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In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau

The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer’s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscop...

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Autores principales: Zhang, Shengnan, Wang, Chuchu, Lu, Jinxia, Ma, Xiaojuan, Liu, Zhenying, Li, Dan, Liu, Zhijun, Liu, Cong
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337406/
https://www.ncbi.nlm.nih.gov/pubmed/30587819
http://dx.doi.org/10.3390/ijms20010090
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author Zhang, Shengnan
Wang, Chuchu
Lu, Jinxia
Ma, Xiaojuan
Liu, Zhenying
Li, Dan
Liu, Zhijun
Liu, Cong
author_facet Zhang, Shengnan
Wang, Chuchu
Lu, Jinxia
Ma, Xiaojuan
Liu, Zhenying
Li, Dan
Liu, Zhijun
Liu, Cong
author_sort Zhang, Shengnan
collection PubMed
description The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer’s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the conformations of two different isoforms of Tau, Tau40 and k19, in mammalian cells. Combined with immunofluorescence imaging and western blot analyses, we found that the isotope-enriched Tau, which was delivered into the cultured mammalian cells by electroporation, is partially colocalized with MT and actin filaments (F-actin). We acquired the NMR spectrum of Tau in human embryonic kidney 293 (HEK-293T) cells, and compared it with the NMR spectra of Tau added with MT, F-actin, and a variety of crowding agents, respectively. We found that the NMR spectrum of Tau in complex with MT best recapitulates the in-cell NMR spectrum of Tau, suggesting that Tau predominantly binds to MT at its MT-binding repeats in HEK-293T cells. Moreover, we found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293T cells, implying a potential cellular protection mechanism under stressful conditions. Collectively, the results of our study reveal that Tau utilizes its MT-binding repeats to bind MT in mammalian cells and highlight the potential of using in-cell NMR to study protein structures at the residue level in mammalian cells.
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spelling pubmed-63374062019-01-22 In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau Zhang, Shengnan Wang, Chuchu Lu, Jinxia Ma, Xiaojuan Liu, Zhenying Li, Dan Liu, Zhijun Liu, Cong Int J Mol Sci Article The intrinsically disordered protein, Tau, is abundant in neurons and contributes to the regulation of the microtubule (MT) and actin network, while its intracellular abnormal aggregation is closely associated with Alzheimer’s disease. Here, using in-cell Nuclear Magnetic Resonance (NMR) spectroscopy, we investigated the conformations of two different isoforms of Tau, Tau40 and k19, in mammalian cells. Combined with immunofluorescence imaging and western blot analyses, we found that the isotope-enriched Tau, which was delivered into the cultured mammalian cells by electroporation, is partially colocalized with MT and actin filaments (F-actin). We acquired the NMR spectrum of Tau in human embryonic kidney 293 (HEK-293T) cells, and compared it with the NMR spectra of Tau added with MT, F-actin, and a variety of crowding agents, respectively. We found that the NMR spectrum of Tau in complex with MT best recapitulates the in-cell NMR spectrum of Tau, suggesting that Tau predominantly binds to MT at its MT-binding repeats in HEK-293T cells. Moreover, we found that disease-associated phosphorylation of Tau was immediately eliminated once phosphorylated Tau was delivered into HEK-293T cells, implying a potential cellular protection mechanism under stressful conditions. Collectively, the results of our study reveal that Tau utilizes its MT-binding repeats to bind MT in mammalian cells and highlight the potential of using in-cell NMR to study protein structures at the residue level in mammalian cells. MDPI 2018-12-26 /pmc/articles/PMC6337406/ /pubmed/30587819 http://dx.doi.org/10.3390/ijms20010090 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
Zhang, Shengnan
Wang, Chuchu
Lu, Jinxia
Ma, Xiaojuan
Liu, Zhenying
Li, Dan
Liu, Zhijun
Liu, Cong
In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau
title In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau
title_full In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau
title_fullStr In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau
title_full_unstemmed In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau
title_short In-Cell NMR Study of Tau and MARK2 Phosphorylated Tau
title_sort in-cell nmr study of tau and mark2 phosphorylated tau
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337406/
https://www.ncbi.nlm.nih.gov/pubmed/30587819
http://dx.doi.org/10.3390/ijms20010090
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