Protein Profiling of Active Cysteine Cathepsins in Living Cells Using an Activity-Based Probe Containing a Cell-Penetrating Peptide

Abstract

Cell-permeable activity-based probes (ABPs) are capable of labeling target proteins in living cells, thereby providing a powerful tool for profiling active enzymes in their native environment. In this study, we describe the synthesis and use of a novel trifunctional cell-permeable activity-based probe (TCpABP) for proteomic profiling of active cysteine cathepsins in living cells. We demonstrate that although TCpABP contains cell-impermeable tags, it was able to enter living cells efficiently via the delivery of a cell-penetrating peptide. TCpABP also allowed simultaneous detection and affinity isolation of labeled proteins with a fluorophore and a biotin motif, respectively. We optimized the enrichment protocol to minimize contaminants and identified 7 cathepsins, 2 of which have never been identified using existing ABPs. We also used a label-free quantification approach to quantify the relative abundances of active cathepsins and compared them with their previously published mRNA expression levels. A high degree of correlation between the mRNA expression levels and protein relative activities was observed for most of the identified cathepsins except cathepsin H. The results herein indicate that TCpABP is valuable for the detection of active cathepsins in living cells and provides useful guidelines for designing novel cell-permeable ABPs for in vivo labeling and their applications in in vivo proteomics studies.

A general strategy for developing cell-permeable photo-modulatable organic fluorescent probes for live-cell super-resolution imaging

“…1 , Supplementary Fig. 1 ) using standard solid-phase peptide synthesis 18 19 20 . The probes 1–3 have the following four components: (a) Lifeact (MGVADLIKKFESISKEE), a 17-amino-acid peptide, which can specifically and reversibly bind to the F-actin cytoskeleton without compromising cellular processes 21 ; (b) an (rR) 3 R 2 peptide to engender cellular permeability of the probe 17 ; (c) six glycine residues as a spacer and (d) a fluorophore (fluorescein isothiocyanate (FITC), rhodamine B (RhB) or a photo-activatable rhodamine B derivative (PA-RhB) that has a high fluorescence quantum yield, high photochemical stability and a large contrast ratio between the fluorescent and the dark state 22 ).…”

“…1 ). Compared with probe 3, probes 5 and 6 contain a different recognition unit, an epoxysuccinyl scaffold, which can selectively form covalent bonds with cysteine cathepsins (typically localized in lysosomes) 19 20 and a different fluorophore (a caged rhodamine 110 derivative (Caged-Rh110) or 5(6)-carboxyfluorescein (FAM)) 23 .…”

“…1 ), which contains a different recognition unit and a fluorophore from probe 3. The recognition unit of probe 5 is an epoxysuccinyl scaffold, which can selectively form covalent bonds with cysteine cathepsins (typically localized in lysosomes) 19 20 . We used Caged-Rh110 instead of PA-RhB in probe 5 because Caged-Rh110 is stable under acidic conditions 23 (pH≈5 in lysosomes), while PA-RhB is pH sensitive 22 .…”

Organelle‐Specific Activity‐Based Protein Profiling in Living Cells

“…High molecular weight probes containing fluorophores and/or enrichment tags are known, but require cell penetrating peptides or receptor motifs, in addition to long labeling times, in order to enter live cells and accumulate. [5] Smaller ABPs can reach their enzyme target quickly, but any organelle accumulation is due to enzyme localization rather than defined probe properties. [2b, 6] Our envisioned ABP requires a moiety with specific physicochemical properties, [7] and an alkyne for CuAAC mediated conjugation of a fluorophore or enrichment tag.…”

“…[10] Furthermore, the DAMP primary amine provides a sufficient handle to append 1) an alkyne for CuAAC, [11] and 2) the broadly reactive papain cysteine protease inhibitor ethyl succinate epoxide, extensively used for labeling cathepsins. [2b, 5–6, 11] The synthesis of organelle-targeting DAMP epoxide probes ( DEX-1 and DEX-2 ) is described in the supporting information (Figure 1, Figure S1 and S2). An ABP lacking DAMP ( DEX-3 ) was synthesized as a weak acid negative control (Figure 1, Figure S2).…”