The Effect of Modification on the Intracellular Distribution of Zyxin in Xenopus laevis Embryos

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Дәйексөз келтіру

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Аннотация

Zyxin is a conserved mechanosensitive LIM domain protein that regulates F-actin filament assembly at cell junctions. In response to cell stretching, zyxin can either move into the nucleus and regulate gene expression, or it can exit the nucleus. Zyxin is recognized as an oncomarker, which makes studying its modifications and how it moves between nucleus and cytoplasm useful for diagnosing diseases at the molecular level. An effect of site-directed mutagenesis at palmitylation sites, O-GlcNAcylation sites, and amino acids at the N- and C-terminus on the ability of zyxin to enter the nucleus was demonstrated using Xenopus laevis embryos at gastrula stage. By adding the Flag epitope to the C-terminus of the zyxin molecule, it was found that the zyxin molecule loses its ability to move into the nucleus as a result. When palmitylation sites are targeted for mutation, the amount of zyxin in the nucleus decreases, whereas when amino acids are mutated to cause O-GlcNAcylation, the amount of zyxin increases. The first data obtained on the influence of these modifications on the movement of zyxin support global research on mechanisms behind changes in the localization of mechanosensitive proteins of the zyxin family. Since disruption of their intracellular localization leads to cancerous tumors and cardiovascular diseases, these investigations have both fundamental and medical importance.

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Авторлар туралы

E. Parshina

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: martnat61@gmail.com
Ресей, ul. Miklukho-Maklaya 16/10, Moscow, 117997

E. Ivanova

Pirogov Russian National Research Medical University

Email: martnat61@gmail.com
Ресей, ul. Ostrovitianova 1, Moscow, 117997

A. Zaraisky

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Email: martnat61@gmail.com
Ресей, ul. Miklukho-Maklaya 16/10, Moscow, 117997

N. Martynova

Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences

Хат алмасуға жауапты Автор.
Email: martnat61@gmail.com
Ресей, ul. Miklukho-Maklaya 16/10, Moscow, 117997

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2. Fig. 1. Sites for targeted mutagenesis of zyxin and schemes of mutant proteins. (a) – Comparison of amino acid sequences of zyxin homologues from frog (henZYX), chicken (galZYX) and human (homZYX) showed high conservatism in proline-rich and LIM-domain regions (areas highlighted in black). The diagram shows the positions that have undergone mutagenesis; (b) the schemes of Myc (1-3) and Flag (1) tagged epitopes and mutated palmithylation (3), glucosaminylation (4) and shortened (5) versions of zyxin.

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3. 2. Intracellular distribution of hybrid forms of zyxin in the N- and C-terminal domains. (a) – Experimental scheme; (b) – the presence of the Flag peptide at the C-terminus prevents its movement into the nucleus. Above is a diagram of hybrid zyxin, below are Western blots with anti-Myx (top blot) and anti-Flag (bottom blot) antibody detection, two tracks per sample; (c) - removal of the Flag peptide from the C–terminus of the zyxin molecule restores its ability to move into the nucleus. Above is a hybrid protein diagram, below is a Western blot with anti–Myc antibody detection, with two tracks per sample.

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4. 3. Analysis of the distribution of zyxin mutated at the sites of palmithylation, O-glucosaminylation, and deletion mutant in embryo cells at the gastrula stage. (a) – Mutations of full-length zyxin at palmithylation sites lead to a decrease in zyxin in the nucleus. Above is the scheme of mutant zyxin, Cys531, 532, 555, 558 and 593 replaced by alanins. On the left is a western blot with anti-Mus antibody detection, the reference load band on the track is actin. On the right is a check of the statistical reliability of the change in the amount of zixin, conl. units (* p < 0.05); (b) – mutations of full-length zyxin in amino acids undergoing O-glucosaminylation lead to a decrease in the amount of zyxin in the nucleus. Above is the scheme of mutant zyxin, Ser216 and 217 replaced by alanins. On the left is a western blot, detection by antibodies to C–zyxin, and the reference load band on the track is actin. On the right is a check of the statistical reliability of changes in the amount of zyxin, conl. units (* p < 0.05); (c) is an analysis of the distribution between the nucleus and cytoplasm of the shortened form of zyxin (334-664 au). Above is a diagram of the deletion mutant of zyxin, zyxin (334-664 au). On the left is a western blot, detection by antibodies to C-zyxin. On the right is a check of the statistical reliability of the change in the amount of zixin, conl. units (* p < 0.05).

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