Photoproximity Profiling of Protein–Protein Interactions in Cells

We report a novel photoproximity protein interaction
(PhotoPPI)
profiling method to map protein–protein interactions in vitro
and in live cells. This approach utilizes a bioorthogonal, multifunctional
chemical probe that can be targeted to a genetically encoded protein
of interest (POI) through a modular SNAP-Tag/benzylguanine covalent
interaction. A first generation photoproximity probe, PP1, responds
to 365 nm light to simultaneously cleave a central nitroveratryl linker
and a peripheral diazirine group, resulting in diffusion of a highly
reactive carbene nucleophile away from the POI. We demonstrate facile
probe loading, and subsequent interaction- and light-dependent proximal
labeling of a model protein–protein interaction (PPI) in vitro.
Integration of the PhotoPPI workflow with quantitative LC–MS/MS
enabled unbiased interaction mapping for the redox regulated sensor
protein, KEAP1, for the first time in live cells. We validated known
and novel interactions between KEAP1 and the proteins PGAM5 and HK2,
among others, under basal cellular conditions. By contrast, comparison
of PhotoPPI profiles in cells experiencing metabolic or redox stress
confirmed that KEAP1 sheds many basal interactions and becomes associated
with known lysosomal trafficking and proteolytic proteins like SQSTM1,
CTSD, and LGMN. Together, these data establish PhotoPPI as a method
capable of tracking the dynamic subcellular and protein interaction
“social network” of a redox-sensitive protein in cells
with high temporal resolution.