title: (19)F NMR monitoring of the eukaryotic 20S proteasome chymotrypsin-like activity: an investigative tool for studying allosteric regulation.
abstract: The proteasome displays three distinct proteolytic activities. Currently, proteasome inhibitors are evaluated using specific fluorescent substrates for each of the individual active sites. However, the photophysical properties of the commonly used fluorophores are similar and thus, the simultaneous monitoring of the three proteolytic activities is not possible. We have developed a bimodal fluorescent fluorinated substrate as a novel tool to study the chymotrypsin-like (ChT-L) proteolytic activity and its regulation by inhibitors and by substrates of trypsin-like (T-L) and caspase-like sites (PA). We demonstrate that this substrate is reliable to evaluate the ChT-L inhibitory activity of new molecules either by fluorescence or (19)F NMR spectroscopy. We have found that the ChT-L activity is dramatically reduced in the presence of T-L and PA substrates. This work provides a proof of concept that the fluorinated substrate enables investigation of the allosteric regulation of the ChT-L activity.
authors.affiliation: Molécules Fluorées et Chimie Médicinale, BioCIS UMR-CNRS 8076, LabEx LERMIT, Université Paris-Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, France. sandrine.ongeri@u-psud.fr julia.kaffy@u-psud.fr.
authors.forename: M
authors.initials: M
authors.lastname: Keita
authors.forename: J
authors.initials: J
authors.lastname: Kaffy
authors.forename: C
authors.initials: C
authors.lastname: Troufflard
authors.forename: E
authors.initials: E
authors.lastname: Morvan
authors.forename: B
authors.initials: B
authors.lastname: Crousse
authors.forename: S
authors.initials: S
authors.lastname: Ongeri
mesh_terms: D000494:Allosteric Regulation; D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism*; D003374:Coumarins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D005461:Fluorine / Q000378:metabolism*; D006834:Hydrazines / Q000737:chemistry; D020128:Inhibitory Concentration 50; D009682:Magnetic Resonance Spectroscopy; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D006834:Hydrazines; C114703:N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amino-4-methylcoumarin; D009842:Oligopeptides; D010455:Peptides; D005461:Fluorine; C055467:7-amino-4-trifluoromethylcoumarin; D002918:Chymotrypsin; D046988:Proteasome Endopeptidase Complex