Poster abstracts
Poster number 104 submitted by Swapnil Mukherjee
Dissecting the Mechanism of IRE1 Activation with Orthogonal Control over Oligomerization and Phosphorylation
Swapnil Mukherjee (Department of Chemistry and Biochemistry, The Ohio State University), Vladislav Belyy (Department of Chemistry and Biochemistry, Center of RNA Biology, The Ohio State University)
Abstract:
Inositol-requiring enzyme 1 (IRE1) is an ER-localized bifunctional kinase/RNase membrane receptor that primarily senses ER stress. When activated, mammalian IRE1 cleaves a specific mRNA encoding the transcription factor X-box binding protein (XBP1), a key step in ER stress alleviation. 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 optogenetic platform to study the consequence of oligomerization on the activity of IRE1. This system comprises the cytosolic domain of mammalian IRE1 fused to components of a light-inducible heterodimerization system. Phosphorylation or dephosphorylation of the protomers allows us to assemble dimers of well-defined phosphorylation states. We used in vitro RNA cleavage as a readout to study how the activity of IRE1 is modulated by change in protein state. In agreement with previous reports, we find that the activity of IRE1 increases on dimerization and phosphorylation. Surprisingly, our initial results suggest that while phosphorylated monomers exhibit higher activity than dephosphorylated monomers, dimeric forms of IRE1 exhibits similar rates of XBP1 cleavage, regardless of phosphorylation state. This suggests that the role of phosphorylation might be to primarily stabilize the active dimeric state of IRE1. Moreover, our preliminary results highlight unexpected kinetic differences between the processing of canonical substrates (XBP1) and non-canonical RNA substrates by distinct forms of IRE1. These findings are especially relevant in the context of IRE1’s additional proposed role in regulated degradation of a variety of ER-targeted mRNAs in response to stress. Taken together, our results build towards a model of how IRE1-dependent signaling is modulated in cells.
Keywords: IRE1, RNase, Optogenetics