BAX Inhibitor-1 suppresses Endoplasmic Reticulum (ER) and Protein stress 

Author(s): Isabella Meerzaman

Mentor(s): Dr. Geraldine Grant, Biology

Abstract
Rationale: Idiopathic pulmonary fibrosis causes chronic inflammation and permanent scarring of the lungs due to the presence of excessive senescent fibroblasts which are resistant to endoplasmic reticulum (ER) and oxidative stress. The unfolded protein response (UPR) resolves excessive ER stress caused by unfolded protein. IRE1
Audio Transcript
For my project I looked at the role BAX-1 plays in suppressing ER and protein stress. We wanted to explore the role of BAX-1 because of its possibility as a therapeutic target for patients with IPF. IPF is a disease that causes chronic inflammation and excessive permanent scarring of the lungs due to the presence of excessive senescent or aged fibroblasts. 

If we look at center of the poster I have outline the unfolded protein response pathway This pathway is initiated when there is an excess amount of protein found in the cell. It tries to reduce the amount of unfolded protein present by increasing the amount of chaperones that can fold the new and misfolded protein. We specifically focused on the IRE1 arm of this pathway. As you can see, once IRE1a is dimerized (activated) it splices XBP1 into an active which promotes the synthesis of chaperone proteins and up-regulates pro-apoptotic genes and BIM. The increased expression of BIM and CHOP increase the expression of BAX which then expels cytochrome C into the cell and eventually lead to apoptosis. In this pathway BI-1, a pro-survival gene, is a regulator of IRE1. When BI-1 binds to IRE1 it prevents the up-regulation of chaperones and process of apoptosis since it prevents the dimerization of IRE1.
In order to evaluate the role of BAX, BI-1, and IRE1 in the cell we grew A549 lung epithelial cells and transfected them with siRNAs which silenced BI-1, IRE1, and BAX. Half of these cells were then treated with tunicamycin to observe the effects under ER stress. The results were measured through QPCR which analyzed pro-apoptotic mRNA expression and a cellTiter-Glo assay which measures for cell apoptosis or cell death. 

From this we found that in the BI-1 silenced cells there was a large increase in BI-1 gene expression and decrease in the presence of chaperones and therefore a decrease in pro-apoptotic genes. In BAX silenced cells there was a decrease in pro-apototic genes and in IRE1 silenced cells there was an up-regulation of pro-apoptotic genes. We also found that BI-1 silenced cells had an increase in cell viability after the tunicamycin challenge.

We expected our data to show an increase in pro apoptotic genes in BI-1 silenced cells and a decrease in pro-apoptotic genes when IRE1 was silenced. We also believed that cells after the tunicamycin challenge there would be less viable cells for all silenced SIRNA cells.

We believe that these trends were due to the presence of an abundance of chaperones and an increased use of the other UPR arms. If there are more chaperones present then there is a smaller amount of unfolded protein in the cell so apoptosis may not have to occur if it is effectively controlled by the chaperones. The increased expression of pro apoptotic genes in iRE1 silenced cells indicates that another arm is activated and producing these genes. From this we concluded that chaperones play an important role in the regulation of protein. When less chaperones are present the cell is more likely to undergo apoptosis. effectively reduce the presence of unfolded protein and therefore reduce the unfolded protein response and the number of cells that undergo apoptosis 

In the future we will replicate this experiment with IPF fibroblasts since there is a higher abundance of chaperone proteins in cancer cells compared to IPF. 

Thank you for listening and thank you, Dr Lee and Dr grant for helping me out with my project.

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