Ubiquitin Receptor-Induced Proximity is Sufficient for Ubiquitin-Independent Targeted Protein Degradation via the 26S Proteasome

The 26S proteasome engages with ubiquitinated substrates primarily through its constituent ubiquitin (Ub) receptors, which initiates a cascade of events that unfold, translocate, and hydrolyze them. Leveraging this recognition mechanism, we developed a targeted protein degradation (TPD) strategy that recruits substrates directly to the proteasome, thereby bypassing the ubiquitination step. Our proteasome-targeting chimera, Protea-Tac, is a heterobifunctional protein degrader composed of a Ub receptor (PSMD4/Rpn10 or ADRM1/Rpn13) and a target-specific, intracellular antibody (single-chain variable fragment or nanobody). This chimera integrates into 26S proteasomes without altering their structural or functional integrity. Localization of target proteins, including c-Fos, BRD4, Flag-TDP43, HA-tau, and GFP-ODC, to the proteasome via Protea-Tac with cognate antibodies resulted in their induced degradation. We demonstrated that this platform is 1) modular, requiring both essential components but allowing for facile switching between targets; 2) Ub-independent through direct proteasomal targeting; and 3) highly target-specific. Protea-Tac degraded c-Fos in vivo and substantially delayed tumor growth in mouse xenograft models. Overall, these findings identify Protea-Tac as a distinct TPD modality capable of directly degrading intracellular proteins via engineered 26S proteasomes.