A regulatory circuit operated by KDELR1 and KDELR3 fine-tunes the composition of the early secretory pathway
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE289834 | GEO | 2026/03/11
REPOSITORIES: GEO
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Project description:Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells, but the function of AGR2 has been obscure. Here we show that AGR2 is expressed in the ER of secretory cells and is essential for in vivo production of intestinal mucin, a large cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, consistent with a direct role for AGR2 in mucin processing. Despite a complete absence of intestinal mucin, mice lacking AGR2 appeared healthy but were highly susceptible to dextran sodium sulfate-induced experimental colitis, indicating a critical role for AGR2 in protection from environmental insults. We conclude that AGR2 is a unique member of the PDI family that has a specialized and non-redundant role in intestinal mucus production. Keywords: small intestine and colon gene expression profiles for Agr2-/- and littermate control mice
2009-05-15 | GSE12503 | GEO
Project description:Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells, but the function of AGR2 has been obscure. Here we show that AGR2 is expressed in the ER of secretory cells and is essential for in vivo production of intestinal mucin, a large cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, consistent with a direct role for AGR2 in mucin processing. Despite a complete absence of intestinal mucin, mice lacking AGR2 appeared healthy but were highly susceptible to dextran sodium sulfate-induced experimental colitis, indicating a critical role for AGR2 in protection from environmental insults. We conclude that AGR2 is a unique member of the PDI family that has a specialized and non-redundant role in intestinal mucus production. Keywords: small intestine and colon gene expression profiles for Agr2-/- and littermate control mice DNA miocroarrays were used to analyze small intenstine and colon mRNA expression of AGR2 KO and littermate control mice. The experiment incorporated a 1 color design and used Agilent arrays that contained roughly 44,00 60mer probes that provide complete coverage of the mouse genome. 12 arrays were hybridized and represent 8 small intestine samples ( 4 each KO and WT) and 4 colon samples (2 each KO and WT)
2010-05-19 | E-GEOD-12503 | biostudies-arrayexpress
Project description:AGR2 is an oncogenic endoplasmic reticulum (ER)-resident protein disulfide isomerase. AGR2 protein has a relatively unique property for a chaperone in that it can bind sequence-specifically to a peptide motif (TTIYY). A synthetic TTIYY-containing peptide column can be used to affinity-purify AGR2 from crude lysates highlighting peptide selectivity in complex mixtures. Hydrogen-deuterium exchange mass spectrometry localized the dominant region in AGR2 that interacts with the TTIYY peptide to within a structural loop from amino acids 131-135 (VDPSL). A peptide binding site consensus of Tx[IL][YF][YF] was developed for AGR2 by measuring its activity against a alanine mutagenized synthetic peptide library. Screening the human proteome for proteins harboring this consensus motif revealed an enrichment in transmembrane proteins and we focus on validating EpCAM as one such oncogenic protein. Recombinant AGR2 and EpCAM proteins formed a dose-dependent protein-protein interaction in vitro. Proximity ligation assays demonstrated that endogenous AGR2 and EpCAM protein associate in cells. Introducing a single alanine mutation in EpCAM at Tyr251 attenuated its binding to AGR2 in vitro and in cells. Hydrogen-deuterium exchange mass spectrometry was used to identify a stable binding site for AGR2 on EpCAM, adjacent to the TILYY motif and surrounding EpCAM’s detergent binding site. Together, these data define a dominant peptide-binding site on AGR2 that mediates its specific peptide-binding function. A model client protein, EpCAM, is proposed for AGR2 to study how an ER-resident chaperone can dock specifically and regulate assembly of a protein destined for the secretory pathway.
2018-01-22 | PXD005782 | Pride
Project description:Background and aims: Intestinal fibrosis is a common complication of Crohn’s disease (CD). It is characterised by an excessive accumulation of fibroblasts differentiating into activated myofibroblasts secreting excessive extracellular matrix. The potential role of the intestinal epithelium in this fibrotic process remains poorly defined. Methods: A total of 113 CD and control subjects were studied. We performed a pilot proteomic study comparing the proteome of surface epithelium isolated by laser capture microdissection in normal and fibrotic zones of resected ileal CD strictures (n=5). Selected proteins were validated by immunohistochemistry (IHC) in colonic and ileal samples of stricturing CD patients (n=44), pure inflammatory CD (n=29) and control subjects (n=40). Functional assays with cell lines cultures and a fibroblast to myofibroblast differentiation model were used to assess the role of the highlighted epithelial proteins in CD fibrosis. Results: Proteomic study revealed an endoplasmic reticulum (ER) stress and unfolded protein response (UPR) markers increase in the epithelium overlying ileal fibrotic strictures, involving Anterior gradient protein 2 homolog (AGR2) and Binding immunoglobulin protein (BiP). This was confirmed by IHC. The ER stress induction in intestinal epithelial cells increased AGR2 as well as BiP expression and led to an extracellular secreted AGR2. A fibroblast to myofibroblast differentiation was obtained with the supernatant of intestinal epithelial cells pre-conditioned by ER stress and with recombinant AGR2. Conclusions: AGR2 and other ER stress markers are increased within the intestinal epithelium overlying fibrotic strictures and might contribute to profibrotic signals involved in CD fibrosis.
2022-04-04 | PXD022214 | Pride
Project description:The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature proteasomal degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.
2018-05-29 | GSE112891 | GEO
Project description:The endoplasmic reticulum (ER) functions in protein and lipid synthesis, calcium ion flux, and inter-organelle communication, all of which are driven by the ER proteome landscape. ER is remodeled in part through autophagy-dependent protein turnover involving membrane-embedded ER-phagy receptors1,2. A refined tubular ER network is formed in neurons within highly polarized dendrites and axons3,4. Autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic ER boutons, associated with hyper-excitability,5 and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy6,7. However, mechanisms and receptor selectivity underlying ER remodeling by autophagy in neurons is limited. Here, we employ genetic, proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy during conversion of stem cells to induced neurons in vitro. Through analysis of single and combinatorial ER-phagy receptor mutants coupled with an allelic series computational framework, we delineate the extent to which each of five receptors contributes to both the magnitude of ER turnover by autophagy and the selectivity of clearance for individual ER proteins. We define specific subsets of reticulon-domain containing ER-tubule shaping proteins or luminal proteins as preferred clients for autophagic turnover via distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, which correlates with aberrant accumulation of ER in axonal structures in ER-phagy receptor or autophagy-deficient cells. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping this critical organelle during transitions in cell states.
2024-01-08 | PXD046646 | Pride
Project description:The endoplasmic reticulum (ER) functions in protein and lipid synthesis, calcium ion flux, and inter-organelle communication, all of which are driven by the ER proteome landscape. ER is remodeled in part through autophagy-dependent protein turnover involving membrane-embedded ER-phagy receptors1,2. A refined tubular ER network is formed in neurons within highly polarized dendrites and axons3,4. Autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic ER boutons, associated with hyper-excitability,5 and the ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy6,7. However, mechanisms and receptor selectivity underlying ER remodeling by autophagy in neurons is limited. Here, we employ genetic, proteomic and computational tools to create a quantitative landscape of ER proteome remodeling via selective autophagy during conversion of stem cells to induced neurons in vitro. Through analysis of single and combinatorial ER-phagy receptor mutants coupled with an allelic series computational framework, we delineate the extent to which each of five receptors contributes to both the magnitude of ER turnover by autophagy and the selectivity of clearance for individual ER proteins. We define specific subsets of reticulon-domain containing ER-tubule shaping proteins or luminal proteins as preferred clients for autophagic turnover via distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, which correlates with aberrant accumulation of ER in axonal structures in ER-phagy receptor or autophagy-deficient cells. This molecular inventory of ER proteome remodeling and versatile genetic toolkit provides a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping this critical organelle during transitions in cell states.
2024-01-08 | PXD041069 | Pride
Project description:Canine mammary tumors (CMTs) in intact female dogs serve as a valuable natural model for understanding human cancers. Our previous findings identified the overexpression of Anterior Gradient 2 (AGR2) in CMT tissues compared to normal mammary glands, highlighting its association with CMT progression. We observed that enhanced AGR2 expression actively promotes CMT cell chemotaxis by modulating the extracellular milieu. To decipher the AGR2-affected secretome associated with chemotaxis, we utilized gel-enhanced liquid chromatography-tandem mass spectrometry (GeLC-MS/MS) to scrutinize the conditioned media (CM) of AGR2-expressing CMT-U27 and CF41.Mg cells in comparison to the respective vector-expressing controls. In total, 798 proteins in CMT-U27 and 788 proteins in CF41.Mg were successfully identified and quantified. Among these proteins, 51 and 40 CM proteins were identified as AGR2-increased, while 28 and 50 CM proteins were designated as AGR2-decreased, respectively. Intriguingly, seven CM proteins exhibited increased abundance in both CMT-U27 and CF41.Mg cells, including AGR2, 14-3-3ε (gene name: YWHAE), and α-Actinin-4 (ACTN4). Ectopic AGR2 expression significantly increases the release of 14-3-3ε and ACTN4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduces their release. AGR2 controls the release of 14-3-3ε and ACTN4, responsive to activation of ER stress and autophagy. Finally, depleting extracellular 14-3-3ε or ACTN4 reduces the AGR2-modulated CM chemotaxis. Our study uncovers the novel extracellular pro-oncogenic functions of 14-3-3ε and ACTN4 in the AGR2-modulated microenvironment.
2024-06-16 | PXD047008 | Pride
Project description:We and others have shown that AGR2 is frequently upregulated during the development of pancreatic cancer. We used microarray to look at the target genes regulated by AGR2 in pancreatic cancer cell lines FA6 and MiaPaCa2. Keywords: gene knock-down, overexpression We transiently down-regulated AGR2 expression in FA6 pancreatic cancer cells using INTERFERin transfection reagent and either AGR2 siRNA or non-targeting control siRNA for 48 hours. RNA was extracted and hybridized on Affymetrix microarrays. We looked for new target genes regulated by AGR2. We generated stable cell lines by introducing control vector pCEP4 or AGR2 overexpressing vector pCEP4-AGR2 into the pancreatic cancer cell line MiaPaCa2, single cell clones were then isolated. RNA was extracted and hybridized on Affymetrix microarrays.We looked for new target genes regulated by AGR2.
2013-07-28 | E-GEOD-12513 | biostudies-arrayexpress
Project description:The in vivo function of AGR2 was examined by generating an AGR2 null mouse. The mice died prematurely due to hyperplasia and dysplasia of the glandular stomach. Gene expression profiling was performed to compare differences in transcription between wild-type and null AGR2 mice.
2012-12-01 | GSE40062 | GEO