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. 2019 Jul 31;10(36):8461-8477.
doi: 10.1039/c9sc00997c. eCollection 2019 Sep 28.

Fluorescent Probes Towards Selective Cathepsin B Detection and Visualization in Cancer Cells and Patient Samples

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Free PMC article

Fluorescent Probes Towards Selective Cathepsin B Detection and Visualization in Cancer Cells and Patient Samples

Marcin Poreba et al. Chem Sci. .
Free PMC article

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.

Figures

Fig. 1
Fig. 1. Human cathepsin B specificity in the P1 position. P1 preference of human cathepsin B was determined using Ac-Ala-Arg-Leu-P1-ACC fluorogenic substrate library containing 19 natural and over 100 unnatural amino acids. The x axis shows abbreviated amino acids, and the y axis displays relative activity of each substrate adjusted to Ac-Ala-Arg-Leu-Arg-ACC substrate (100%) which served as a control. The P1 specificity screening was performed in triplicate. The substrate hydrolysis (RFU/s) data are presented as an average (S.D. for each substrate was below 10%). The best recognized amino acids (Lys(2CL-Z), Cys(Bzl), Cys(MeBzl), Cys(Me)Bzl and Nle(OBzl)) are shown in red.
Fig. 2
Fig. 2. Human cathepsin B specificity in the P4–P2 positions. The P4–P2 preferences of human cathepsin B were determined using the P1-Arg HyCoSuL library. The x axis shows abbreviated amino acids, and the y axis displays relative activity of each substrate adjusted to the best recognized amino acid (marked in red; hSer(Bzl) in P2, Phg in P3, and hCha in P4). Each sub-library (P4, P3, P2) screening was performed in triplicate and the substrate hydrolysis data (RFU/s, %) are presented as average (S.D. for each substrate was below 10%).
Fig. 3
Fig. 3. Selective substrates and ABPs for cathepsin B. (A) The structures and kinetic parameters of hydrolysis (kcat/KM) for the most selective cathepsin B ACC-labeled substrates developed through HyCoSuL profiling and for two reference substrates (*): cathepsin B selective (Cbz-Arg-Arg-AMC) and broad spectrum (Cbz-Phe-Arg-AMC). (B) Structures of three cathepsin B potent and selective ABPs containing the Cha-Leu-Glu(Bzl)-Arg sequence. Each ABP is labeled with either Cy5 or Cy7 fluorescence tag. (C) Labeling of recombinant cathepsins using three cathepsin B selective ABPs. Enzymes (100 nM final concentration) were incubated separately with various probe concentrations for 30 min and then subjected to SDS-PAGE analysis. Fluorescence was scanned using the 700 nm channel (Cy5) or 800 nm channel (Cy7) (LI-COR instrument). (D) The inhibition constant (kobs/I) of three cathepsin B ABPs determined for the five recombinant cathepsins.
Fig. 4
Fig. 4. Detection of cathepsin B activity is cell lysates using fluorescent substrate. (Panel A) The structure of cathepsin B selective fluorogenic substrate used in this study. (Panel B) The potency (kobs/I) of two widely used cathepsin B inhibitors (CA-074 and CA-074Me) towards human recombinant cathepsins B and L. (Panel C) The inhibition profile of cathepsin B activity in selected cell lysates at 100 μM of protease inhibitors. (Panel D) The inhibition profile of cathepsin B activity in selected cell lysates as a function of various inhibitors concentrations.
Fig. 5
Fig. 5. Cathepsin B labeling in human cancer cells using MP-CB-2 ABP. Eighteen human cancer cell lines were incubated with 1 μM Cy5-labeled MP-CB-2 ABP for various time (1, 4, 8, 24 hours), followed by SDS-PAGE and Western blot analysis. The red band indicates cathepsin B (29 kDa single chain form – active enzyme) labeling with probe, and the green band is the anti-cathepsin B antibody staining (∼43 kDa proenzyme, ∼29 kDa single chain and ∼26 kDa heavy chain). The MP-CB-2 probe is highly-selective towards cathepsin B and in most cell lines it can detect cathepsin B after 1 hour after incubation. After protein transfer to the membrane, molecular weight marker was marked with fluorescent ink to visualize it on both 700 nm (red) and 800 nm (green) channels.
Fig. 6
Fig. 6. Cathepsin B localization in A-431 cell line. (A) Cathepsin B was selectively labeled in A-431 cell line using 1 μM MP-CB-2 ABP (left panel). This probe labeled single chain cathepsin B form (∼29 kDa) and also the light chain (∼6 kDa), which dissociated from the heavy chain after sample boiling. The cathepsin B labeling could be prevented using E-64d inhibitor. MP-CB-2 probe did not label cathepsin L even after prolonged incubation (24 hours). Color code for the overlay Western blot: MP-CB-2 probe is red, and cathepsin antibody is green. After protein transfer to the membrane, molecular weight marker was marked with fluorescent ink to visualize it on both 700 nm (red) and 800 nm (green) channels. (B) Localization of cathepsin B in A-431 cells using 1 μM of ABP (overnight incubation) and anti-cathepsin B antibody. ABP and catB antibody greatly overlapped, which demonstrates the ABP selectivity. However, the signal from the probe also overlapped with anti-cathepsin L antibody, demonstrating that both enzymes share the same subcellular localization. MP-CB-2 probe could also label nuclear cathepsin B. (C) The colocalization plot of cathepsin, Cy5 probe and nucleus from randomly selected area (line). Data were captured from a line drawn through the cell (white line on Panel B, overlay). The y axis shows the relative intensity of each signal, and the x axis displays the distance (in pixels). These plots demonstrate the high MP-CB-2 probe selectivity (left panel) and nuclear localization of cathepsin B (left panel). Scale bar is 20 μm. (D) Colocalization of cathepsin B MP-CB-2 ABP with cathepsin B (left) and cathepsin L (right) in seven cancer cell lines (A-431, U2-OS, PC-3, OVCAR-5, SK-OV-3, HCT-116, and MDA-MB-231) presented on box plots. The weighted colocalization coefficients were calculated for ABP/cat B and ABP/cat L based on the analysis of at least seven randomly selected images taken for each cell line.
Fig. 7
Fig. 7. Cathepsin B detection in lysosomes of cancer cells. Cy5 MP-CB-2 probe (red) labels active cathepsin B in the lysosomes of three cancer cell lines (HCT-116, OVCAR-5 and SK-OV-3). The application of anti-cathepsin B antibody (green) demonstrates high degree of ABP selectivity. Staining cells with anti-cathepsin L antibody shows very similar distribution of this enzyme in tested cancer cells (right panel).
Fig. 8
Fig. 8. Cathepsin B detection in membranes and lysosomes of cancer cells. Application of anti-cathepsin B antibody (green) demonstrates the lysosomal and membrane-bound cathepsin B localization in MDA-MB-231, U2-OS and PC-3 cancer cells. However, MP-CB-2 probe (red) labels cathepsin B only in lysosomes. Staining cells with anti-cathepsin L antibody shows distinct cathepsin L localization, as this enzyme is present only in the lysosomes, but not in the membrane.
Fig. 9
Fig. 9. Labeling of cathepsin B in non-small lung cancer sample. (Panel A) The workflow of the experiment. Cells were collected from a cancer patient and cultured on a Petri dish. After 4 weeks cells were harvested, counted and used for experiments. (Panel B) Cathepsin B was selectively labeled in cancer cells using 1 μM MP-CB-2 ABP (left panel). This probe labeled single chain cathepsin B form (∼29 kDa) and also the light chain (∼6 kDa), which dissociated from the heavy chain after sample boiling. (Panel C) Localization of cathepsin B in cells using 1 μM of ABP (overnight incubation) and anti-cathepsin B antibody. ABP and catB antibody greatly overlapped, which demonstrates the ABP selectivity. When cells were pretreated with E64d inhibitor, the cathepsin B staining was significantly reduced.

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