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A Cell-Permeable Photosensitizer for Selective Proximity Labeling and Crosslinking of Aggregated Proteome

ABSTRACT: Intracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, we report on a cell-permeable photosensitizer (P8, Rose Bengal derivative) for selective photo-induced proximity labeling, crosslinking and identification of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect. Further acetylation permeabilizes Rose Bengal for selective imaging, labeling and crosslinking aggregated proteome in live stressed cells. A combination of photo-chemical, tandem mass spectrometry and protein biochemistry characterizations reveal the complexity in photosensitizing pathways (Type I & II), modification sites and labeling mechanisms. The diverse labeling reaction types result in highly effective enrichment and identification of aggregated proteome. Finally, aggregated proteomics and interaction analyses thereby reveal extensive entangling of proteostasis network components mediated by HSP70 chaperone (HSPA1B) and active participation of autophagy pathway in combating proteasome inhibition. Overall, this work exemplifies the first photo-induced proximity labeling and crosslinking method (namely AggID) to profile intracellular aggregated proteome.

ORGANISM(S): Homo Sapiens

SUBMITTER: Lihua Zhang

PROVIDER: PXD044948 | iProX | Wed Aug 30 00:00:00 BST 2023

REPOSITORIES: iProX

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Project description:PRIDE > Archive > PXD013420 Project PXD013420 Download Project Files Summary Title A binding site hotspot map of the FKBP12–rapamycin–FRB ternary complex by photo-affinity labeling and mass spectrometry-based proteomics Description Structural characterization of small molecule binding site hotspots within the global proteome is uniquely enabled by photo-affinity labeling (PAL) coupled with chemical enrichment and unbiased analysis by mass spectrometry (MS). MS-based binding site hotspot maps provide structural resolution of interaction sites in conjunction with identification of target proteins. However, binding site hotspot mapping has been confined to relatively simple small molecules to date; extension to more complex compounds would enable the structural definition of new binding modes in the proteome. Here, we extend PAL and MS methods to derive a binding site hotspot map for the immunosuppressant rapamycin, a complex macrocyclic natural product that forms a ternary complex with the proteins FKBP12 and FRB. Photo-rapamycin was developed as a diazirine-based PAL probe for rapamycin, and the FKBP12–photo-rapamycin–FRB ternary complex formed readily in vitro. Photo-irradiation, digestion, and MS analysis of the ternary complex revealed a McLafferty rearrangement product of photo-rapamycin conjugated to specific surfaces on FKBP12 and FRB. Molecular modeling of the ternary complex based on the binding site map revealed a 5.0 Å minimum distance constraint between the conjugated residues and the diazirine carbon. Molecular dynamics further predicted a 9.0 Å labeling radius for the diazirine upon photo-activation that may be useful in the interpretation of binding site measurements from PAL more broadly. Thus, in characterizing the ternary complex of photo-rapamycin by MS, we applied binding site hotspot mapping to a macrocyclic natural product and extracted a precise structural measurement for interpretation of PAL products that may enable the discovery of new ligand space in the “undruggable” proteome.

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A Cell-Permeable Photosensitizer for Selective Proximity Labeling and Crosslinking of Aggregated Proteome

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