Poster abstracts
Poster number 54 submitted by Swapnil Mukherjee
Dissecting the mechanism of IRE1 activation with orthogonal control over oligomerization and phosphorylation
Swapnil Mukherjee (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University), Vladislav Belyy (Department of Chemistry and Biochemistry, Center for RNA Biology, The Ohio State University)
Abstract:
The accumulation of misfolded proteins in the lumen of the endoplasmic reticulum is known as ER stress, a condition that has widespread implications in several metabolic and neurological disorders and cancer. Inositol-requiring enzyme 1 (IRE1) is an ER membrane-localized bifunctional kinase/RNase receptor that senses ER stress. When activated, mammalian IRE1 initiates a nonconventional cytosolic splicing reaction, which results in the production of the transcription factor X-box binding protein (XBP1s) and constitutes a key step in ER stress alleviation. The activation of IRE1 has been shown to rely on some form of oligomeric transition. However, the precise mechanism by which oligomerization activates IRE1 is still unknown, in part due to the substantial experimental challenges associated with studying subtle oligomeric transitions in low-affinity protein clusters. To overcome this problem, we engineered an in vitro platform to study the consequences of oligomerization and phosphorylation on the activity of IRE1. Having verified oligomeric state transitions using mass photometry, we used in vitro RNA cleavage as a readout to study how the activity of IRE1 is modulated by different forms of IRE1. We find that the RNase activity of IRE1 robustly increases upon dimerization and phosphorylation. However, we do not see any noticeable increase in RNase activity upon tetramerization, suggesting that dimerization of IRE1 is sufficient for inducing XBP1 mRNA cleavage. Initial results show that tetramerization speeds up IRE1 kinase activity, indicating that transient oligomerization is an intermediate step leading to the formation of RNase-active phosphorylated dimers. Interestingly, we notice that the phosphorylation-mediated enhancement of IRE1’s RNase activity becomes far more prominent after pre-incubation with physiological concentrations of ADP . This suggests that, in addition to oligomerization and phosphorylation state, the activity of IRE1 is also influenced by its kinase active site occupancy. Overall, our results contribute to building a comprehensive molecular model of IRE1 activation thereby overcoming potential bottlenecks in precise therapeutic targeting of ER stress.
Keywords: ER Stress, RNase, Unfolded Protein Response