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.
Highlights
IntroductionCysteine cathepsins are a structurally-related group of enzymes belonging to the papain superfamily of proteases (C1 family, CA clan in the Merops nomenclature).[1,2] These enzymes tend to be optimally active in reducing, acidic conditions, typi ed by those found in the endolysosomal compartment, where they degrade proteins, thereby maintaining cellular homeostasis,[3] over the past few decades the roles of cathepsins expanded and it is evident that these enzymes are assigned to more speci c functions including lipid metabolism, antigen presentation, autophagy, senescence/ageing and cellular stress signaling.[4,5,6,7] Their localization is limited to acidic vacuoles, as active forms of these enzymes are found in theIn cancer, elevated activities of cathepsins are associated with tumor development and progression, and poor patient prognosis in response to chemotherapy.[13,14] As cathepsins are widely expressed and secreted into the extracellular milieu in a number of cancers by various tumor and tumor-associated immune cells, they are very attractive molecular targets for theranostic applications.[15,16,17] The contribution of cathepsins to cancer progression is mainly due to the processing of extracellular matrix proteins, chemokine processing and shedding of cell surface molecules thereby enabling the cancer cells to migrate and invade other organs.[18,19,20] it has been demonstrated that the activity of intracellular cathepsins may contribute to tumorigenesis, as inhibition of intracellular cathepsins resulted in increased tumor cell death and a reduced tumors size in several mice models.[21]
We found that cathepsin B has a preference for large, hydrophobic amino acids (hSer(Bzl), Glu(Bzl), hCha) in the P2 position, which were signi cantly better recognized (>5-fold) than the best natural amino acid, valine
Conclusions a highly selective cathepsin B activitybased probes (ABPs) that allows accurate detection of cathepsin B activity in various cancer cells, regardless of the cathepsin B expression level, and the activities of other cathepsins. This ABP can serve as a cathepsin Bselective inhibitor that may be useful in further studies of biological functions of this enzyme
Summary
Cysteine cathepsins are a structurally-related group of enzymes belonging to the papain superfamily of proteases (C1 family, CA clan in the Merops nomenclature).[1,2] These enzymes tend to be optimally active in reducing, acidic conditions, typi ed by those found in the endolysosomal compartment, where they degrade proteins, thereby maintaining cellular homeostasis,[3] over the past few decades the roles of cathepsins expanded and it is evident that these enzymes are assigned to more speci c functions including lipid metabolism, antigen presentation, autophagy, senescence/ageing and cellular stress signaling.[4,5,6,7] Their localization is limited to acidic vacuoles, as active forms of these enzymes are found in theIn cancer, elevated activities of cathepsins are associated with tumor development and progression, and poor patient prognosis in response to chemotherapy.[13,14] As cathepsins are widely expressed and secreted into the extracellular milieu in a number of cancers by various tumor and tumor-associated immune cells, they are very attractive molecular targets for theranostic applications.[15,16,17] The contribution of cathepsins to cancer progression is mainly due to the processing of extracellular matrix proteins, chemokine processing and shedding of cell surface molecules thereby enabling the cancer cells to migrate and invade other organs.[18,19,20] it has been demonstrated that the activity of intracellular cathepsins may contribute to tumorigenesis, as inhibition of intracellular cathepsins resulted in increased tumor cell death and a reduced tumors size in several mice models.[21].
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