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This work was a massive collaborations between Fletcher, Clausen, and Virdee Labs, and I was lucky enough to be involved in this complex yet beautiful RNF213 story.

Overall, RNF213 E3 activity is tightly coupled to the cellular ATP/ADP ratio. Interferon stimulation causes an increase in ATP levels, which induces RNF213 E3 activity and substrate ubiquitination.

Indeed, reducing ATP levels in cells via 2DG treatment also lowered RNF213 activity while interferon treatment massively increasing the activity. Importantly, the expression levels of the protein was barely different!

Interesting observation was that interferons increase ATP levels in cells. So we wanted to test if IFN-mediated ATP boost also modulates RNF213 activity.

We also showed that ATP-dependent RNF213 activation was due to ATP binding function of RNF213 instead of hydrolysis.

Indeed, we showed that RNF213 activity is dependent on ATP concentration inside of living cells. Reduced ATP levels (via 2DG treatment) lowered active RNF213 whereas supplementing cells with ATPgS significantly boosted RNF213 activity.

Once inside of the cells, ABP binds to activated E3 ligases, including RNF213, allowing a readout of the activity status of intracellular, endogenous protein.

We next asked the question; does RNF213 require ATP inside of cells?. We developed a method where we delivered the ABP into living cells using electroporation.

Indeed, addition of AMP could hinder RNF213 activity under high ATP conditions.

Nucleotide-dependent regulation of RNF213 E3 activity. We showed that recombinant RNF213 protein binds to activity-based probe, which reads out the activity status of the protein, only after ATPgS treatment but not ADP or AMP, suggesting that RNF213 requires ATP to be activated.

A summary of our findings….

And it's out! :).

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