Spatiotemporal-resolved protein networks profiling with photoactivation dependent proximity labeling

Abstract

Enzymatic-based proximity labeling approaches based on activated esters or phenoxy radicals have been widely used for mapping subcellular proteome and protein interactors in living cells. However, activated esters are poorly reactive which leads to a wide labeling radius and phenoxy radicals generated by peroxide treatment may disturb redox-sensitive pathways. Herein, we report a photoactivation-dependent proximity labeling (PDPL) method designed by genetically attaching photosensitizer protein miniSOG to a protein of interest. Triggered by blue light and tunned by irradiation time, singlet oxygen is generated, thereafter enabling spatiotemporally-resolved aniline probe labeling of histidine residues. We demonstrate its high-fidelity through mapping of organelle-specific proteomes. Side-by-side comparison of PDPL with TurboID reveals more specific and deeper proteomic coverage by PDPL. We further apply PDPL to the disease-related transcriptional coactivator BRD4 and E3 ligase Parkin, and discover previously unknown interactors. Through over-expression screening, two unreported substrates Ssu72 and SNW1 are identified for Parkin, whose degradation processes are mediated by the ubiquitination-proteosome pathway.

Profiling stress-triggered RNA condensation with photocatalytic proximity labeling

“…Future study that combines proteomic and transcriptomic data in the same cell line with the same bait protein could help elaborate the relationship between m 6 A-mRNA enrichment and m 6 A-binding protein localization during various stages of SG assembly/disassembly. Such multipronged approach would require photoactivatable protein labeling with miniSOG, which has been recently demonstrated by us and other groups [41][42][43] .…”

Light Activated BioID (LAB): an optically activated proximity labeling system to study protein-protein interactions

“…Another PL tool using light-activated miniSOG has been recently developed (PDPL), which uses singlet oxygen to create electrophilic residues on prey proteins, which can be subsequently bound to an alkyne chemical probe and pulled down using click chemistry (Zhai et al 2022). However, the aniline probe used with PDPL can be toxic to cells (Wang et al 2016, Zhai et al 2022). Furthermore, miniSOG has a labeling radius of up to 70nm, which is much larger than TurboID’s 10nm labelling radius (Kim et al 2014).…”

“…Furthermore, miniSOG has a labeling radius of up to 70nm, which is much larger than TurboID’s 10nm labelling radius (Kim et al 2014). While a smaller labeling radius is more suited for discovering direct PPIs, a larger labeling radius is more appropriate for identifying proteins that may be present in the same cellular compartment (Cho et al 2020, Zhai et al 2022). Another light-activated PL tool, MicroMap, which uses an antibody conjugated to an iridium photocatalyst to label nearby proteins via carbene intermediates, has also recently been developed (Geri et al 2020, Seath et al 2021).…”

“…Furthermore, miniSOG has a labeling radius of up to 70nm, which is much larger than TurboID's 10nm labelling radius (Kim et al 2014). While a smaller labeling radius is more suited for discovering direct PPIs, a larger labeling radius is more appropriate for identifying proteins that may be present in the same cellular compartment (Cho et al 2020, Zhai et al 2022.…”

“…These enzymes are genetically fused to a protein of interest (the bait ), and generate short-lived intermediate reactive biotins that diffuse away from the bait site and covalently tag neighboring proteins (the prey ), except in PUP-IT, where the biotin handle does not leave the bait. Other methods of tagging include using photosensitizers such as the enzyme miniSOG (Zhai et al 2022) or iridium-conjugated antibodies to generate singlet oxygen (Müller et al 2021) or activated carbenes (Geri et al 2020), as alternate intermediates for targeting biotin-tagged probes. Subsequently, biotin-tagged prey proteins are enriched using streptavidin-coated beads and identified using mass spectrometry (MS).…”