Role of the ubiquitin-proteasome pathway in the inner ear : identification of an E3 ubiquitin ligase for Atoh1

[摘要] Atoh1, the proneural basic-helix-loop-helix transcription factor, is critical for the differentiation of inner ear hair cells. Hair cells do not develop in mice that lack Atoh1, and overexpression of the transcription factor in embryonic ears induces differentiation of extra hair cells. The level of Atoh1 expression is under the control of a Wnt and Notch transcriptional regulatory network to keep the level of mRNA within a narrow range. Once the protein is made, it activates its own expression through an interaction with the Atoh1 enhancer, such that Atoh1 transcription is self-perpetuating. Because of this autoregulatory loop, halting transcription of the gene to maintain Atoh1 at an appropriate level would require that the amount of protein be decreased. Since the ubiquitin-proteasome pathway regulates catabolism of key regulatory proteins, we assessed its role in the degradation of Atoh1. E3 ubiquitin ligases confer substrate specificity to degradation of proteins by transferring a ubiquitin tag to a specific protein substrate. Using an immunoprecipitation/mass spectrometry screening approach, we identified Huwe1, a HECT domain E3 ubiquitin ligase, as an Atoh1 binding partner. We validated the binding between Atoh1 and Huwe1 through reciprocal co-immunoprecipitation and mass spectrometry. We found that Huwe1 promoted polyubiquitylation of Atoh1 through a lysine 48-linked polyubiquitin chain. Mutation at a catalytic cysteine within the HECT domain of Huwe1 reduced the polyubiquitylation. We also defined a motif in the C-terminus of Atoh1 responsible for interaction with Huwe1. Inhibition of proteasomal activity, as well as Huwe1 depletion, stabilized Atoh1 in the cochlea and resulted in generation of new hair cells in the newborn cochlea.

[发布机构] Massachusetts Institute of Technology