Non-canonical ubiquitination sites direct degradation of the shortest known cholesterol-dependent degron

Chua NK and Brown AJ

School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW 2052, Australia.

Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol synthesis. Excess cholesterol triggers SM degradation via the ubiquitin-proteasome system. The E3 ligase, MARCH6, is responsible for mediating the ubiquitination of SM. Furthermore, this process requires the first 100 amino acids of SM (termed SM N100), which represents the shortest known cholesterol-regulated degron. Ubiquitination is one of the most common post-translational modifications and they often occur on lysine residues, but we have shown that lysine residues are not crucial for the cholesterol-mediated degradation of SM. Despite being an important rate-limiting enzyme in cholesterol synthesis, the precise ubiquitination sites within the cholesterol-regulated degron of SM remain elusive. In this study, we mutated non-canonical ubiquitination residues to alanine. We observed that serine residues are required for the cholesterol-dependent degradation of SM N100. In addition, loss of the key serine residues boosted SM N100 protein levels and MARCH6 knockdown did not further enhance protein levels. Our results reveal non-canonical ubiquitination in the shortest known cholesterol-regulated degron. This finding characterises the mechanism by which SM exerts its role as a rate-limiting enzyme and how cholesterol modulates the stability of SM.