Kelch‐like Proteins Have A Sweet Spot: Site‐specific Glycosylation Influences Metabolic Regulation and Protein Homeostasis

Abstract

The Kelch‐like (KLHL) family of proteins, a conserved group of 42 human members, binds to E3 ubiquitin ligase complexes to target protein substrates for proteasomal degradation. Although mutations in KLHL genes cause various human diseases—from cancer to neurodegeneration—their functional regulation remains understudied. Post‐translational modifications in general influence protein fates, yet how they modulate KLHL proteins is not fully explored. In this context, our laboratory has examined O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc), a nutrient‐sensing, intracellular glycosylation governed by O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA) that decorates serines (S) or threonine (T) residues in response to cellular stimuli. Using global transcriptional responses to OGT or OGA inhibition and chemical biology approaches, we previously reported that O‐GlcNAcylation of KEAP1, a KLHL protein, is required for the ubiquitination and regulation of the transcription factor NRF2 and the cellular response to oxidative stress. Given the functional conservation of the KLHL family, we hypothesized similar roles for O‐GlcNAcylation in other KLHL proteins, such as gigaxonin that promotes the degradation of intermediate filament (IF) proteins. Human loss‐of‐function gigaxonin mutations are known to cause IF accumulation in neurons, resulting in neuronal swelling and ultimately giant axonal neuropathy, a fatal neurodegenerative disease. By mass spectrometry, immunofluorescence and co‐immunoprecipitation assays, we showed that gigaxonin glycosylation sites at residues S272 and T277 are important for clearing IF aggregates in gigaxonin−/− cells. Taken together, our findings implicate O‐GlcNAc modification of KLHL proteins in governing protein homeostasis of E3 ligase substrates, suggest O‐GlcNAcylation as a general mode of regulation for other KLHL proteins, and inspire future tool developments to detect site‐specific glycosylation changes in human health and disease.Support or Funding InformationResearch on this project in the Boyce Lab is supported by NIH grants R01GM118847 and R01NS111588 and a gift from the Hannah’s Hope Foundation