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The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells

Transmembrane channel-like protein 1 (TMC1) is a transmembrane protein forming mechano-electrical transduction (MET) channel, which transduces mechanical stimuli into electrical signals at the top of stereocilia of hair cells in the inner ear. As an unexpected phenomenon, we found that the cytosolic...

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Autores principales: Yamaguchi, Soichiro, Kamino, Maho, Hamamura, Maho, Otsuguro, Ken-ichi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10289374/
https://www.ncbi.nlm.nih.gov/pubmed/37352201
http://dx.doi.org/10.1371/journal.pone.0287249
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author Yamaguchi, Soichiro
Kamino, Maho
Hamamura, Maho
Otsuguro, Ken-ichi
author_facet Yamaguchi, Soichiro
Kamino, Maho
Hamamura, Maho
Otsuguro, Ken-ichi
author_sort Yamaguchi, Soichiro
collection PubMed
description Transmembrane channel-like protein 1 (TMC1) is a transmembrane protein forming mechano-electrical transduction (MET) channel, which transduces mechanical stimuli into electrical signals at the top of stereocilia of hair cells in the inner ear. As an unexpected phenomenon, we found that the cytosolic N-terminal (Nt) region of heterologously-expressed mouse TMC1 (mTMC1) was localized in nuclei of a small population of the transfected HEK293 cells. This raised the possibility that the Nt region of heterologously-expressed mTMC1 was cleaved and transported into the nucleus. To confirm the cleavage, we performed western blot analyses. The results revealed that at least a fragment of the Nt region was produced from heterologously-expressed mTMC1. Site-directed mutagenesis experiments identified amino acid residues which were required to produce the fragment. The accumulation of the heterologously-expressed Nt fragment into the nuclei depended on nuclear localization signals within the Nt region. Furthermore, a structural comparison showed a similarity between the Nt region of mTMC1 and basic region leucine zipper (bZIP) transcription factors. However, transcriptome analyses using a next-generation sequencer showed that the heterologously-expression of the Nt fragment of mTMC1 hardly altered expression levels of genes. Although it is still unknown what is the precise mechanism and the physiological significance of this cleavage, these results showed that the cytosolic Nt region of heterologously-expressed mTMC1 could be cleaved in HEK293 cells. Therefore, it should be taken into account that the cleavage of Nt region might influence the functional analysis of TMC1 by the heterologous-expression system using HEK293 cells.
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spelling pubmed-102893742023-06-24 The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells Yamaguchi, Soichiro Kamino, Maho Hamamura, Maho Otsuguro, Ken-ichi PLoS One Research Article Transmembrane channel-like protein 1 (TMC1) is a transmembrane protein forming mechano-electrical transduction (MET) channel, which transduces mechanical stimuli into electrical signals at the top of stereocilia of hair cells in the inner ear. As an unexpected phenomenon, we found that the cytosolic N-terminal (Nt) region of heterologously-expressed mouse TMC1 (mTMC1) was localized in nuclei of a small population of the transfected HEK293 cells. This raised the possibility that the Nt region of heterologously-expressed mTMC1 was cleaved and transported into the nucleus. To confirm the cleavage, we performed western blot analyses. The results revealed that at least a fragment of the Nt region was produced from heterologously-expressed mTMC1. Site-directed mutagenesis experiments identified amino acid residues which were required to produce the fragment. The accumulation of the heterologously-expressed Nt fragment into the nuclei depended on nuclear localization signals within the Nt region. Furthermore, a structural comparison showed a similarity between the Nt region of mTMC1 and basic region leucine zipper (bZIP) transcription factors. However, transcriptome analyses using a next-generation sequencer showed that the heterologously-expression of the Nt fragment of mTMC1 hardly altered expression levels of genes. Although it is still unknown what is the precise mechanism and the physiological significance of this cleavage, these results showed that the cytosolic Nt region of heterologously-expressed mTMC1 could be cleaved in HEK293 cells. Therefore, it should be taken into account that the cleavage of Nt region might influence the functional analysis of TMC1 by the heterologous-expression system using HEK293 cells. Public Library of Science 2023-06-23 /pmc/articles/PMC10289374/ /pubmed/37352201 http://dx.doi.org/10.1371/journal.pone.0287249 Text en © 2023 Yamaguchi et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://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
Yamaguchi, Soichiro
Kamino, Maho
Hamamura, Maho
Otsuguro, Ken-ichi
The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells
title The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells
title_full The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells
title_fullStr The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells
title_full_unstemmed The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells
title_short The cytosolic N-terminal region of heterologously-expressed transmembrane channel-like protein 1 (TMC1) can be cleaved in HEK293 cells
title_sort cytosolic n-terminal region of heterologously-expressed transmembrane channel-like protein 1 (tmc1) can be cleaved in hek293 cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10289374/
https://www.ncbi.nlm.nih.gov/pubmed/37352201
http://dx.doi.org/10.1371/journal.pone.0287249
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