The new findings just add to the uniqueness of the Ire1 pathway, which responds to unfolded proteins. “This pathway is really a sort of duckbilled platypus,” says Papa. “There have been so many surprises.”
The response starts when unfolded proteins recruit chaperones away from Ire1; this is thought to expose a patch on Ire1 that mediates oligomerization. Transphosphorylation by Ire1 oligomers then somehow activates the RNase activity of Ire1, which excises an inhibitory intron from the HAC1u mRNA. After tRNA ligase joins the mRNA fragments, the active Hac1 transcriptional activator can be produced.
The new activation discovery began with an attempt to create a specific inhibitor of the Ire1 kinase. That inhibitor, 1NM-PP1, binds only mutant versions of Ire1 that have a redesigned nucleotide-binding site. Although 1NM-PP1 inhibited the kinase activity of Ire1, there was a surprise further downstream. “The drug is working exactly as you would expect as a kinase inhibitor,” says Papa, “yet the RNase is still activated.”
This activation by 1NM-PP1 also worked in Ire1 mutants that were dead for kinase activity or lacked transphosphorylation sites. When kinase-dead and constitutive mutations were combined in a single mutant version of Ire1, the 1NM-PP1 could now be used as an instructive chemical that could, by itself, activate the entire pathway.
In wild-type cells, the UCSF team thinks that an occasional molecule of ATP can sneak into the nucleotide-binding site of oligomerized Ire1. This allows transphosphorylation, which further opens the nucleotide-binding site so that nucleotide can bind more easily and activate the RNase activity of Ire1.
A nucleotide signal, in particular the efficient Ire1 activator ADP, may help Ire1 to do its job, because high concentrations of ADP indicate low energy status and thus incipient problems in the energy-intensive process of protein folding. 1NM-PP1 revealed this unusual pathway because the drug's small size allowed it to sneak into Ire1's nucleotide-binding site even without Ire1 transphosphorylation, and its particular binding properties may allow it to stay around long enough to effect RNase activation. ▪