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.