Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2

Adams, Benjamin M.; Canniff, Nathan P; Guay, Kevin P; Larsen, Ida Signe Bohse; Hebert, Daniel N.

doi:10.7554/elife.63997

Cited by 47 publications

(48 citation statements)

References 81 publications

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“…KEGG enrichment analysis of the differentially expressed proteins showed that the downregulated proteins UGGT1, HSPA5, HSPA8, and HSP90AA1 are enriched in protein processing in ER, the PI3K–AKT signaling pathway, RNA transport, and the estrogen signaling pathway in Lec1 cells, and in regulating the actin cytoskeleton via focal adhesion molecules in Lec4 cells ( Figure 3 B). UGGT1 is a carbohydrate-dependent chaperone which facilitates proper folding and maturation of the cellular N-glycoproteome [ 84 ]. The downregulated expression of heat shock proteins HSP90AA1, HSPA5, and HSPA8 was involved in apoptosis-related protein, antigen presentation, and the estrogen signaling pathway.…”

Section: Discussionmentioning

confidence: 99%

N-Linked Glycosylation in Chinese Hamster Ovary Cells Is Critical for Insulin-like Growth Factor 1 Signaling

Salvi

Kumar

Brahmbhatt

et al. 2022

IJMS

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Cell surface proteins carrying N-glycans play important roles in inter- and intracellular processes including cell adhesion, development, and cellular recognition. Dysregulation of the glycosylation machinery has been implicated in various diseases, and investigation of global differential cell surface proteome effects due to the loss of N-glycosylation will provide comprehensive insights into their pathogenesis. Cell surface proteins isolated from Parent Pro–5 CHO cells (W5 cells), two CHO mutants with loss of N-glycosylation function derived from Pro–5 CHO (Lec1 and Lec4 cells), were subjected to proteome analysis via high-resolution LCMS. We identified 44 and 43 differentially expressed membrane proteins in Lec1 and Lec4 cells, respectively, as compared to W5 cells. The defective N-glycosylation mutants showed increased abundance of integrin subunits in Lec1 and Lec4 cells at the cell surface. We also found significantly reduced levels of IGF-1R (Insulin like growth factor-1 receptor); a receptor tyrosine kinase; and the GTPase activating protein IQGAP1 (IQ motif-containing GTPase activating protein), a highly conserved cytoplasmic scaffold protein) in Lec1 and Lec4 cells. In silico docking studies showed that the IQ domain of IQGAP1 interacts with the kinase domain of IGF-1R. The integrin signaling and insulin growth factor receptor signaling were also enriched according to GSEA analysis and pathway analysis of differentially expressed proteins. Significant reductions of phosphorylation of ERK1 and ERK2 in Lec1 and Lec4 cells were observed upon IGF-1R ligand (IGF-1 LR3) stimulation. IGF-1 LR3, known as Long arginine3-IGF-1, is a synthetic protein and lengthened analog of insulin-like growth factor 1. The work suggests a novel mechanism for the activation of IGF-1 dependent ERK signaling in CHO cells, wherein IQGAP1 plausibly functions as an IGF-1R-associated scaffold protein. Appropriate glycosylation by the enzymes MGAT1 and MGAT5 is thus essential for processing of cell surface receptor IGF-1R, a potential binding partner in IQGAP1 and ERK signaling, the integral components of the IGF pathway.

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Section: Discussionmentioning

confidence: 99%

N-Linked Glycosylation in Chinese Hamster Ovary Cells Is Critical for Insulin-like Growth Factor 1 Signaling

Salvi

Kumar

Brahmbhatt

et al. 2022

IJMS

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“…Folding and processing of native polypeptides, the primary function of the Endoplasmic Reticulum (ER), are mediated by a cadre of ER-resident protein chaperones. ER dysfunction leads to an increase in misfolded proteins in the ER lumen, a condition known as ER stress [ 1 ]. ER stress activates ER-transmembrane proteins, including Protein RNA-like Endoplasmic Reticulum Kinase (PERK), Inositol-Regulated Enzyme 1-alpha (IRE1α), and Activating-Transcription Factor 6-α (ATF6α).…”

Section: Introductionmentioning

confidence: 99%

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response

et al. 2022

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Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of newly synthesized RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.

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“…Folding and processing of native polypeptides, the primary function of the Endoplasmic Reticulum (ER), are mediated by a cadre of ER-resident protein chaperones. ER dysfunction leads to an increase in misfolded proteins in the ER lumen, a condition known as ER stress (Adams et al 2020). ER stress activates ER-transmembrane proteins, including Protein RNA-like Endoplasmic Reticulum Kinase (PERK), Inositol-Regulated Enzyme 1-alpha (IRE1α), and Activating-Transcription Factor 6-α (ATF6α).…”

Section: Introductionmentioning

confidence: 99%

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response

Alzahrani

Guan

Zagore

et al. 2022

Preprint

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Endoplasmic Reticulum (ER) stress, caused by the accumulation of misfolded proteins in the ER, elicits a homeostatic mechanism known as the Unfolded Protein Response (UPR). The UPR reprograms gene expression to promote adaptation to chronic ER stress. The UPR comprises an acute phase involving inhibition of bulk protein synthesis and a chronic phase of transcriptional induction coupled with the partial recovery of protein synthesis. However, the role of transcriptional regulation in the acute phase of the UPR is not well understood. Here we analyzed the fate of newly synthesized mRNA encoding the protective and homeostatic transcription factor X-box binding protein 1 (XBP1) during this acute phase. We have previously shown that global translational repression induced by the acute UPR was characterized by decreased translation and increased stability of XBP1 mRNA. We demonstrate here that this stabilization is independent of new transcription. In contrast, we show XBP1 mRNA newly synthesized during the acute phase accumulates with long poly(A) tails and escapes translational repression. Inhibition of nascent RNA polyadenylation during the acute phase decreased cell survival with no effect in unstressed cells. Furthermore, during the chronic phase of the UPR, levels of XBP1 mRNA with long poly(A) tails decreased in a manner consistent with co-translational deadenylation. Finally, additional pro-survival, transcriptionally-induced mRNAs show similar regulation, supporting the broad significance of the pre-steady state UPR in translational control during ER stress. We conclude that the biphasic regulation of poly(A) tail length during the UPR represents a previously unrecognized pro-survival mechanism of mammalian gene regulation.

show abstract

Quantitative glycoproteomics reveals cellular substrate selectivity of the ER protein quality control sensors UGGT1 and UGGT2

Cited by 47 publications

References 81 publications

N-Linked Glycosylation in Chinese Hamster Ovary Cells Is Critical for Insulin-like Growth Factor 1 Signaling

N-Linked Glycosylation in Chinese Hamster Ovary Cells Is Critical for Insulin-like Growth Factor 1 Signaling

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response

Newly synthesized mRNA escapes translational repression during the acute phase of the mammalian unfolded protein response

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