Mapping Dynamic Changes in Protein Interactome using Proximity Labeling with High Temporal Resolution - PROJECT SUMMARY Physiological and oncogenic signals are propagated through a chain of protein interactions. The assembly and disassembly of key signaling complexes are often transient and can occur within minutes or even seconds. Mapping dynamic changes in protein interactions is one of the main approaches for deciphering the regulation of critical biological processes and for the identification of potential therapeutic targets in the treatment of cancer. However, the interrogation of rapid changes in protein interactomes remains a significant challenge due to the poor sensitivity and/or insufficient temporal resolution of current tools. We propose to develop a set of tools that will enable the facile analysis of changes in protein interactions occurring within one minute or less and will significantly improve the sensitivity of existing approaches. Currently, the most comprehensive characterization of protein interactomes is achieved through the application of the biotin proximity labeling approach. This method uses promiscuous biotin ligases, such as BioID or TurboID, that can biotinylate any protein within a 10Å radius. By attaching a biotin ligase to a protein of interest, researchers can detect even weakly associated protein complexes in living cells. However, detection of dynamic changes in the protein interactome using this strategy has been challenging due to a poor signal-to-noise ratio when cells are labeled with biotin for short periods of time (1-2 minutes). We will develop three orthogonal methods that dramatically reduce the background signal during biotinylation and enable fast labeling of samples within one minute or less. One approach will employ a light-regulated biotin ligase that demonstrates no activity in the dark, thus minimizing the background biotin signal to the lowest level. Two other strategies will employ engineered biotin ligases capable of labeling proteins with biotin analogs, desthiobiotin and iminobiotin. These two analogs offer a significant advantage because they can be separated from biotin by affinity chromatography using selective elution condition. This will allow us to reduce the background signal caused by endogenous biotin present in mammalian cells and thus significantly improve signal-to-noise ratio. Furthermore, since all three technologies utilize different biotin analogs, we can apply three different labels within the same cell and separate them during the sample preparation. The application of this novel strategy will enable the labeling of different time points in the same biological sample or the analysis of interactions for three different proteins within the same cells. Overall, the combined advantages of the proposed tools will significantly enhance our capabilities for dissecting the signaling processes driving tumor formation and progression.
