Determination of α‑Synuclein Protein Interactions by μMap Photoproximity Labeling

Fibrillar aggregates of the natively disordered protein
α-synuclein
(αS) are hallmarks of Parkinson’s disease and related
neurodegenerative disorders termed synucleinopathies. Here, we used
micromap (μMap) photoproximity labeling to determine the interactomes
of αS monomers and fibrils in mouse brain lysate to better understand
both the loss of healthy function and gain of toxic function aspects
of synucleinopathies. Several αS variants were synthesized and
characterized, showing that the small size (1 kDa) of the Ir catalyst
attached through a Cys-maleimide linkage makes it minimally perturbing
to αS, with a narrow labeling radius that allows one to identify
interactome differences between different regions of αS. Monomer
and fibril interactomes were compared to each other and to previous
proximity labeling data sets for validation, and several examples
of further investigations are demonstrated, including Western blotting,
affinity pulldowns, fluorescence and super-resolution microscopy,
and μMap in primary neurons.