Week 3- The Pathway that Bridges the Two: Introducing mTORC-1

Anita M -

The past few days consisted of signing my DCC form and completing online lab training. In the meantime, I am still reading through human physiology and pathology, as well as expanding my knowledge on GRASP55 (the protein of interest for my project). Here’s a more detailed look at the link between GRASP55 and ATF6:

When someone experiences a heart attack, ischemia, or hypoxia (basically any lack of oxygen to heart tissues), some heart tissue dies. The cells surrounding the dead tissue become stressed. More specifically, their ER becomes stressed and starts to misfold/not fold proteins.

ER stress inhibits a pathway called the mechanistic target of rapamycin complex 1 (mTORC1). mTORC1 is largely involved in maintaining cellular metabolism and basic cellular functions (such as growth, autophagy, etc.). When mTORC1 is inhibited under stressful conditions, GRASP55 relocalizes and becomes involved in unconventional protein secretion. As opposed to conventional protein secretion (Ribosome–> Rough ER–> Golgi Apparatus), unconventional protein secretion is protein secretion that bypasses the Golgi Apparatus (basically, the proteins don’t get “packaged” and leave the cell in vesicles that stay intact).

Under normal stress-free conditions, mTORC1 is activated and GRASP55 remains as a structure of the Golgi Apparatus and does not become a part of unconventional protein secretion.  Proteins secreted unconventionally are found in the plasma membrane of the cell and the extracellular matrix (ECM), and some secreted ECM proteins have been found to play vital roles in various physiological conditions such as cancer, inflammation, angiogenesis (development of blood vessels), and neurological diseases.
ER Stress also leads to activation of ATF6, which activates the UPR and attempts to bring the cell back to homeostasis. Furthermore, ATF6 also has the ability to activate mTORC1. Remember that when mTORC1 is activated, GRASP55 is “turned off” and unconventional protein secretion doesn’t occur. So therein lies the question: why doesn’t ATF6 activate mTORC1 under stressful conditions? And how does inhibition of mTORC1 and activation of GRASP55 effect ATF6?


We are trying to investigate the link between GRASP55 and ATF6 and since mTORC1 is a pathway that links the two, it is important to understand the role mTORC1 plays in each pathway. Hopefully by discovering if/how GRASP55 and ATF6 are related, we can also find answers to how the role of mTORC1 is altered for patients who have experienced a heart attack.

 

Starting next week, I will began experiments on HEK293 cells to further investigate the relationship between GRASP55 and ATF6 for patients who have experienced heart attacks.

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    shreyash_p
    Incredible job Amita! I really like how you explained mTORC1’s role in connecting GRASP55 and ATF6, especially under stress conditions like a heart attack. Your plan to start cell experiments on HEK293 cells sounds very promising.
    Arnab
    Nice developments, can you explain why your lab chooses to use the HEK cell line as opposed to an iPSC differentiated cardiac cell line or AC16 cells which are another very popular human cell line used in Cardiology Research? Thanks!
    anita_m
    Thanks for reading, Shrevash! Hopefully, the experiments this week will go smoothly and I'll have some cool updates next week!
    Akash Joseph
    Hi Anita! I like how you broke down mTORC1 and GRASP55, and the link between GRASP55 and ATF6! The visual representations aided my understanding. I am excited to hear about your experience from the experiments with the HEK293 cells next week :)
    anita_m
    Thanks, Arnab! There are a couple of reasons for using HEK293 rather than iPSC or AC16. The first is feasibility reasons. Our lab does not have iPSC cell lines and preparing AC16 lines are time and labor intensive. AC16 cells do not passage since myocytes reach senescence upon maturation. This means after extracting myocytes from mice, which is extremely costly money-wise and time-wise to the lab, we get pretty much one experiment out of it. If we want to repeat it, we have to go through the whole process of extracting cells again. HEK293 cells are immortalized, so we can easily passage some from an existing cell line and repeat experiments on them if necessary. The second is I'm overexpressing GRASP55 in these cells using a plasmid. Transfecting HEK293 is a simpler process, whereas transfecting myocytes involves the use of an adenovirus to ensure that the gene of interest is actually expressed within the cells. Adenoviruses have to be specific to the gene of interest within the plasmid and we do not currently have one that is related to GRASP55. Finally, previous studies have shown that GRASP55 relocalization is not cell-type specific and the underlying mechanisms and gene expression remains consistent across cell-types. Because of this, if we were to overexpress GRASP55 on HEK293s or AC16s we would expect to see similar results despite them being different cell lines. Typically, experiments begin on HEK293s and will be repeated/moved to AC16s if the data shows that the experiment is worth further investigation.
    anita_m
    Hi Mr. Joseph, I'm glad the visuals helped! I'm excited to start working with HEK293 cells and hopefully get some good data. Thanks for reading and stay tuned for updates!
    camille_bennett
    Hi Anita, great work. Do you have any hypotheses as to why ATF6 doesn't activate mTORC1 under stressful conditions?
    anita_m
    Hi Ms. Bennett, Thank you for your question! My hypothesis is ATF6's activation of the Unfolded Protein Response (UPR) also inhibits mTORC1 through some kind of negative feedback loop. It would also make sense that ATF6 essentially "switches its attention" to activating the UPR instead of activating mTORC1 since mTORC1 is responsible for multiple metabolic pathways in the cell. When the cell experiences ER stress, it prioritizes getting rid of the stress over maintaining metabolic function.

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