title: Activity-Based Lipid Esterase Profiling of M. bovis BCG at Different Metabolic States Using Tetrahydrolipstatin (THL) as Bait.
abstract: This chapter provides a step-by-step protocol using activity-based protein profiling (ABPP) as a chemical-proteomic tool to survey the antibiotic properties of a small molecule. Here, we investigate the molecular mechanism behind the bactericidal activity of tetrahydrolipstatin (THL). ABPP relies on small molecule probes that target the active site of specific enzymes in complex proteomes. These probes in turn are equipped with a reporter tag that allows capturing, visualization, enrichment, identification, and quantification of its targets either in vitro or in situ. THL possesses bactericidal activities, but its precise spectrum of molecular targets is poorly characterized. Here, we used THL analogs functionalized to enable Huisgen-base cycloaddition, commonly known as "click chemistry," to identify target proteins after enrichment from mycobacterial cell lysates obtained from different physiological conditions.
authors: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore (NUS), Singapore, 117456, Singapore.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore, 117456, Singapore.|Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, 48109, USA.
authors: Madhu Sudhan
authors: MS
authors: Ravindran
authors: NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore (NUS), Singapore, 117456, Singapore. markus_wenk@nuhs.edu.sg.|Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore, 117456, Singapore. markus_wenk@nuhs.edu.sg.|Department of Biological Sciences, National University of Singapore (NUS), Singapore, 117456, Singapore. markus_wenk@nuhs.edu.sg.|Swiss Tropical and Public Health Institute, University of Basel, 4051, Basel, Switzerland. markus_wenk@nuhs.edu.sg.
authors: Markus R
authors: MR
authors: Wenk
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D057930:Click Chemistry; D061565:Cycloaddition Reaction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004950:Esterases / Q000378:metabolism*; D007783:Lactones / Q000494:pharmacology*; D050356:Lipid Metabolism; D009163:Mycobacterium bovis / Q000201:enzymology*; D000077403:Orlistat; D040901:Proteomics
chemical_list: D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D007783:Lactones; D000077403:Orlistat; D004950:Esterases
keywords: Activity-based protein profiling; Cycloaddition reaction; Lipases; Lipid esterases; Tetrahydrolipstatin