Deciphering molecular interactions by proximity labeling

Abstract

Many biological processes are executed and regulated through the molecular interactions of proteins and nucleic acids. Proximity labeling (PL) is a technology for tagging the endogenous interaction partners of specific protein 'baits', via genetic fusion to promiscuous enzymes that catalyze the generation of diffusible reactive species in living cells. Tagged molecules that interact with baits can then be enriched and identified by mass spectrometry or nucleic acid sequencing. Here we review the development of PL technologies and highlight studies that have applied PL to the discovery and analysis of molecular interactions. In particular, we focus on the use of PL for mapping protein-protein, protein-RNA and protein-DNA interactions in living cells and organisms.

Figure 1.

Peroxidase- and biotin ligase-based proximity labeling methods for PPI mapping. (a) Peroxidase-based approaches, such as APEX or HRP, oxidize biotin phenol into reactive phenoxyl radicals using hydrogen peroxide, which preferentially labels proximal over distal endogenous proteins. (b) Biotin ligase-based approaches, such as BioID or TurboID, utilize ATP and biotin to catalyze the formation of reactive biotin-5’-AMP intermediates, which diffuse and label proximal proteins. (c) Schematic of example proteomic workflow for mapping PPI. PL enzymes fused to the bait of interest and a spatial reference control are expressed in separate samples. Biotinylated proteins from each sample are enriched and analyzed via quantitative mass spectrometry. Proteins that preferentially interact with the bait of interest can be identified by ratiometric analysis.

Figure 2.

PL-based methods to investigate protein-nucleic acid interactions. (a) Schematic of APEX-RIP and Proximity-CLIP. APEX targeted to a specific subcellular location catalyzes the biotinylation of proximal proteins, and the RNA-protein interactions are subsequently crosslinked by either UV or formaldehyde (FA). The subcellular RBP-occupied RNA can be captured via streptavidin-based enrichment of the biotinylated RBPs. (b) Schematic of APEX-seq. APEX directly biotinylates proximal RNA (yellow), but not distal RNA (grey), of a protein bait. (c) Schematic of Cap-seq. Upon blue light illumination, miniSOG generates ROS that react with guanine nucleobases in RNA. The photo-oxidation intermediates are intercepted by amine probes (R-NH2) to form covalent adducts. (d) Schematic of RaPID. An RNA of interest is tagged with a BoxB aptamer to recruit a fusion protein of λ-N and a promiscuous biotin ligase, which can biotinylate associated RBPs. (e) PL strategies based on MS2 tags and MCP to capture RBPs associated with an RNA of interest. (f) dCas13-based PL strategies to biotinylate RBPs associated with an endogenous RNA of interest. (g) dCas9-based PL strategies to biotinylate DNA-binding proteins at specific genomic locus. (h) Schematic of ChromID. BASU is fused to engineered chromatin readers that can specifically recognize particular chromatin marks, leading to the biotinylation of chromatin-binding proteins.