Cargando…
VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells
VivosX is an in vivo disulfide crosslinking approach that utilizes a pair of strategically positioned cysteines on two proteins to probe physical interactions within cells. Histone H2A.Z, which often replaces one or both copies of H2A in nucleosomes downstream of promoters, was used to validate Vivo...
Autores principales: | , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
eLife Sciences Publications, Ltd
2018
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107336/ https://www.ncbi.nlm.nih.gov/pubmed/30091702 http://dx.doi.org/10.7554/eLife.36654 |
_version_ | 1783349959950598144 |
---|---|
author | Mohan, Chitra Kim, Lisa M Hollar, Nicole Li, Tailai Paulissen, Eric Leung, Cheuk T Luk, Ed |
author_facet | Mohan, Chitra Kim, Lisa M Hollar, Nicole Li, Tailai Paulissen, Eric Leung, Cheuk T Luk, Ed |
author_sort | Mohan, Chitra |
collection | PubMed |
description | VivosX is an in vivo disulfide crosslinking approach that utilizes a pair of strategically positioned cysteines on two proteins to probe physical interactions within cells. Histone H2A.Z, which often replaces one or both copies of H2A in nucleosomes downstream of promoters, was used to validate VivosX. Disulfide crosslinks between cysteine-modified H2A.Z and/or H2A histones within nucleosomes were induced using a membrane-permeable oxidant. VivosX detected different combinations of H2A.Z and H2A within nucleosomes in yeast cells. This assay correctly reported the change in global H2A.Z occupancy previously observed when the deposition and eviction pathways of H2A.Z were perturbed. Homotypic H2A.Z/H2A.Z (ZZ) nucleosomes accumulated when assembly of the transcription preinitiation complex was blocked, revealing that the transcription machinery preferentially disassembles ZZ nucleosomes. VivosX works in human cells and distinguishes ZZ nucleosomes with one or two ubiquitin moieties, demonstrating that it can be used to detect protein-protein interactions inside cells from different species. |
format | Online Article Text |
id | pubmed-6107336 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | eLife Sciences Publications, Ltd |
record_format | MEDLINE/PubMed |
spelling | pubmed-61073362018-08-27 VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells Mohan, Chitra Kim, Lisa M Hollar, Nicole Li, Tailai Paulissen, Eric Leung, Cheuk T Luk, Ed eLife Biochemistry and Chemical Biology VivosX is an in vivo disulfide crosslinking approach that utilizes a pair of strategically positioned cysteines on two proteins to probe physical interactions within cells. Histone H2A.Z, which often replaces one or both copies of H2A in nucleosomes downstream of promoters, was used to validate VivosX. Disulfide crosslinks between cysteine-modified H2A.Z and/or H2A histones within nucleosomes were induced using a membrane-permeable oxidant. VivosX detected different combinations of H2A.Z and H2A within nucleosomes in yeast cells. This assay correctly reported the change in global H2A.Z occupancy previously observed when the deposition and eviction pathways of H2A.Z were perturbed. Homotypic H2A.Z/H2A.Z (ZZ) nucleosomes accumulated when assembly of the transcription preinitiation complex was blocked, revealing that the transcription machinery preferentially disassembles ZZ nucleosomes. VivosX works in human cells and distinguishes ZZ nucleosomes with one or two ubiquitin moieties, demonstrating that it can be used to detect protein-protein interactions inside cells from different species. eLife Sciences Publications, Ltd 2018-08-09 /pmc/articles/PMC6107336/ /pubmed/30091702 http://dx.doi.org/10.7554/eLife.36654 Text en © 2018, Mohan et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited. |
spellingShingle | Biochemistry and Chemical Biology Mohan, Chitra Kim, Lisa M Hollar, Nicole Li, Tailai Paulissen, Eric Leung, Cheuk T Luk, Ed VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells |
title | VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells |
title_full | VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells |
title_fullStr | VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells |
title_full_unstemmed | VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells |
title_short | VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells |
title_sort | vivosx, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells |
topic | Biochemistry and Chemical Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107336/ https://www.ncbi.nlm.nih.gov/pubmed/30091702 http://dx.doi.org/10.7554/eLife.36654 |
work_keys_str_mv | AT mohanchitra vivosxadisulfidecrosslinkingmethodtocapturesitespecificproteinproteininteractionsinyeastandhumancells AT kimlisam vivosxadisulfidecrosslinkingmethodtocapturesitespecificproteinproteininteractionsinyeastandhumancells AT hollarnicole vivosxadisulfidecrosslinkingmethodtocapturesitespecificproteinproteininteractionsinyeastandhumancells AT litailai vivosxadisulfidecrosslinkingmethodtocapturesitespecificproteinproteininteractionsinyeastandhumancells AT paulisseneric vivosxadisulfidecrosslinkingmethodtocapturesitespecificproteinproteininteractionsinyeastandhumancells AT leungcheukt vivosxadisulfidecrosslinkingmethodtocapturesitespecificproteinproteininteractionsinyeastandhumancells AT luked vivosxadisulfidecrosslinkingmethodtocapturesitespecificproteinproteininteractionsinyeastandhumancells |