Organelle-specific activity-based protein profiling in living cells.

ABSTRACT: A multimodal activity-based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin-reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in?vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins?B and Z, two lysosomal cysteine proteases. Analysis of starvation-induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle-targeting of activity-based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.

SUBMITTER: Wiedner SD 

PROVIDER: S-EPMC4023867 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature