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Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins

The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering...

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Autores principales: Zhao, Jianhua, Makhija, Suraj, Zhou, Chenyu, Zhang, Hanxiao, Wang, YongQiang, Muralidharan, Monita, Huang, Bo, Cheng, Yifan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9388094/
https://www.ncbi.nlm.nih.gov/pubmed/35858375
http://dx.doi.org/10.1073/pnas.2207200119
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author Zhao, Jianhua
Makhija, Suraj
Zhou, Chenyu
Zhang, Hanxiao
Wang, YongQiang
Muralidharan, Monita
Huang, Bo
Cheng, Yifan
author_facet Zhao, Jianhua
Makhija, Suraj
Zhou, Chenyu
Zhang, Hanxiao
Wang, YongQiang
Muralidharan, Monita
Huang, Bo
Cheng, Yifan
author_sort Zhao, Jianhua
collection PubMed
description The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ–20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.
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spelling pubmed-93880942022-08-19 Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins Zhao, Jianhua Makhija, Suraj Zhou, Chenyu Zhang, Hanxiao Wang, YongQiang Muralidharan, Monita Huang, Bo Cheng, Yifan Proc Natl Acad Sci U S A Biological Sciences The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αβ–20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4β engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources. National Academy of Sciences 2022-07-19 2022-08-16 /pmc/articles/PMC9388094/ /pubmed/35858375 http://dx.doi.org/10.1073/pnas.2207200119 Text en Copyright © 2022 the Author(s). Published by PNAS. https://creativecommons.org/licenses/by/4.0/This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Biological Sciences
Zhao, Jianhua
Makhija, Suraj
Zhou, Chenyu
Zhang, Hanxiao
Wang, YongQiang
Muralidharan, Monita
Huang, Bo
Cheng, Yifan
Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins
title Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins
title_full Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins
title_fullStr Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins
title_full_unstemmed Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins
title_short Structural insights into the human PA28–20S proteasome enabled by efficient tagging and purification of endogenous proteins
title_sort structural insights into the human pa28–20s proteasome enabled by efficient tagging and purification of endogenous proteins
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9388094/
https://www.ncbi.nlm.nih.gov/pubmed/35858375
http://dx.doi.org/10.1073/pnas.2207200119
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