Abstract
Human cysteine cathepsins constitute an 11-membered family of proteases responsible for degradation of proteins in cellular endosomal-lysosomal compartments as such, they play important roles in antigen processing, cellular stress signaling, autophagy, and senescence. Moreover, for many years these enzymes were also linked to tumor growth, invasion, angiogenesis and metastasis when upregulated. Individual biological roles of each cathepsin are difficult to establish, because of their redundancy and similar substrate specificities. Selective chemical tools that enable imaging of individual cathepsin activities in living cells, tumors, and the tumor microenvironment may provide a better insight into their functions. In this work, we used HyCoSuL technology to profile the substrate specificity of human cathepsin B. The use of unnatural amino acids in the substrate library enabled us to uncover the broad cathepsin B preferences that we utilized to design highly-selective substrates and fluorescent activity-based probes (ABPs). We further demonstrated that Cy5-labeled MP-CB-2 probe can selectively label cathepsin B in eighteen cancer cell lines tested, making this ABP highly suitable for other biological setups. Moreover, using Cy5-labelled MP-CB-2 we were able to demonstrate by fluorescence microscopy that in cancer cells cathepsins B and L share overlapping, but not identical subcellular localization.