The Unfolded Protein response: Or how to keep your Christmas present production line in order

Season’s Greetings and welcome back to my blog dear readers. Hopefully you had a wonderful Christmas day and boxing day as I did, I found it very relaxing cuddled up to my dogs and watching Paddington 2. But there “ain’t no rest for the wicked” even over Christmas, so back to the science we go.

What is the Unfolded Protein Response?

As I mentioned in the first post, AAG has been inferred to activate ER stress and the associated unfolded protein response (UPR). The unfolded protein response is a series of events that activate during ER stress to restore the ER balance and restore order from the chaos that occurs during the ER stress.

What are the key players in the UPR?

The master regulator in the UPR is binding immunoglobulin protein or Bip, and it controls the activity of IRE1α, PERK and ATF6. You can think of the ER as Santa’s present workshop and the ER stress as the mess of incorrectly made presents produced when the elves in the workshop are overworked. Whilst Bip, IRE1α, PERK and ATF6 are like Santa and 3 naughty elves in the workshop.

Bip is Santa and when it is near the others he gives them a telling off for being naughty elves, this inhibits them from doing anything. However, once a crisis occurs in the present making production line (the protein making ER) he is called away to deal with it, this involves acting as a chaperone to ensure that the proteins are correctly folded into function proteins or in this analogy ensure that the presents are correctly wrapped up. (In this analogy: mRNA is the present parts, unfolded proteins are the presents, folded proteins are the wrapped presents)

In his absence the naughty elves (IRE1α, PERK and ATF6) start working to ensure they get back on the nice list.

IRE1α acts to degrade mRNA to reduce the amount of proteins produced whilst it also splices (or cuts) XBP1 into an active form that encourages the expression of genes from the DNA that chaperone the unfolded proteins to ensure they fold properly and destroy those that are folded improperly. This means that the IRE1α elf gets to work removing excess parts from the production line in order to reduce the number of presents produced and workload of the elves downstream, whilst he also briefs another elf (the XBP1 elf) to train those that need to wrap up the presents and destroy the incorrectly wrapped presents.

PERK acts to phosphorylate the proteins (such as eIF2α) that translate the mRNA into proteins in order to lessen total and incorrectly folded protein production. This PERK elf essentially gives some present making elves a break to both reduce their stress levels and to lessen the number of presents being produced, thereby reducing the number of incorrectly wrapped presents being produced by the rushed wrapping elves.

ATF6 is transported and cut into an active form before it reaches the DNA and encourages the expression of genes that chaperone the protein folding. Therefore, this elf heads off to get a new look, a haircut perhaps, so the other elves will listen to him before he then instructs other elves how to correctly wrap the presents.

Figure 1
An overview of the IRE1α, PERK and ATF6 mediated pathways of the unfolded protein response
IRE1α is a splicing molecule, causing MRNA degradation, however its main role is to activate transcription factors, such as XBP1, which bind DNA to induce production of chaperones that aid protein folding and proteins that destroy the unfolded ones.
PERK phosphorylates proteins, such as eIF2α, to prevent initiation of translation and protein production.
ATF6 is transported and cleaved in the Golgi before reaching the DNA where it acts as a transcription factor to produce chaperones.
Taken from Hetz, 2012

Therefore, by reducing the rate of protein production and reducing incorrect protein folding by increasing the ratio of folding chaperones to unfolded proteins- or alternatively reducing present production and amount of incorrectly wrapped presents, this reduces the stress in both the ER and Santa’s production line. (A slightly more in-depth and scientific overview can be seen in figure 1) (Wang et al., 2009; Hetz, 2012; Yadav et al., 2014).

So the UPR is solely beneficial?

Well if you remember my first blog post then you will recall that prolonged or excessive activation of the UPR can lead to cell death. This is because:

IRE1α degradation of mRNA can destroy the mRNA needed to keep the cell alive, whereas its activation of JNK can lead to downstream activation of BAX and BAK which cause organised cell death (apoptosis).

PERK activation of the ATF4 transcription factor enhances this as it produces expression of CHOP, another transcription factor, which alters the expression of several proteins. I shan’t bore you with all their details, however CHOP increases the quantity of proteins that increase the activity of BAX/BAK and decrease the quantity of proteins that prevent apoptosis. Thus, this tips the balance of the cell survival scales in favour of cell death (Hetz, 2012; Sano and Reed, 2013).

But how do we know that AAG is associated with ER stress and the UPR?

Unfortunately, we must leave Christmas behind and return once more to the science. One of the reasons we know that AAG is involved with the UPR is thanks to my supervisor Dr Meira and her previous PhD student Dr Forrer Charlier. They undertook research utilising cell lines that would upon ER stress and ATF6 and IRE1 production, but not general stress, produce an enzyme (luciferase) that can catalyse a reaction to produce light at a specific wavelength. This light emission was found to be significantly reduced in the AAG lacking cell lines, indicating that at least the ATF6 and IRE1 arms of the UPR are impaired in AAG lacking cells (seen figure 2).

Figure 2
How the expression of luciferase, indicating ER stress, is altered following MMS induced DNA alkylation damage in normal, AAG lacking and AAG restored cells.
These graphs reveal that the AAG lacking cells show greatly reduced ER stress following alkylation compared to the normal wildtype cells.
Taken from Forrer Charlier, 2018

This was further supported by a western blot that measured other UPR molecules, it indicated that BiP, spliced Xbp1 and phosphorylated-eIF2a were almost absent in the AAG lacking cells following alkylation that should have increased them. This suggested that all aspects of the UPR, the Bip regulation and its 3 arms require AAG to function (Forrer Charlier, 2018).

However, whilst we know that the UPR depends on AAG we do not know how.

But in my next and lamentably final blog post I shall summarize, with yet more awful analogies, the research I shall be undertaking and how it should further illuminate how AAG activity results in ER stress and the UPR.

Until next time, Tom.

Bibliography

Forrer Charlier, Clara (2018) Deciphering the crosstalk: characterisation of the regulation of the ER stress response by the DNA repair enzyme AAG. Doctoral thesis, University of Surrey.

Hetz, C. (2012). The unfolded protein response: controlling cell fate decisions under ER stress and beyond. Nature Reviews Molecular Cell Biology, 13(2), pp.89-102.

Sano, R., & Reed, J. C. (2013). ER stress-induced cell death mechanisms. Biochimica et biophysica acta1833(12), pp.3460–3470.

Wang, M., Wey, S., Zhang, Y., Ye, R. and Lee, A. (2009). Role of the Unfolded Protein Response Regulator GRP78/BiP in Development, Cancer, and Neurological Disorders. Antioxidants & Redox Signaling, 11(9), pp.2307-2316.

Yadav, R. K., Chae, S. W., Kim, H. R., & Chae, H. J. (2014). Endoplasmic reticulum stress and cancer. Journal of cancer prevention19(2), pp.75–88.