Assessing the ability of the ER stress sensor ERN1/IRE1a to act as a molecular chaperone
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Abstract
BACKGROUND: The endoplasmic reticulum (ER) plays a crucial role in the biosynthesis of membrane, organelle and secreted proteins, which serve important roles in maintaining cellular structure and communication. Disruption in protein synthesis and homeostatic mechanisms, causes proteins to misfold and accumulate resulting in a state of ER stress. ER stress sensors such as IRE1alpha, detect aspects of protein misfolding and signal the unfolded protein response (UPR) to restore protein homeostasis. Among structures that may induce ER stress, IRE1alpha was shown to recognize a structural sequence localized to edge strands of beta-sheets in the enzymatic A1 subunit of cholera toxin. It is possible that unfolded beta-strands have a propensity to self-assemble and recognition of these sequences by IRE1alpha may prevent aggregation.
AIMS: In our study we sought to characterize the ability of cholera toxin peptides and other peptides that bind IRE1alphaLD to self-assemble and form aggregates. Our goal was to test if recombinant IRE1alphaLD can block self-assembly of aggregation-prone peptides.
METHODS: We will monitor aggregation of synthetic peptides in solution using absorbance, fluorescence polarization, and thioflavin T binding assays. We will monitor aggregation kinetics of CTx peptides that bind IRE1alpha using thioflavin T binding to peptides in the presence and absence of IRE1alphaLD.
RESULTS: We were able to observe that these isolated peptides that bind to IRE1alpha are prone to self-assemble into aggregates. Peptides that did not bind IRE1alpha were less prone to aggregation. Unfortunately, we were unable to determine if IRE1alpha blocks aggregation due to experimental limitations - where IRE1alpha itself interfered with the probes for aggregation.
CONCLUSION: It remains to be tested if, in addition to signaling the UPR pathway, IRE1alpha can serve as a molecular chaperone to directly prevent protein aggregation in the ER. We suggest alternative strategies such as Dynamic Light Scattering (DLS) and Negative Stain Electron Microscopy (NSEM) to determine the aggregation kinetics of unfolded proteins and test the ability of IRE1a to block aggregation.
Description
2025