title: A new fluorogenic peptide determines proteasome activity in single cells.
abstract: The ubiquitin-proteasome system plays a critical role in many diseases, making it an attractive biomarker and therapeutic target. However, the impact of results obtained in vitro using purified proteasome particles or whole cell extracts is limited by the lack of efficient methods to assess proteasome activity in living cells. We have engineered an internally quenched fluorogenic peptide with a proteasome-specific cleavage motif fused to TAT and linked to the fluorophores DABCYL and EDANS. This peptide penetrates cell membranes and is rapidly cleaved by the proteasomal chymotrypsin-like activity, generating a quantitative fluorescent reporter of in vivo proteasome activity as assessed by time-lapse or flow cytometry fluorescence analysis. This reporter is an innovative tool for monitoring proteasomal proteolytic activities in physiological and pathological conditions.
authors.affiliation: Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, Milan 20156, Italy.
authors.forename: Silvana A M
authors.initials: SA
authors.lastname: Urru
authors.forename: Pietro
authors.initials: P
authors.lastname: Veglianese
authors.forename: Ada
authors.initials: A
authors.lastname: De Luigi
authors.forename: Elena
authors.initials: E
authors.lastname: Fumagalli
authors.forename: Eugenio
authors.initials: E
authors.lastname: Erba
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Gonella Diaza
authors.forename: Andrea
authors.initials: A
authors.lastname: Carrà
authors.forename: Enrico
authors.initials: E
authors.lastname: Davoli
authors.forename: Tiziana
authors.initials: T
authors.lastname: Borsello
authors.forename: Gianluigi
authors.initials: G
authors.lastname: Forloni
authors.forename: Niccolò
authors.initials: N
authors.lastname: Pengo
authors.forename: Enrico
authors.initials: E
authors.lastname: Monzani
authors.forename: Paolo
authors.initials: P
authors.lastname: Cascio
authors.forename: Simone
authors.initials: S
authors.lastname: Cenci
authors.forename: Roberto
authors.initials: R
authors.lastname: Sitia
authors.forename: Mario
authors.initials: M
authors.lastname: Salmona
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D009474:Neurons / Q000201:enzymology; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021122:Protein Subunits / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D010455:Peptides; D021122:Protein Subunits; C553269:TAT-EDANS-DABCYL peptide; D046988:Proteasome Endopeptidase Complex