Engineered unnatural ubiquitin for optimal detection of deubiquitinating enzymes
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Abstract
Deubiquitinating enzymes (DUBs) are responsible for removing ubiquitin (Ub) from its protein conjugates. DUBs have been implicated as attractive therapeutic targets in the treatment of viral diseases, neurodegenerative disorders and cancer. The lack of selective chemical tools for the exploration of these enzymes significantly impairs the determination of their roles in both normal and pathological states. Commercially available fluorogenic substrates are based on the C-terminal Ub motif or contain Ub coupled to a fluorophore (Z-LRGG-AMC, Ub-AMC); therefore, these substrates suffer from lack of selectivity. By using a hybrid combinatorial substrate library (HyCoSuL) and a defined P2 library containing a wide variety of nonproteinogenic amino acids, we established a full substrate specificity profile for two DUBs—MERS PLpro and human UCH-L3. Based on these results, we designed and synthesized Ub-based substrates and activity-based probes (ABPs) containing selected unnatural amino acids located in the C-terminal Ub motif. Biochemical analysis and cell-based experiments confirmed the activity and selectivity of engineered Ub-based substrates and probes. Using this approach, we propose that for any protease that recognizes Ub and Ub-like substrates, a highly active and selective unnatural substrate or probe can be engineered.
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SciScore for 10.1101/2020.01.30.926881: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Biotinylated Ub-based probes were detected with a fluorescent streptavidin Alexa Fluor 647 conjugate (1:10 000) in TBS-T with 1% BSA, and UCH-L3 was detected with a mouse anti-human monoclonal IgG1 antibody (1:1000) and fluorescent goat anti-mouse (1:10 000) using an Azure Biosystems Sapphire™ Biomolecular Imager and Azure Spot Analysis Software. anti-human monoclonal IgG1suggested: Noneanti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resour… SciScore for 10.1101/2020.01.30.926881: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Biotinylated Ub-based probes were detected with a fluorescent streptavidin Alexa Fluor 647 conjugate (1:10 000) in TBS-T with 1% BSA, and UCH-L3 was detected with a mouse anti-human monoclonal IgG1 antibody (1:1000) and fluorescent goat anti-mouse (1:10 000) using an Azure Biosystems Sapphire™ Biomolecular Imager and Azure Spot Analysis Software. anti-human monoclonal IgG1suggested: Noneanti-mousesuggested: NoneExperimental Models: Cell Lines Sentences Resources DUB labeling in cell lysates: A-431 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin) in a humidified 5% CO2 atmosphere at 37°C. A-431suggested: NoneSoftware and Algorithms Sentences Resources Kinetic parameters were calculated using GraphPad Prism software with the Michaelis-Menten equation. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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