A high-throughput genetic screening protocol to measure lipid bilayer stress-induced unfolded protein response in Saccharomyces cerevisiae

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2α/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An ˜66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

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Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae

Proteome Science ◽

10.1186/1477-5956-11-36 ◽

2013 ◽

Vol 11 (1) ◽

pp. 36 ◽

Cited By ~ 9

Author(s):

Rimantas Slibinskas ◽

Raimundas Ražanskas ◽

Rūta Zinkevičiūtė ◽

Evaldas Čiplys

Keyword(s):

Saccharomyces Cerevisiae ◽

Unfolded Protein Response ◽

Gel Electrophoresis ◽

Two Dimensional ◽

Two Dimensional Gel Electrophoresis ◽

Unfolded Protein ◽

Protein Response

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Yeast Bax Inhibitor, Bxi1p, Is an ER-Localized Protein That Links the Unfolded Protein Response and Programmed Cell Death in Saccharomyces cerevisiae

PLoS ONE ◽

10.1371/journal.pone.0020882 ◽

2011 ◽

Vol 6 (6) ◽

pp. e20882 ◽

Cited By ~ 34

Author(s):

James Cebulski ◽

Joshua Malouin ◽

Nathan Pinches ◽

Vincent Cascio ◽

Nicanor Austriaco

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Death ◽

Programmed Cell Death ◽

Unfolded Protein Response ◽

Unfolded Protein ◽

The Unfolded Protein Response ◽

Protein Response

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The Unfolded Protein Response: An Overview

Biology ◽

10.3390/biology10050384 ◽

2021 ◽

Vol 10 (5) ◽

pp. 384

Author(s):

Adam Read ◽

Martin Schröder

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Unfolded Protein Response ◽

Protein Homeostasis ◽

Unfolded Proteins ◽

Unfolded Protein ◽

Altered Glycosylation ◽

Stressed Cells ◽

The Unfolded Protein Response ◽

Protein Response

The unfolded protein response is the mechanism by which cells control endoplasmic reticulum (ER) protein homeostasis. Under normal conditions, the UPR is not activated; however, under certain stresses, such as hypoxia or altered glycosylation, the UPR can be activated due to an accumulation of unfolded proteins. The activation of the UPR involves three signaling pathways, IRE1, PERK and ATF6, which all play vital roles in returning protein homeostasis to levels seen in non-stressed cells. IRE1 is the best studied of the three pathways, as it is the only pathway present in Saccharomyces cerevisiae. This pathway involves spliceosome independent splicing of HAC1 or XBP1 in yeast and mammalians cells, respectively. PERK limits protein synthesis, therefore reducing the number of new proteins requiring folding. ATF6 is translocated and proteolytically cleaved, releasing a NH2 domain fragment which is transported to the nucleus and which affects gene expression. If the UPR is unsuccessful at reducing the load of unfolded proteins in the ER and the UPR signals remain activated, this can lead to programmed cell death.

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