Targeted protein degradation (TPD) approaches including PROTAC and molecular glue have revolutionized drug discovery. However, in part due to complication of the ubiquitination and related deubiquitination processes, these ubiquitination-dependent approaches face several challenges in their therapeutic uses. Ubiquitination-independent proteasomal degradation approaches may be a paradigm-shifting solution to key challenges of the field. Recently, midnolin was discovered to promote degradation of non-ubiquitinated proteins by directly recruiting substrates to proteasomes, providing an ideal platform for developing novel TPD strategy through direct 26S proteasome recruitment. However, how midnolin interacts with proteasome and promotes substrate degradation remains enigmatic, hindering its potential application.

This study determined cryo-EM structures of midnolin-proteasome complexes, providing mechanistic insights to midnolin-catalyzed substrate degradation. Based on structural and mechanistic understanding, authors have engineered the midnolin system for targeted degradation of desired substrates, and provide the first proof-of-principle for degrading a non-native therapeutically high-profile target that is otherwise undruggable.

Key findings from the study include:

1. Cryo-EM structures of midnolin-26S proteasome complexes, in three functional states: The highly conserved C-terminal α-helix of midnolin anchors tightly to the proteasome RPN1 subunit, while the N-terminal ubiquitin-like domain of midnolin interacts with RPN11 subunit of 19S regulatory particle (RP). This ‘two-arms’ interaction scheme aligns the substrate-binding Catch domain of midnolin above the proteasome AAA-ATPase motor, thus facilitates efficient substrate degradation. These results laid solid structural basis for understanding midnolin-promoted proteasomal protein degradation.
2. Characterization of midnolin degrons: Efficiencies of degrons of native midnolin-dependent proteasome substrates were characterized. This work demonstrated that proteasome can degrade substrates directly or indirectly recruited by midnolin. As it is overexpressed in several cancers, midnolin may be used for degradation of desired target proteins via indirect recruitment in these cancer cells.
3. Development of engineered midnolin targeting chimeras (MidTAC): Based on mechanistic insights, a strategy for targeted protein degradation has been developed, in which the Catch domain of midnolin is replaced with a designed target-recruiting domain. Using a fully reconstituted system, it was shown that the MidTAC approach can indeed promote ubiquitination-independent proteasomal degradation of desired substrates.
4. Degradation of undruggable nuclear β-catenin using this MidTAC strategy: Targeted degradation of nuclear β-catenin without interfering with cytosolic β-catenin has been achieved using MidTAC. Nuclear β-catenin is a high-profile drug target for cancers and fibrosis, and considered by many as undruggable since adherens junction-attached β-catenin has a function distinct from nuclear β-catenin and should not be degraded. This MidTAC approach may be useful for targeted degradation of other proteins in subcellular location-specific manners.

The midnolin-proteasome pathway degrades proteins in a ubiquitination-independent manner. This study determined cryo-EM structures of midnolin-proteasome complexes and developed engineered midnolin-based targeting chimeras (MidTAC) for targeted protein degradation.
DOI：https://doi.org/10.1093/procel/pwaf069