Limits...
Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

Samali A, Fitzgerald U, Deegan S, Gupta S - Int J Cell Biol (2010)

Bottom Line: Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field.There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems.We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Ireland, Galway, Galway, Ireland.

ABSTRACT
The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

No MeSH data available.


Related in: MedlinePlus

Detection of transcript levels of UPR target genes by RT-PCR. (a) Upper panel, cartoon of XBP1 splicing during ER stress. Lower panel, schematic representation of various mutant constructs of IRE1. (b) Modulation of XBP1 splicing by mutant IRE1. Total RNA was isolated from HEK 293 cells that were transfected with IRE1 mutants, either untreated or treated with thapsigargin (0.5 μM) 6 hours, and RT-PCR analysis of total RNA was performed to simultaneously detect both spliced and unspliced XBP1 mRNA and GADPH. (c) Induction of UPR target genes upon exposure to thapsigargin. Total RNA was isolated from indicated cells after treatment thapsigargin (Tg), and the expression levels of the indicated genes were determined by real-time RT-PCR, normalizing against GAPDH expression.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2821749&req=5

fig1: Detection of transcript levels of UPR target genes by RT-PCR. (a) Upper panel, cartoon of XBP1 splicing during ER stress. Lower panel, schematic representation of various mutant constructs of IRE1. (b) Modulation of XBP1 splicing by mutant IRE1. Total RNA was isolated from HEK 293 cells that were transfected with IRE1 mutants, either untreated or treated with thapsigargin (0.5 μM) 6 hours, and RT-PCR analysis of total RNA was performed to simultaneously detect both spliced and unspliced XBP1 mRNA and GADPH. (c) Induction of UPR target genes upon exposure to thapsigargin. Total RNA was isolated from indicated cells after treatment thapsigargin (Tg), and the expression levels of the indicated genes were determined by real-time RT-PCR, normalizing against GAPDH expression.

Mentions: In response to accumulation of unfolded proteins in the ER, IRE1 oligomerizes in the plane of the membrane, allowing for transautophosphorylation of juxtaposed kinase domains. The transautophosphorylation of the kinase domain of IRE1 activates its unusual effector function, which causes the unconventional splicing of the mRNA that encodes a transcription factor named XBP1 [15]. In metazoans, a 26-nucleotide intron is spliced out by activated IRE1, leading to a shift in the codon reading frame (Figure 1(a)). The XBP1 protein encoded by the spliced mRNA is more stable and is a potent transcription factor of the basic-leucine zipper (bZIP) family and one of the key regulators of ER folding capacity [15, 16]. The splicing of XBP1 mRNA can be detected by semiquantitative RT-PCR using primers specific for XBP1 which will detect both unspliced and spliced isoforms. The 5′ to 3′ sequences of primers used to detect unspliced and spliced XBP1 mRNA are as indicated below.


Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.

Samali A, Fitzgerald U, Deegan S, Gupta S - Int J Cell Biol (2010)

Detection of transcript levels of UPR target genes by RT-PCR. (a) Upper panel, cartoon of XBP1 splicing during ER stress. Lower panel, schematic representation of various mutant constructs of IRE1. (b) Modulation of XBP1 splicing by mutant IRE1. Total RNA was isolated from HEK 293 cells that were transfected with IRE1 mutants, either untreated or treated with thapsigargin (0.5 μM) 6 hours, and RT-PCR analysis of total RNA was performed to simultaneously detect both spliced and unspliced XBP1 mRNA and GADPH. (c) Induction of UPR target genes upon exposure to thapsigargin. Total RNA was isolated from indicated cells after treatment thapsigargin (Tg), and the expression levels of the indicated genes were determined by real-time RT-PCR, normalizing against GAPDH expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821749&req=5

fig1: Detection of transcript levels of UPR target genes by RT-PCR. (a) Upper panel, cartoon of XBP1 splicing during ER stress. Lower panel, schematic representation of various mutant constructs of IRE1. (b) Modulation of XBP1 splicing by mutant IRE1. Total RNA was isolated from HEK 293 cells that were transfected with IRE1 mutants, either untreated or treated with thapsigargin (0.5 μM) 6 hours, and RT-PCR analysis of total RNA was performed to simultaneously detect both spliced and unspliced XBP1 mRNA and GADPH. (c) Induction of UPR target genes upon exposure to thapsigargin. Total RNA was isolated from indicated cells after treatment thapsigargin (Tg), and the expression levels of the indicated genes were determined by real-time RT-PCR, normalizing against GAPDH expression.
Mentions: In response to accumulation of unfolded proteins in the ER, IRE1 oligomerizes in the plane of the membrane, allowing for transautophosphorylation of juxtaposed kinase domains. The transautophosphorylation of the kinase domain of IRE1 activates its unusual effector function, which causes the unconventional splicing of the mRNA that encodes a transcription factor named XBP1 [15]. In metazoans, a 26-nucleotide intron is spliced out by activated IRE1, leading to a shift in the codon reading frame (Figure 1(a)). The XBP1 protein encoded by the spliced mRNA is more stable and is a potent transcription factor of the basic-leucine zipper (bZIP) family and one of the key regulators of ER folding capacity [15, 16]. The splicing of XBP1 mRNA can be detected by semiquantitative RT-PCR using primers specific for XBP1 which will detect both unspliced and spliced isoforms. The 5′ to 3′ sequences of primers used to detect unspliced and spliced XBP1 mRNA are as indicated below.

Bottom Line: Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field.There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems.We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, National University of Ireland, Galway, Galway, Ireland.

ABSTRACT
The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.

No MeSH data available.


Related in: MedlinePlus