Mechanistic characterization of disulfide bond reduction of an ERAD substrate mediated by cooperation between ERdj5 and BiP

Xiaohan Cai; Shogo Ito; Kentaro Noi; Michio Inoue; Ryo Ushioda; Yukinari Kato; Kazuhiro Nagata; Kenji Inaba

doi:10.1016/j.jbc.2023.105274

Mechanistic characterization of disulfide bond reduction of an ERAD substrate mediated by cooperation between ERdj5 and BiP

J Biol Chem. 2023 Nov;299(11):105274. doi: 10.1016/j.jbc.2023.105274. Epub 2023 Sep 21.

Authors

Xiaohan Cai¹, Shogo Ito¹, Kentaro Noi², Michio Inoue³, Ryo Ushioda⁴, Yukinari Kato⁵, Kazuhiro Nagata⁴, Kenji Inaba⁶

Affiliations

¹ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi, Japan; Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan.
² Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.
³ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi, Japan.
⁴ Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan.
⁵ Graduate School of Medicine, Tohoku University, Sendai, Japan.
⁶ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Miyagi, Japan; Department of Molecular and Chemical Life Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan; Department of Chemistry, Graduate School of Science, Tohoku University, Sendai, Miyagi, Japan; Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Agency for Medical Research and Development (AMED), Tokyo, Japan. Electronic address: kenji.inaba.a1@tohoku.ac.jp.

Abstract

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality control process that eliminates misfolded proteins from the ER. DnaJ homolog subfamily C member 10 (ERdj5) is a protein disulfide isomerase family member that accelerates ERAD by reducing disulfide bonds of aberrant proteins with the help of an ER-resident chaperone BiP. However, the detailed mechanisms by which ERdj5 acts in concert with BiP are poorly understood. In this study, we reconstituted an in vitro system that monitors ERdj5-mediated reduction of disulfide-linked J-chain oligomers, known to be physiological ERAD substrates. Biochemical analyses using purified proteins revealed that J-chain oligomers were reduced to monomers by ERdj5 in a stepwise manner via trimeric and dimeric intermediates, and BiP synergistically enhanced this action in an ATP-dependent manner. Single-molecule observations of ERdj5-catalyzed J-chain disaggregation using high-speed atomic force microscopy, demonstrated the stochastic release of small J-chain oligomers through repeated actions of ERdj5 on peripheral and flexible regions of large J-chain aggregates. Using systematic mutational analyses, ERAD substrate disaggregation mediated by ERdj5 and BiP was dissected at the molecular level.

Keywords: BiP; ER proteostasis; ER-associated degradation; ERdj5; J-chain; high-speed AFM.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Endoplasmic Reticulum Chaperone BiP* / chemistry
Endoplasmic Reticulum Chaperone BiP* / genetics
Endoplasmic Reticulum Chaperone BiP* / metabolism
Endoplasmic Reticulum-Associated Degradation*
HEK293 Cells
Immunoglobulin J-Chains / metabolism
Molecular Chaperones* / chemistry
Molecular Chaperones* / genetics
Molecular Chaperones* / metabolism
Protein Domains
Protein Folding

Substances

Endoplasmic Reticulum Chaperone BiP
Molecular Chaperones
DNAJC10 protein, human
Immunoglobulin J-Chains