Mechanism-Based Probe for the Analysis of Cathepsin Cysteine Proteases in Living Cells

Abstract

Mechanism-based probes are providing new tools to evaluate the enzymatic activities of protein families in complex mixtures and to assign protein function. The application of these chemical probes for the visualization of protein labeling in cells and proteomic analysis is still challenging. As a consequence, imaging and proteomic analysis often require different sets of chemical probes. Here we describe a mechanism-based probe, azido-E-64, that can be used for both imaging and proteomics. Azido-E-64 covalently modifies active Cathepsin (Cat) B in living cells, an abundant cysteine protease involved in microbial infections, apoptosis, and cancer. Furthermore, azido-E-64 contains an azide chemical handle that can be selectively derivatized with phosphine reagents via the Staudinger ligation, which enables the imaging and proteomic analysis of Cat B. We have utilized azido-E-64 to visualize active Cat B during infection of primary macrophages with Salmonella typhimurium , an facultative intracellular bacterial pathogen. These studies demonstrated that active Cat B is specifically excluded from Salmonella -containing vacuoles, which suggests that inhibition of protease activity within bacteria-containing vacuoles may contribute to bacterial virulence.

A chemical approach for detecting sulfenic acid-modified proteins in living cells

“…The azide moiety is a unique chemical handle that permits selective coupling with bio-orthogonal phosphine or alkyne-derivatized reagents for detection and isolation by affinity purification for subsequent proteomic analysis. [33][34][35][36][37][38] The advantage of DAz-1 over existing probes for sulfenic acid detection is its small size, which contributes to its favorable uptake in cells and facilitates protein labeling. Moreover, once introduced into proteins the azide group can be tagged by a wide variety of affinity and fluorescent reagents ex vivo.…”

“…In addition, the ability to 'tag' proteins with biotin or a range of other affinity and fluorescent reagents via the azide chemical reporter on DAz-1 provides an opportunity for enrichment and proteomic analysis of oxidized proteins. 33,34 These studies are currently underway and will be reported in due course. This new method to detect sulfenic acid-modified proteins provides a powerful means to detect changes in cysteine oxidation in vivo and should find a wide variety of applications for the study of biological processes that are central to human health and disease.…”

“…Two-step labeling methods analogous to this approach have been successfully employed to profile protein glycosylation, 38 farnesylation 63 and protease activity. 34 Once the specificity of DAz-1 was validated in vitro we proceeded to test the azido-probe, and our overall strategy, in cultured human cells. In these experiments we employed the human T lymphoma cell line Jurkat, which are well characterized and resemble naive primary T-cells in their response to stimulation.…”

Bioorthogonal Click Chemistry: Covalent Labeling in Living Systems

“…Following precedents established by Cravatt and Speers [29], Overkleeft and coworkers [30], and Taunton and coworkers [31], Ploegh and coworkers recently demonstrated the use of an azido probe and the Staudinger ligation to visualize active cathepsins, an important class of cysteine proteases, in cells [32].…”

Identifying the Cellular Targets of Bioactive Small Molecules with Activity-Based Probes

“…These qualities make them valuable tools for cellular localization study [51,52] or activity analysis [53,99,100] of related proteins in intact cells once their targets become known. Besides, they are also helpful in understanding the roles of their targets in physiological and pathological processes.…”