Ubiquitin-AMC (R&D systems) hydrolysis was measured in a QuantaMaster spectrofluorimeter (PTI) by monitoring an increase of fluorescence emission at 435 nm with an excitation at 380 nm. Reactions using reconstituted proteasome used 100 nM Ubp6, 150 nM Rpn1, 150 nM recombinant base, 300 nM CP, 300 nM Lid, 300 nM Rpn10, 20 µM unfolded substrate, and 3–10 µM Ub-AMC. Reactions using proteasomes purified from yeast used 100 nM proteasome. Reactions were carried out either in the presence GF buffer (see above) with 1mM DTT and 1× ATP regeneration system or 1 mM ATPgS. Samples were incubated at 30 °C for 5–10 minutes prior to the addition of substrate to ensure Ubp6 association and nucleotide exchange.
Ubiquitin amc
Manufactured by R&D Systems
Sourced in United States
Ubiquitin-AMC is a synthetic substrate used in fluorescence-based assays to measure the activity of deubiquitinating enzymes (DUBs). It consists of the ubiquitin protein covalently linked to the fluorogenic reporter molecule 7-amino-4-methylcoumarin (AMC). Upon cleavage of the substrate by a DUB, the release of the AMC moiety results in an increase in fluorescence, which can be detected and quantified.
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Lab products found in correlation
Sars cov 2 plpro, by R&D Systems (2 mentions)
Black 384 well plate, by Corning (2 mentions)
Polarstar omega, by BMG Labtech (2 mentions)
Silver stain ms kit, by Fujifilm (2 mentions)
Lys63 linked ubiquitin tetramers, by R&D Systems (2 mentions)
Varioskan lux, by Thermo Fisher Scientific (2 mentions)
Prism 9, by GraphPad (2 mentions)
Ebselen 2 phenyl 1 2 benzisoselenazol 3 2h one, by Merck Group (1 mentions)
Gs 441524, by MedChemExpress (1 mentions)
Remdesivir, by MedChemExpress (1 mentions)
Grl0617, by MedChemExpress (1 mentions)
Ub vs ha, by R&D Systems (1 mentions)
Celltiter glo cell viability assay kit, by Promega (1 mentions)
Ubiquitin vinyl sulfone, by R&D Systems (1 mentions)
Synergy neo2, by Agilent Technologies (1 mentions)
Synergy 4, by Agilent Technologies (1 mentions)
Victor x5, by PerkinElmer (1 mentions)
Varioskan flash 3001, by Thermo Fisher Scientific (1 mentions)
Normal goat igg sc 2028, by Santa Cruz Biotechnology (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
24 protocols using ubiquitin amc
1
Proteasome-Mediated Deubiquitination Dynamics
2
Characterization of SARS-CoV Proteases
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Recombinant SARS-CoV-1 PLpro, SARS-CoV-2 PLpro and Ubiquitin-AMC were purchased as 32, 11 and 250 μM solutions, respectively, from R&D Systems.
Ebselen (2-Phenyl-1,2-benzisoselenazol-3(2H)-one) is commercially available from Sigma Aldrich.
All compounds were obtained and fully characterized in previous studies39 (link). Their purity and homogeneity were confirmed by HRMS and 77Se NMR (see Supplementary data).
Enzyme and inhibition assays were designed based on the procedures described in14 (link),16 (link),39 (link),40 (link).
Ebselen (2-Phenyl-1,2-benzisoselenazol-3(2H)-one) is commercially available from Sigma Aldrich.
All compounds were obtained and fully characterized in previous studies39 (link). Their purity and homogeneity were confirmed by HRMS and 77Se NMR (see Supplementary data).
Enzyme and inhibition assays were designed based on the procedures described in14 (link),16 (link),39 (link),40 (link).
3
Screening Small-Molecule Inhibitors of ISG15
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The SmartTM chemical library was purchased from Chemdiv (San Diego, USA), which contains 50,080 small-molecule compounds with enhanced diversity and drug-like properties. Remdesivir and its prodrug GS-441524, as well as GRL0617 were purchased from MedChemExpress (NJ, USA). Arg-Leu-Arg-Gly-Gly-AMC (RLRGG-AMC) was purchased from Bachem Bioscience (Bubendorf, Switzerland). ISG15-AMC, ubiquitin-AMC, and HA-Ub-VS were purchased from R&D systems (Minneapolis, USA). Cellular protease assay kit (Cat# 539125) was purchased from Sigma-Aldrich. CellTiter-Glo cell viability assay kit was purchased from Promega. Other chemicals were purchased from Sigma-Aldrich unless specified.
4
Proteasome-Mediated Deubiquitination Dynamics
5
Characterization of SARS-CoV Proteases
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1.General SARS-CoV-1 PLpro, SARS-CoV-2 PLpro and Ubiquitin-AMC were purchased as 32, 11 and 3 μM solutions, respectively, from R&D Systems.
All compounds were obtained and fully characterized in previous studies [19] [20] [21] .
Their purity and homogeneity were confirmed by HRMS and 77 Se NMR.
All compounds were obtained and fully characterized in previous studies [19] [20] [21] .
Their purity and homogeneity were confirmed by HRMS and 77 Se NMR.
6
Enzymatic Activity Assay of UCH-L1 Mutants
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Qualitative catalytic activity of wild-type and UCH-L1 mutant constructs was assayed using the activity-based probe ubiquitin vinylsulfone (Boston Biochem, Cambridge, MA) as previously described. Quantitative catalytic activity for purified wild-type and C220S mutants was assayed using the fluorogenic substrate ubiquitin-AMC (Boston Biochem) using a molecular devices automated plate reader. The reaction was conducted in a buffer containing 50 mM HEPES, 0.5 mM EDTA, 1 mg/ml ovalbumin, 1 mM DTT, 100 nM UbAMC, pH 7.8. The final concentration of the UCH-L1 mutants is unknown, but equal volumes of enzyme are added from stocks with equal (by SDS-PAGE) amounts of protein. Proteins were purified using a small-scale HA-purification system (MBL International, Woburn, MA) from cells expressing the respective constructs.
7
Enzymatic Assay of ChlaDUB Constructs
8
UCH-L1 Hydrolase Activity Assay
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The UCH-L1 hydrolase activity assay was performed as previously described with slight modifications.14 (link) Briefly, 2 μM UCH-L1 protein was incubated with 12.5 μM 15dPGJ2 or vehicle for 2 h before dilution with UCH-L1 hydrolase buffer. Samples were then mixed with 500 nM ubiquitin-AMC (BostonBiochem, Cambridge, MA, USA). The final concentration for UCH-L1 was 100 nM. Free AMC fluorophore was read using a fluorescent plate reader (ex 360 nm, em 460 nm).
9
Selective Inhibitor Screening of UCH Enzymes
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To test
for selectivity,
two human ubiquitin C-terminal hydrolases (UCH-L1 and UCH-L3) and
two unrelated enzymes (Hepatitis C Virus NS3 serine protease and B. anthracis dihydroorotase) were tested with the top hit
compound from HTS and a lead SARS-PLpro inhibitor (I-1 ) using a fluorometric assay. The fluorogenic substrate used in this
study was ubiquitin-AMC (Boston Biochem). All assays were performed
in 384-well black plates (Corning) in a total volume of 24 μL
of the assay buffer containing 50 mM HEPES (pH 7.5), 5 mM DTT, 0.1
mg mL–1 BSA, and 0.01% Triton X-100 (v/v) in triplicate.
A series of compound concentrations (0 to 200 μM final concentration
at 2-fold serial dilution) in 100% DMSO was prepared in a 384-well
plate. Then 3× compound solutions were prepared in the assay
buffer prior to assays. A total of 8 μL of each enzyme solution
was distributed into wells, and 8 μL of varying concentrations
of compounds was added and incubated for 10 min. The enzyme reaction
was initiated by adding 8 μL of the substrate (50 μM final
concentration), and fluorescence intensity was continuously monitored
at excitation/emission wavelengths of 350 nm/460 nm for 10 min.
for selectivity,
two human ubiquitin C-terminal hydrolases (UCH-L1 and UCH-L3) and
two unrelated enzymes (Hepatitis C Virus NS3 serine protease and B. anthracis dihydroorotase) were tested with the top hit
compound from HTS and a lead SARS-PLpro inhibitor (
study was ubiquitin-AMC (Boston Biochem). All assays were performed
in 384-well black plates (Corning) in a total volume of 24 μL
of the assay buffer containing 50 mM HEPES (pH 7.5), 5 mM DTT, 0.1
mg mL–1 BSA, and 0.01% Triton X-100 (v/v) in triplicate.
A series of compound concentrations (0 to 200 μM final concentration
at 2-fold serial dilution) in 100% DMSO was prepared in a 384-well
plate. Then 3× compound solutions were prepared in the assay
buffer prior to assays. A total of 8 μL of each enzyme solution
was distributed into wells, and 8 μL of varying concentrations
of compounds was added and incubated for 10 min. The enzyme reaction
was initiated by adding 8 μL of the substrate (50 μM final
concentration), and fluorescence intensity was continuously monitored
at excitation/emission wavelengths of 350 nm/460 nm for 10 min.
10
Ubiquitin-AMC Cleavage Assay for Lid Variants
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All Ubiquitin-AMC cleavage experiments were performed at 30°C in lid buffer. Because Rpn11’s Km for various ubiquitin substrates ranges from ~20 to ~300 μM, we assayed our WT and mutant lid variants at a constant, sub-Km Ubiquitin-AMC concentration. For all lid variants and the Rpn11/Rpn8 MPN-domain dimer, 500 nM enzyme was incubated with 2.5 μM Ubiquitin-AMC (Boston Biochem), and Rpn11-catalyzed ubiquitin cleavage was monitored by the increase in AMC fluorescence (Ex: 360 nm, Em: 435 nm) using a QuantaMaster spectrofluorometer (PTI). The slopes of individual time traces were translated to initial cleavage rates using a standard curve for Ubiquitin-AMC (ranging from 0.5–2.5 μM) that had been completely cleaved by the DUB Yuh1. Ubiquitin-AMC cleavage rates for all variants were measured in triplicate except for WT lid, Rpn11/Rpn8 dimer, Rpn5 (H282A, K283A) and Rpn8 (Q115A), where n = 11, n = 6, n = 4, and n = 4, respectively.
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