The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells

Neidhardt, Lisa; Cloots, Eva; Friemel, Natalie; Weiss, Caroline A M; Harding, Heather P; McLaughlin, Stephen H; Janssens, Sophie; Ron, David

doi:10.1038/s44318-023-00014-z

Cited by 13 publications

(4 citation statements)

References 72 publications

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“…This result could represent a decreased response to ER stress in mice without Fmo5. Considering how imperative it is for goblet cells to effectively complete the many steps involved in producing and secreting mucus, the ER of GCs has evolved to include an additional ER-UPR arm in AGR2/IRE-1β that is specifically sensitive to Muc2 31, 32 . We measured protein levels of the chaperone for this arm, AGR2, and find that AGR2 levels are significantly higher in Fmo5 -/- mice than wildtype ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Fmo5 plays a sex-specific role in goblet cell maturation and mucus barrier formation

Schaller,

Sykes,

Mecano

et al. 2024

Preprint

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The intestine plays a key role in metabolism, nutrient and water absorption, and provides both physical and immunological defense against dietary and luminal antigens. The protective mucus lining in the intestine is a critical component of intestinal barrier function that when compromised, can lead to dysfunctional intestinal barriers that are a defining characteristic of inflammatory bowel disease (IBD), among other intestinal diseases. Here, we define a new role for the flavin-containing monooxygenase family of enzymes in maintaining a healthy intestinal epithelium. In nematodes, we find that Cefmo-2 is necessary and sufficient for proper intestinal barrier function, intestinal actin expression, and is induced by intestinal damage. In mice, we utilize an intestine-specific, inducible knockout model of the prevalent gut Fmo (Fmo5) and find striking phenotypes within two weeks of knockout. These phenotypes include sex-dependent changes in colon epithelial histology, goblet cell localization and maturation factors, and mucus barrier formation. Each of these changes are significantly more severe in female mice, plausibly mirroring differences observed in some types of IBD in humans. Looking further at these phenotypes, we find increased protein folding stress in Fmo5 knockout animals and successfully rescue the severe female phenotype with addition of a chemical ER chaperone. Together, our results identify a new role for Fmo5 in the mammalian intestine and support a key role for Fmo5 in maintenance of ER/protein homeostasis and proper mucus barrier formation.

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Section: Resultsmentioning

confidence: 99%

“…A previous study showed that the BiP/IRE-1α arm of the UPR senses a wide variety of ligands that cause ER stress but does not respond to excess Muc2 presence 31 . A mucin-sensitive UPR ER arm made up of AGR2 and IRE1β is hypothesized to take on the responsibility for the folding of mucins in goblet cells 31, 47 . A study by Grey et al .…”

Section: Discussionmentioning

confidence: 99%

Fmo5 plays a sex-specific role in goblet cell maturation and mucus barrier formation

Schaller,

Sykes,

Mecano

et al. 2024

Preprint

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“…XBP1s and XBP1u fragments were amplified from cDNA by PCR using primers flanking the splicing site identified by IRE1 with NEB Q5® High-Fidelity 2X Master Mix, as previously reported (Neidhardt et al, 2023). Briefly, XBP1u (255bp) and XBP1s (229bp) were separated in a 3 % agarose gel by electrophoresis and stained with SYBR Safe nucleic acid gel stain (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α

Tung,

Huang,

George

et al. 2024

Preprint

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Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (⍺) signalling remains poorly understood, possibly because its activation involves ER to Golgi and nuclear trafficking. Here, we generated an ATF6⍺/IRE1 dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6⍺ signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6⍺ activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6⍺ signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6⍺-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6⍺. Purified CRT interacts with the luminal domain of ATF6⍺ in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6⍺ in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6⍺ to selectively regulate one arm of the UPR.

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“…XBP1s and XBP1u fragments were amplified from cDNA by PCR using primers flanking the splicing site identified by IRE1 with NEB Q5 High-Fidelity 2X Master Mix, as previously reported ( Neidhardt et al, 2024 ). Briefly, XBP1u (255 bp) and XBP1s (229 bp) were separated in a 3% agarose gel by electrophoresis and stained with SYBR Safe nucleic acid gel stain (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α

Tung,

Huang,

George

et al. 2024

eLife

Self Cite

View full text Add to dashboard Cite

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (⍺) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6⍺/IRE1 dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6⍺ signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6⍺ activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6⍺ signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6⍺-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6⍺. Purified CRT interacted with the luminal domain of ATF6⍺ in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6⍺ in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6⍺ to selectively regulate one arm of the UPR.

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The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells

Cited by 13 publications

References 72 publications

Fmo5 plays a sex-specific role in goblet cell maturation and mucus barrier formation

Fmo5 plays a sex-specific role in goblet cell maturation and mucus barrier formation

A genome wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α

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