Comparing the BAD Protein Interactomes in 2D and 3D Cell Culture Using Proximity Labeling

Protein–protein interaction studies using proximity
labeling
techniques, such as biotin ligase-based BioID, have become integral
in understanding cellular processes. Most studies utilize conventional
2D cell culture systems, potentially missing important differences
in protein behavior found in 3D tissues. In this study, we investigated
the protein–protein interactions of a protein, Bcl-2 Agonist
of cell death (BAD), and compared conventional 2D culture conditions
to a 3D system, wherein cells were embedded within a 3D extracellular
matrix (ECM) mimic. Using BAD fused to the engineered biotin ligase
miniTurbo (BirA*), we identified both overlapping and distinct BAD
interactomes under 2D and 3D conditions. The known BAD binding proteins
14-3-3 isoforms and Bcl-XL interacted with BAD in both 2D and 3D.
Of the 131 BAD-interactors identified, 56% were specific to 2D, 14%
were specific to 3D, and 30% were common to both conditions. Interaction
network analysis demonstrated differential associations between 2D
and 3D interactomes, emphasizing the impact of the culture conditions
on protein interactions. The 2D–3D overlap interactome encapsulated
the apoptotic program, which is a well-known role of BAD. The 3D unique
pathways were enriched in ECM signaling, suggestive of hitherto unknown
functions for BAD. Thus, exploring protein–protein interactions
in 3D provides novel clues into cell behavior. This exciting approach
has the potential to bridge the knowledge gap between tractable 2D
cell culture and organoid-like 3D systems.