zip14) from common freshwater teleost yellow cat...

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Autores principales: Liu, Sheng-Zan, Xu, Yi-Chuang, Tan, Xiao-Ying, Zhao, Tao, Zhang, Dian-Guang, Yang, Hong, Luo, Zhi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9321221/
https://www.ncbi.nlm.nih.gov/pubmed/35887381
http://dx.doi.org/10.3390/ijms23148034
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author Liu, Sheng-Zan
Xu, Yi-Chuang
Tan, Xiao-Ying
Zhao, Tao
Zhang, Dian-Guang
Yang, Hong
Luo, Zhi
author_facet Liu, Sheng-Zan
Xu, Yi-Chuang
Tan, Xiao-Ying
Zhao, Tao
Zhang, Dian-Guang
Yang, Hong
Luo, Zhi
author_sort Liu, Sheng-Zan
collection PubMed
description Zip family proteins are involved in the control of zinc (Zn) ion homeostasis. The present study cloned the promoters and investigated the transcription responses and protein subcellular localizations of three LIV-1 subfamily members (zip10, zip13, and zip14) from common freshwater teleost yellow catfish, Pelteobagrus fulvidraco, using in vitro cultured HEK293T model cells. The 2278 bp, 1917 bp, and 1989 bp sequences of zip10, zip13, and zip14 promoters, respectively, were subcloned into pGL3-Basic plasmid for promoter activity analysis. The pcDNA3.1 plasmid coding EGFP tagged pfZip10, pfZip13, and pfZip14 were generated for subsequent confocal microscope analysis. Several potential transcription factors’ binding sites were predicted within the promoters. In vitro promoter analysis in the HEK293T cells showed that high Zn administration significantly reduced the transcriptional activities of the zip10, zip13, and zip14 promoters. The −2017 bp/−2004 bp MRE in the zip10 promoter, the −360 bp/−345 bp MRE in the zip13 promoter, and the −1457 bp/−1442 bp MRE in the zip14 promoter were functional loci that were involved in the regulation of the three zips. The −606 bp/−594 bp KLF4 binding site in the zip13 promoter was a functional locus responsible for zinc-responsive regulation of zip13. The −1383 bp/−1375 bp STAT3 binding site in the zip14 promoter was a functional locus responsible for zinc-responsive regulation of zip14. Moreover, confocal microscope analysis indicated that zinc incubation significantly reduced the fluorescence intensity of pfZip10-EGFP and pfZip14-EGFP but had no significant influence on pfZip13-EGFP fluorescence intensity. Further investigation found that pfZip10 localizes on cell membranes, pfZip14 colocalized with both cell membranes and lysosome, and pfZip13 colocalized with intracellular ER and Golgi. Our research illustrated the transcription regulation of zip10, zip13, and zip14 from P. fulvidraco under zinc administration, which provided a reference value for the mechanisms involved in Zip-family-mediated control of zinc homeostasis in vertebrates.
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spelling pubmed-93212212022-07-27 Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model Liu, Sheng-Zan Xu, Yi-Chuang Tan, Xiao-Ying Zhao, Tao Zhang, Dian-Guang Yang, Hong Luo, Zhi Int J Mol Sci Article Zip family proteins are involved in the control of zinc (Zn) ion homeostasis. The present study cloned the promoters and investigated the transcription responses and protein subcellular localizations of three LIV-1 subfamily members (zip10, zip13, and zip14) from common freshwater teleost yellow catfish, Pelteobagrus fulvidraco, using in vitro cultured HEK293T model cells. The 2278 bp, 1917 bp, and 1989 bp sequences of zip10, zip13, and zip14 promoters, respectively, were subcloned into pGL3-Basic plasmid for promoter activity analysis. The pcDNA3.1 plasmid coding EGFP tagged pfZip10, pfZip13, and pfZip14 were generated for subsequent confocal microscope analysis. Several potential transcription factors’ binding sites were predicted within the promoters. In vitro promoter analysis in the HEK293T cells showed that high Zn administration significantly reduced the transcriptional activities of the zip10, zip13, and zip14 promoters. The −2017 bp/−2004 bp MRE in the zip10 promoter, the −360 bp/−345 bp MRE in the zip13 promoter, and the −1457 bp/−1442 bp MRE in the zip14 promoter were functional loci that were involved in the regulation of the three zips. The −606 bp/−594 bp KLF4 binding site in the zip13 promoter was a functional locus responsible for zinc-responsive regulation of zip13. The −1383 bp/−1375 bp STAT3 binding site in the zip14 promoter was a functional locus responsible for zinc-responsive regulation of zip14. Moreover, confocal microscope analysis indicated that zinc incubation significantly reduced the fluorescence intensity of pfZip10-EGFP and pfZip14-EGFP but had no significant influence on pfZip13-EGFP fluorescence intensity. Further investigation found that pfZip10 localizes on cell membranes, pfZip14 colocalized with both cell membranes and lysosome, and pfZip13 colocalized with intracellular ER and Golgi. Our research illustrated the transcription regulation of zip10, zip13, and zip14 from P. fulvidraco under zinc administration, which provided a reference value for the mechanisms involved in Zip-family-mediated control of zinc homeostasis in vertebrates. MDPI 2022-07-21 /pmc/articles/PMC9321221/ /pubmed/35887381 http://dx.doi.org/10.3390/ijms23148034 Text en © 2022 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, Sheng-Zan
Xu, Yi-Chuang
Tan, Xiao-Ying
Zhao, Tao
Zhang, Dian-Guang
Yang, Hong
Luo, Zhi
Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model
title Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model
title_full Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model
title_fullStr Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model
title_full_unstemmed Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model
title_short Transcriptional Regulation and Protein Localization of Zip10, Zip13 and Zip14 Transporters of Freshwater Teleost Yellow Catfish Pelteobagrus fulvidraco Following Zn Exposure in a Heterologous HEK293T Model
title_sort transcriptional regulation and protein localization of zip10, zip13 and zip14 transporters of freshwater teleost yellow catfish pelteobagrus fulvidraco following zn exposure in a heterologous hek293t model
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9321221/
https://www.ncbi.nlm.nih.gov/pubmed/35887381
http://dx.doi.org/10.3390/ijms23148034
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