Activity-Based Protein Profiling Of Cysteine Cathepsins Using Cell-Permeable Fluorescent Probes

In the postgenomic era new strategies are emerging for global characterization ofprotein expression and function. Activity-based protein profiling (ABPP) has emerged as apowerful method to display enzyme activities which are often regulated on apost-translational level. In ABPP research, the detection of protein activity can befacilitated using activity-based probes (ABPs) which specifically labeled the targetproteases.Cell membrane is a natural barrier that can restrict ABPP application of probes inliving cells. Biotin motifs and most fluorophores are not cell permeable, thereby limitingmost ABPs to in vitro work with cell lysates. In vitro proteomic preparations are usuallynot capable of maintaining complex intracellular conditions and therefore do not provideaccurate information about enzyme activites. Developing cell-permeable ABPs and intactcell labeling methods have been applied for ABPP to address this limitation.In this thesis, we have developed two novel ABPs for cysteine cathepsins withmembrane permeability enabled by a cell-penetrating peptide (CPP). The two ABPs werenamed CpFABP and TCpABP. We use TCpABP for proteomic profiling of active cysteinecathepsins in living cells while using CpFABP labeling and tracking lysosomes. The maincontents are shown as follow.(1) We synthesized the cell-permeable activity-based probe (CpFABP and TCpABP)which contain the Tyr-Leu epoxysuccinyl scaffold and a cell-penetrating octa-argininepeptide. These probes specifically labeled cysteine cathepsins. We noted that theses probeswere unstable during the traditional freeze-drying step possibly owing to the occurrence ofnucleophilic oxirane ring opening. we used an improved protocol to get cell-permeableprobes at greater than90%purity.(2) The probe TCpABP consists of four components:(a) a epoxysuccinyl scaffold as reactive group,(b) a fluorophore for visualization,(c) a biotin motif for enrichment, and (d)a cell-penetrating peptide for probe transmembrane delivery. TCpABP is a trifunctionalprobe which contains cell-impermeable tags, it was able to enter living cells efficiently viathe delivery of a cell-penetrating peptide. TCpABP also allowed simultaneous detectionand affinity isolation of labeled proteins with a fluorophore and a biotin motif, respectively.We optimized the enrichment protocol to minimize contaminants and identified7cathepsins,2of which have never been identified using existing ABPs. We also used alabel-free quantification approach to quantify the relative abundances of active cathepsinsand compared them with their previously published mRNA expression levels. A highdegree of correlation between the mRNA expression levels and protein relative activitieswas observed for most of the identified cathepsins except cathepsin H. The results hereinindicate that TCpABP is valuable for the detection of active cathepsins in living cells andprovides useful guidelines for designing novel cell-permeable ABPs for in vivo labelingand their applications in in vivo proteomics studies.(3) We have identified the labeled proteins and quantify the relative abundances ofthem, the results demonstrated that our probes is an efficient tool to label active cathepsinsin their native environment with high sensitivity and high specificity. By exploiting thisremarkable ability, we have take CpFABP as a novel lysosome marker. Co-localizationstudies that utilized LysoTracker probes as standard lysosomal markers demonstrated thatour novel probe is effective in specifically labeling lysosomes in various kinds of live cells.Furthermore, our studies revealed that this probe has the ability to label fxed cells,permeabilized cells, and NH4Cl-treated cells, unlike LysoTracker probes, which showineffective labeling under the same conditions. Remarkably, when applied to monitor theprocess of lysosome-dependent apoptosis, our probe not only displayed the expectedrelease of lysosomal cathepsins from lysosomes into the cytosol but also revealedadditional information about the location of the cathepsins during apoptosis, which is undetectable by other chemical lysosome markers. These results suggest a wide array ofpromising applications for our probe and provide useful guidelines for its use as alysosome marker in lysosome-related studies.