Multiplexed Photo-Cross-Linking Reveals Comprehensive Midnolin Interactome: Insights into Ubiquitin-Independent Degradation and Functional Diversity

The midnolin-proteasome pathway represents a crucial ubiquitin-independent protein degradation mechanism. However, its precise interactome, including both degradative substrates and nondegradative interactors, remains largely uncharacterized due to the limitations of conventional approaches. To address this, we developed a robust multiplexed photo-cross-linking platform for comprehensively profiling the midnolin interactome. This platform uniquely integrates two distinct chemical biology strategies: first, the genetic encoding of a photo-cross-linking unnatural amino acid into midnolin's Catch domain and second, the use of a residue-selective photo-cross-linker, enabling the covalent capture and subsequent proteomic profiling of protein-protein interactions. Using this platform, we successfully identified numerous midnolin interactors, including several newly identified degradative substrates (e.g., ETV3, JUN, PRKD1, FN3KRP) and multiple nondegradative binding interactors, significantly expanding its known interactome repertoire. Collectively, our research establishes an innovative platform for investigating the context-dependent interactome of the midnolin system. This platform offers unprecedented insights into midnolin's multifaceted roles in ubiquitin-independent degradation and diverse cellular processes. Furthermore, leveraging this platform to explore midnolin interactome across various biological contexts holds significant potential. It could accelerate the development of next-generation strategies for degrading pathogenic proteins and aid in the discovery of therapeutic targets, particularly for those undruggable targets that resist conventional ubiquitin-dependent approaches.