Molecular and cellular analyses of GNAO1 disorder mutants uncover disease mechanisms and potential treatment strategies

Heterotrimeric G proteins (Gabg) are molecular switches that reversibly connect cell-surface receptors and intracellular effector molecules. Point mutations in GNAO1, which encodes the Gao subunit, have been implicated in a rare genetic condition known as GNAO1 disorder. Previous efforts classified 55 GNAO1 disorder-associated Gao variants into four distinct mechanistic groups. While the mechanisms for Groups 1 and 2 were well-established, Group 3 had no obvious biochemical defect and Group 4 were nonfunctional enzymes. Here, we evaluate two peptides in the pursuit of a possible pharmacological chaperoning approach to restoring function to the Group 4 Gao variants, which are unstable with a high likelihood of misfolding. Our effort reveals that G protein modulator (GPM)-1 and -1b are not viable pharmacological chaperone candidates. In addition, we seek to uncover the mechanistic defect of Group 3 Gao variants. Here, we implement an auxin-inducible degron (AID) system in yeast, which allows us to rapidly degrade the endogenous Ga protein and determine the isolated effect of each Group 3 mutant on Gbg-induced transcription. Our effort reveals a subset of Group 3 variants that exhibit attenuated interactions with Regulator of G protein Signaling (RGS) proteins. Taken together, the findings extend our understanding of the mechanistic defect of Group 3 Gao variants and serve as a step forward in evaluating potential therapeutic strategies for targeting GNAO1 disorder-associated Gao variants.