title: 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.
authors.affiliation: Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA. hang@wi.mit.edu
authors.forename: Howard C
authors.initials: HC
authors.lastname: Hang
authors.forename: Joana
authors.initials: J
authors.lastname: Loureiro
authors.forename: Eric
authors.initials: E
authors.lastname: Spooner
authors.forename: Adrianus W M
authors.initials: AW
authors.lastname: van der Velden
authors.forename: You-Me
authors.initials: YM
authors.lastname: Kim
authors.forename: Annette M
authors.initials: AM
authors.lastname: Pollington
authors.forename: Rene
authors.initials: R
authors.lastname: Maehr
authors.forename: Michael N
authors.initials: MN
authors.lastname: Starnbach
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002401:Cathepsin B / Q000032:analysis* / Q000235:genetics*; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D015341:Nucleic Acid Probes / Q000235:genetics*
chemical_list: D015341:Nucleic Acid Probes; D003546:Cysteine Endopeptidases; D002401:Cathepsin B