IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response

Mariska S. Simpson; Heidi De Luca; Sarah Cauthorn; Phi Luong; Namrata D. Udeshi; Tanya Svinkina; Stefanie S. Schmieder; Steven A. Carr; Michael J. Grey; Wayne I. Lencer

doi:10.1083/jcb.202402062

IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response

Mariska S. Simpson, Heidi De Luca, Sarah Cauthorn, Phi Luong, Namrata D. Udeshi, Tanya Svinkina, Stefanie S. Schmieder, Steven A. Carr, Michael J. Grey^*, Wayne I. Lencer^*

^*Corresponding author for this work

Faculty of Science

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge β-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.

Original language	English
Article number	e202402062
Number of pages	15
Journal	The Journal of cell biology
Volume	223
Issue number	7
Early online date	5 Apr 2024
DOIs	https://doi.org/10.1083/jcb.202402062
Publication status	Published - 1 Jul 2024

Bibliographical note

Publisher Copyright:
© 2024 Simpson et al.

Funding

We thank members of the Lencer Lab and Ineke Braakman (Utrecht University) for feedback throughout the course of this project; the Harvard Digestive Disease Center for services and instrumentation provided by the Epithelial Cell and Mucosal Immunology Core and the Microscopy and Histopathology Core; the Center for Macromolecular Interactions (Harvard Medical School) for access to microscale thermophoresis instrumentation; the Taplin Mass Spectrometry Facility (Harvard Medical School) for identification/validation of purified proteins, and Francesca Massi (University of Massachusetts Chan School of Medicine) for access to the Fluorlog-3 spectrofluorometer.Funding was provided by National Institutes of Health grants R01DK048106 (W.I. Lencer, M.J. Grey), K01DK119414 (M.J. Grey),and P30DK034854 (W.I. Lencer), and Boston Children's Hospital-Broad Institute Collaboration Grant (W.I. Lencer, P. Luong,N.D. Udeshi, S.A. Carr).

Funders	Funder number
Lencer Lab and Ineke Braakman
Harvard Digestive Diseases Center, Harvard University
Harvard Medical School
Universiteit Utrecht
Taplin Mass Spectrometry Facility
National Institutes of Health	P30DK034854, K01DK119414, R01DK048106

Keywords

Biochemistry
Protein homeostasis

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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Cite this

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title = "IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response",

abstract = "IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge β-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.",

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author = "Simpson, {Mariska S.} and {De Luca}, Heidi and Sarah Cauthorn and Phi Luong and Udeshi, {Namrata D.} and Tanya Svinkina and Schmieder, {Stefanie S.} and Carr, {Steven A.} and Grey, {Michael J.} and Lencer, {Wayne I.}",

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doi = "10.1083/jcb.202402062",

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AU - Luong, Phi

AU - Udeshi, Namrata D.

AU - Svinkina, Tanya

AU - Schmieder, Stefanie S.

AU - Carr, Steven A.

AU - Grey, Michael J.

AU - Lencer, Wayne I.

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N2 - IRE1α is an endoplasmic reticulum (ER) sensor that recognizes misfolded proteins to induce the unfolded protein response (UPR). We studied cholera toxin (CTx), which invades the ER and activates IRE1α in host cells, to understand how unfolded proteins are recognized. Proximity labeling colocalized the enzymatic and metastable A1 segment of CTx (CTxA1) with IRE1α in live cells, where we also found that CTx-induced IRE1α activation enhanced toxicity. In vitro, CTxA1 bound the IRE1α lumenal domain (IRE1αLD), but global unfolding was not required. Rather, the IRE1αLD recognized a seven-residue motif within an edge β-strand of CTxA1 that must locally unfold for binding. Binding mapped to a pocket on IRE1αLD normally occupied by a segment of the IRE1α C-terminal flexible loop implicated in IRE1α oligomerization. Mutation of the CTxA1 recognition motif blocked CTx-induced IRE1α activation in live cells, thus linking the binding event with IRE1α signal transduction and induction of the UPR.

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IRE1α recognizes a structural motif in cholera toxin to activate an unfolded protein response

Abstract

Bibliographical note

Funding

Keywords

UN SDGs

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