Tracking chromatin state changes using nanoscale photo proximity labeling

ABSTRACT: Interactions between biomolecules underlie all cellular processes, and ultimately control cell fate. Perturbation of native interactions through mutation, changes in expression levels, or external stimuli leads to altered cellular physiology and can result in either disease or therapeutic effects. Mapping these interactions and determining how they respond to stimulus is the genesis of many drug development efforts, leading to new therapeutic targets and improvements in human health. However, in the complex environment of the nucleus it is challenging to determine protein-protein interactions due to low abundance, transient or multi-valent binding, and a lack of technologies that are able to interrogate these interactions without disrupting the protein binding surface under study. Here, we describe a method for the traceless incorporation of Ir-photosensitizers into the nuclear microenvironment using engineered split inteins. These Ir-catalysts can activate diazirine warheads via Dexter energy transfer to form reactive carbenes within a ~10 nm radius, cross-linking with proteins within the immediate microenvironment (a process termed uMap) for analysis via quantitative chemoproteomics. Additionally, we show that this nanoscale proximity labeling method can reveal the critical changes in interactomes in the presence of cancer-associated mutations, as well as treatment with small-molecule inhibitors. uMap improves our fundamental understanding of nuclear protein-protein interactions, and in doing so is expected to have a significant impact on the field of epigenetic drug discovery in both academia and industry.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Tom Muir   David MacMillan  

PROVIDER: MSV000090929 | MassIVE | Mon Dec 19 14:44:00 GMT 2022

SECONDARY ACCESSION(S): PXD038956

REPOSITORIES: MassIVE