Endoplasmic reticulum stress measurement

Commonly we add an ER stress inducer and measure the activation of the UPR and the consequent downstream responses. However these results do not directly reflect the accumulation of misfolded and unfolded protein within the ER lumen. It has been challenging to directly measure ER stress levels in cells. (1)

ER dilation (EM)

Upon ER stress, the ER lumen is remarkably enlarged in cells and tissues, which can be detected by electron microscopy (2,3). This method has been used often to detect ER stress in pancreatic β cells.

Real-time redox measurements during ER stress

The ER maintains an oxidizing environment to promote disulfide bond formation in newly synthesized proteins [4]. An increase in ER protein load could overwhelm oxidative folding enzymes, preventing proper disulfide formation and therefore inducing ER stress

Feroz Papa’s group recently developed a method to monitor the redox state of GFP to reflect ER stress [5]. This reporter named “eroGFP (ER-targeted redox-sensitive GFP)” has been designed to change fluorescence at two maximas, 400 and 490 nm, upon disulfide formation between an engineered cysteine pair. As eroGFP becomes reduced, excitation at 490 nm increases while decreases at 400 nm. The ratio of the fluorescence measured at 490 nm versus 400 nm reports ER redox status in cells.. Currently this method is only available in yeast cells.

 

Studying the UPR master regulators activation

The ability to measure IRE1α and PERK phosphorylation, and ATF6α cleavage would be ideal to determine UPR activation levels. However; endogenous expression levels of these molecules are low and hard to detect with available commercial antibodies.(6)

 

Methods for measuring IRE1α activation

IRE1α is an ER transmembrane serine/threonine kinse undergoing autophosphoryaltion upon ER stress. Thus, the best way to measure activation levels of IRE1α is to measure its phosophorylation levels. Our group developed anti-phospho-IRE1α specific antibody from bulk antiserum by affinity purification followed by adsorption against the nonphospho analog column peptide (Open biosystems, Huntsville, AL). The polymerase chain reaction (PCR) was performed in triplicate for each sample, after which all experiments were repeated twice. Spliced and unspliced XBP-1 mRNA can also be measured by semi-quantitative RT-PCR and XBP-1 protein can be detected by immunoblot using anti-XBP-1 specific antibody (7)

Article by Dr. JOSE PEREIRA, MD

 

Footnotes:

 

1. Christine M. Measuring ER stress and the unfolded protein response using mammalian tissue culture system .Author manuscript; available in PMC 2013 Jul 4. Published in final edited form as: Methods Enzymol. 2011; 490: 71–92. doi:  10.1016/B978-0-12-385114-7.00004-0 PMCID: PMC3701721 NIHMSID: NIHMS457369

2. Yoshida H, Matsui T, Hosokawa N, Kaufman RJ, Nagata K, Mori K. Dev Cell. 2003;4:265–271. [PubMed]

3. Lee AH, Iwakoshi NN, Glimcher LH. Mol Cell Biol. 2003;23:7448–7459. [PMC free article] [PubMed]

4. Urano F, Wang X, Bertolotti A, Zhang Y, Chung P, Harding HP, Ron D. Science (New York, N.Y. 2000;287:664–666. [PubMed]

5. Nishitoh H, Matsuzawa A, Tobiume K, Saegusa K, Takeda K, Inoue K, Hori S, Kakizuka A, Ichijo H. Genes Dev. 2002;16:1345–1355. [PMC free article] [PubMed]

6. Nishitoh H, Saitoh M, Mochida Y, Takeda K, Nakano H, Rothe M, Miyazono K, Ichijo H. Molecular Cell. 1998;2:389–395. [PubMed]

7. Harding HP, Zhang Y, Ron D. Nature. 1999;397:271–274. [PubMed]