A new fluorogenic peptide determines proteasome activity in single cells.
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.
Urru
Silvana A M
SA
Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, Milan 20156, Italy.
Veglianese
Pietro
P
De Luigi
Ada
A
Fumagalli
Elena
E
Erba
Eugenio
E
Gonella Diaza
Rodolfo
R
Carrà
Andrea
A
Davoli
Enrico
E
Borsello
Tiziana
T
Forloni
Gianluigi
G
Pengo
Niccolò
N
Monzani
Enrico
E
Cascio
Paolo
P
Cenci
Simone
S
Sitia
Roberto
R
Salmona
Mario
M
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
D005456:Fluorescent Dyes; D010455:Peptides; D021122:Protein Subunits; C553269:TAT-EDANS-DABCYL peptide; D046988:Proteasome Endopeptidase Complex