Structure-based engineering of the midnolin-proteasome pathway for targeted protein degradation

The midnolin-proteasome pathway promotes degradation of nuclear proteins in a ubiquitination-independent manner. How midnolin recruits substrates to proteasome for direct degradation remains unclear. Here we present cryo-EM structures of midnolin-proteasome complexes and engineered midnolin for targeted protein degradation. Our structural and biochemical analysis reveal that midnolin’s C-terminal α-helix anchors tightly to the proteasome RPN1 subunit and midnolin’s N-terminal ubiquitin-like domain interacts with RPN11 subunit, aligning its substrate-binding Catch domain above the proteasome ATPase motor, thus facilitating substrate degradation. Based on mechanistic insights, we have developed a strategy for targeted protein degradation using engineered midnolin targeting chimeras (MidTAC), in which the Catch domain is replaced with a target-recruiting domain. Using this strategy, we have achieved degradation of nuclear β-catenin, a critical oncoprotein resulting from Wnt pathway dysregulation, without interfering with cytosolic β-catenin involved in cell adhesion. This MidTAC approach may be useful for targeted degradation of other proteins.