A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate.
The dynamic modification of proteins with ubiquitin is a key regulation paradigm in eukaryotic cells that controls stability, localization, and function of the vast majority of intracellular proteins. Here we describe a robust fluorescence intensity assay for monitoring the enzymatic activity of deubiquitinating proteases, which reverse ubiquitin modifications and comprise over 100 members in humans. The assay was developed for the catalytic domain of human ubiquitin-specific protease 2 (USP2) and human ubiquitin carboxyterminal hydrolase L3 (UCH-L3), and makes use of the novel substrate ubiquitin-rhodamine110-glycine. The latter combines the advantages of a high dynamic range and beneficial optical properties. Its enzymatic behavior is characterized by the kinetic constants K(m)=1.5 microM, k(cat) = 0.53s(-1) and k(cat)/K(m) = 3.5 x 10(5)M(-1) s(-1) for USP2 and K(m) = 34 nM, k(cat)=4.72s(-1), and k(cat)/K(m) = 1.4 x 10(8)M(-1) s(-1) for UCH-L3. This new assay is suitable for inhibitor screening and characterizations, and has been established for the 384-well plate format using protease concentrations of 120 pM for USP2 and 1 pM for UCH-L3 and substrate concentrations of 100 nM for both enzymes. Due to the low protease concentrations and high sensitivity, this assay would allow the determination of inhibitory constants in the subnanomolar range.
Hassiepen
Ulrich
U
Novartis Institutes for Biomedical Research, Center for Proteomic Chemistry/Expertise Platform Proteases, Novartis Pharma AG, 4002 Basel, Switzerland. ulrich.hassiepen@novartis.com
Eidhoff
Ulf
U
Meder
Gabriele
G
Bulber
Jean-Francois
JF
Hein
Andreas
A
Bodendorf
Ursula
U
Lorthiois
Edwige
E
Martoglio
Bruno
B
D001681:Biological Assay / Q000295:instrumentation / Q000379:methods; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D047668:Inteins; D007700:Kinetics; D008954:Models, Biological; D012235:Rhodamines / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D043222:Ubiquitin Thiolesterase
D005456:Fluorescent Dyes; D012235:Rhodamines; D025801:Ubiquitin; D010450:Endopeptidases; C509806:UCHL3 protein, human; C108665:USP2 protein, human; D043222:Ubiquitin Thiolesterase; D003546:Cysteine Endopeptidases; D005998:Glycine