title: A novel photoaffinity-based probe for selective detection of cathepsin L active form.
abstract: Detecting the active forms of proteases by using activity-based probes in complex proteomes has become an intensively investigated field of research over the past years because many pathogenic conditions involve alterations in protease activities. The detection of lysosomal cysteine proteases, the cathepsins, has mostly relied on the use of probes that incorporate reactive electrophilic moieties to modify a cysteine in the active site covalently. Here we report the first example of an activity-based probe that targets the cathepsins and incorporates a photoactivatable benzophenone group for covalent labelling. When tested on a set of five cathepsins (B, K, L, S and V), this probe selectively labelled the active site of cathepsin L. Furthermore, when tested on crude cell extracts, the probe specifically detected cathepsin L quantities as low as a few picomoles. This study suggests that photoaffinity labelling is a promising approach for developing highly selective and useful cathepsin L probes. In particular, this probe might allow the detection of small amounts of the secreted active cathepsin L form in the cellular microenvironment in vitro and ex vivo.
authors.affiliation: Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia.
authors.forename: Ana
authors.initials: A
authors.lastname: Torkar
authors.forename: Sarah
authors.initials: S
authors.lastname: Bregant
authors.forename: Laurent
authors.initials: L
authors.lastname: Devel
authors.forename: Marko
authors.initials: M
authors.lastname: Novinec
authors.forename: Brigita
authors.initials: B
authors.lastname: Lenarčič
authors.forename: Tamara
authors.initials: T
authors.lastname: Lah
authors.forename: Vincent
authors.initials: V
authors.lastname: Dive
mesh_terms: D001577:Benzophenones / Q000737:chemistry; D020134:Catalytic Domain; D056668:Cathepsin L / Q000737:chemistry / Q000378:metabolism*; D015195:Drug Design; D057075:Enzyme Assays / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism
chemical_list: D001577:Benzophenones; D019920:Photoaffinity Labels; D020543:Proteome; C047723:benzophenone; D056668:Cathepsin L