title: Activity based subcellular resolution imaging of lipases.
abstract: Lipases play a key role in whole body energy homeostasis. Dysregulation of lipolytic activities affects lipid absorption, mobilization, and transport, and is causative for lipid-related diseases. Regulation of enzymes involved in lipid metabolism is governed by a complex network of protein-protein and protein-small molecule interactions. Thus these enzymes have to be studied under the physiologically most relevant conditions, that is, in vivo. Our latest generation of activity based probes designed for capturing of lipases employs bioorthogonal chemical linker groups, which are membrane permeable and thus allow studying protein activity in living cells. Another advantage is the virtually unlimited choice of reporter tags. Here we report on a novel method combining in vivo activity based labeling of lipases with in situ detection of lipolytic activities by on slide click chemistry and imaging by fluorescence microscopy. We demonstrate that cytosolic as well as organelle resident lipases are specifically labeled in intact living cells. This method will shed light on the (sub)cellular localization of lipolytic proteomes of cells and tissues in health and disease directly at enzymatic activity level without the need of prior knowledge of the identities of the responsible enzymes or dependence on the availability of specific antibodies.
authors.affiliation: Proteomics Core Facility, Center for Medical Research and Institute of Pathology, Medical University of Graz, Stiftingtalstrasse 24, A-8010 Graz, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Viertler
authors.forename: Matthias
authors.initials: M
authors.lastname: Schittmayer
authors.forename: Ruth
authors.initials: R
authors.lastname: Birner-Gruenberger
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000818:Animals; D001386:Azides / Q000737:chemistry; D019556:COS Cells; D002384:Catalysis; D002522:Chlorocebus aethiops; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D050356:Lipid Metabolism; D008856:Microscopy, Fluorescence; D063065:Organophosphonates / Q000138:chemical synthesis / Q000737:chemistry; D020543:Proteome / Q000032:analysis
chemical_list: D000480:Alkynes; D001386:Azides; D063065:Organophosphonates; D020543:Proteome; D003300:Copper; D008049:Lipase