Abstract
Cysteine cathepsins, such as cathepsin S (CTSS), are implicated in the pathology of a wide range of diseases and are of potential utility as diagnostic and prognostic biomarkers. In previous work, we demonstrated the potency and efficiency of a biotinylated diazomethylketone (DMK)-based activity-based probe (ABP), biotin-PEG-LVG-DMK, for disclosure of recombinant CTSS and CTSS in cell lysates. However, the limited cell permeability of both the biotin and spacer groups restricted detection of CTSS to cell lysates. The synthesis and characterisation of a cell permeable ABP to report on intracellular CTSS activity is reported. The ABP, Z-PraVG-DMK, a modified peptidyl diazomethylketone, was based on the N-terminus of human cystatin motif (Leu-Val-Gly). The leucine residue was substituted for the alkyne-bearing proparcylglycine to facilitate conjugation of an azide-tagged reporter group using click chemistry, following irreversible inhibition of CTSS. When incubated with viable Human Embryonic Kidney 293 cells, Z-PraVG-DMK permitted disclosure of CTSS activity following cell lysis and rhodamine azide conjugation, by employing standard click chemistry protocols. Furthermore, the fluorescent tag facilitated direct detection of CTSS u...Continue Reading
References
Jan 1, 1992·Journal of Enzyme Inhibition·A HallF Kasprzykowski
Apr 15, 1992·The Biochemical Journal·B WalkerJ Nelson
Mar 2, 1982·Biochemistry·W X Tian, C L Tsou
Sep 1, 1996·The Biochemical Journal·G LalmanachF Gauthier
May 2, 2001·Journal of Inorganic Biochemistry·A Kr zelW Bal
Dec 14, 2001·AAPS PharmSci·T Maurer, H L Fung
Jun 19, 2002·The Biochemical Journal·Christopher J ScottBrian Walker
Dec 12, 2002·Chemical Reviews·James C PowersKaren Ellis James
Nov 13, 2003·Chembiochem : a European Journal of Chemical Biology·Rolf Breinbauer, Maja Köhn
May 5, 2004·Chemistry & Biology·Anna E Speers, Benjamin F Cravatt
May 30, 2008·Pharmaceutical Research·Christopher D HeinDong Wang
Jul 25, 2008·The Journal of Biological Chemistry·Carlos López-Otín, Judith S Bond
Oct 22, 2008·Expert Review of Proteomics·Marko Fonović, Matthew Bogyo
Nov 22, 2011·Current Opinion in Chemical Biology·Laura E EdgingtonMatthew Bogyo
Mar 18, 2015·Biochemical and Biophysical Research Communications·Thibaut GarenneGilles Lalmanach
Apr 16, 2015·Biological Chemistry·Richard D A WilkinsonRoberta E Burden
Aug 9, 2015·Biochimie·Caroline S HughesChristopher J Scott