Metabolic Regulation of Kelch-like Proteins Through O-glycosylation

Abstract

O-GlcNAcylation is a reversible post-translational modification that decorates an O-linked ß-N-acetylglucosamine (O-GlcNAc) moiety onto the serine/threonine residues of target proteins. In mammals, this modification is regulated by only two enzymes: O-GlcNAc transferase (OGT, the writer) and O-GlcNAcase (OGA, the eraser). Several studies have revealed that O-GlcNAcylation can be responsive to metabolic status or stress stimulation. However, the specific O-GlcNAc targets in response to various nutrient and stress signals are not well defined. We conducted a global transcriptome profiling in triple-negative breast cancer cells to search for signaling events that respond to O-GlcNAc fluctuation. Unexpectedly, we found that the NRF2-dependent stress response positively correlates with lower OGT activity in multiple human tumor gene expression datasets. NRF2, a major transcriptional regulator of redox balance, is usually activated by oxidative stress but degraded by proteasome under basal conditions via the KEAP1-CUL3 ubiquitin ligase-mediated polyubiquitination. Using azidosugar metabolic labeling, bioorthogonal chemistry and mass spectrometry, we determined that the NRF2 negative regulator KEAP1 is O-GlcNAcylated within its BTB and Kelch motifs. KEAP1 belongs to the Kelch-like (KLHL) adaptor protein family, which was known to regulate substrate proteostasis via CUL3-mediated ubiquitination. Of 11 candidate O-GlcNAc sites on KEAP1, serine 104 is responsible for regulating NRF2 activity by promoting the KEAP1-CUL3 interaction. Interestingly, we found that other KLHL protein, gigaxonin, is also O-GlcNAcylated on up to nine candidate sites. Mutation of gigaxonin is known to cause giant axonal neuropathy (GAN), a neurodegenerative disease that is characterized by the accumulation of intermediate filaments in axons. We found gigaxonin O-GlcNAcylation is required for its ability to facilitate the ubiquitination and proteolysis of intermediate filaments. Mutation of specific gigaxonin O-GlcNAcylation sites compromised its optimal interactions with intermediate filament proteins. This finding provides new molecular insight into GAN pathogenesis. The link between proteostasis and nutrient-sensing is fundamentally important yet incompletely understood. Together, my dissertation work has revealed new connections among nutrient-sensitive glycosylation, KLHL protein function, proteostasis and downstream signaling, with relevance for human diseases.