Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion
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Abstract
Coiled-coil (CC) dimer-forming peptides are attractive designable modules for mediating protein association. Highly stable CCs are desired for biological activity regulation and assay. Here, we report the design and versatile applications of orthogonal CC dimer-forming peptides with a dissociation constant in the low nanomolar range. In vitro stability and specificity was confirmed in mammalian cells by enzyme reconstitution, transcriptional activation using a combination of DNA-binding and a transcriptional activation domain, and cellular-enzyme-activity regulation based on externally-added peptides. In addition to cellular regulation, coiled-coil-mediated reporter reconstitution was used for the detection of cell fusion mediated by the interaction between the spike protein of pandemic SARS-CoV2 and the ACE2 receptor. This assay can be used to investigate the mechanism of viral spike protein-mediated fusion or screening for viral inhibitors under biosafety level 1 conditions.
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SciScore for 10.1101/2020.12.10.419440: (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 The primary antibodies were mouse anti-Myc (Cell signaling technologies; diluted 1:2,000), rabbit anti-HA (Sigma H6908; diluted 1:2,000), and mouse anti-β-actin (Cell Signaling 3700; diluted 1:2,000). anti-Mycsuggested: Noneanti-HAsuggested: (Sigma-Aldrich Cat# H6908, RRID:AB_260070)anti-β-actinsuggested: NoneThe secondary antibodies were HRP-conjugated goat anti-rabbit IgG (Abcam ab6721; diluted 1:3,000) and HRP-conjugated goat anti-mouse IgG (Santa Cruz, … SciScore for 10.1101/2020.12.10.419440: (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 The primary antibodies were mouse anti-Myc (Cell signaling technologies; diluted 1:2,000), rabbit anti-HA (Sigma H6908; diluted 1:2,000), and mouse anti-β-actin (Cell Signaling 3700; diluted 1:2,000). anti-Mycsuggested: Noneanti-HAsuggested: (Sigma-Aldrich Cat# H6908, RRID:AB_260070)anti-β-actinsuggested: NoneThe secondary antibodies were HRP-conjugated goat anti-rabbit IgG (Abcam ab6721; diluted 1:3,000) and HRP-conjugated goat anti-mouse IgG (Santa Cruz, sc2005; diluted 1:3,000). anti-rabbit IgGsuggested: (Abcam Cat# ab6721, RRID:AB_955447)anti-mouse IgGsuggested: (Santa Cruz Biotechnology Cat# sc-2005, RRID:AB_631736)Experimental Models: Cell Lines Sentences Resources At 70% confluency, the donor HEK293T cells were transfected with plasmids expressing S protein, syncytia reporter split fluorescent protein GFP1-10 (GFP1-10:N7, GFP1-10:P7 or GFP1-10) and fluorescent protein mCherryNLS, and the acceptor cells were transfected with plasmids expressing the ACE2 receptor, syncytia reporter split protein GFP11 (N8:GFP11, 3×(N8:GFP11), P8:GFP11), and fluorescent protein BFP using PEI transfection reagent. HEK293Tsuggested: NoneSoftware and Algorithms Sentences Resources The exact peptide concentrations were determined based on absorbance at 280 nm, and the extinction coefficients were calculated by the ProtParam web tool (https://web.expasy.org/cgi-bin/protparam/protparam). ProtParamsuggested: (ProtParam Tool, RRID:SCR_018087)Data analysis was carried out with Astra 7.0 software (Wyatt, CA) utilizing the theoretical extinction coefficient calculated by ProtParam. Astrasuggested: (ASTRA, RRID:SCR_016255)The integration of heat pulses and model-fitting were performed with Origin 7.0 using a single-site binding model. Originsuggested: (Origin, RRID:SCR_014212)Data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) and SpectroFlo (Cytek) software. FlowJosuggested: (FlowJo, RRID:SCR_008520)The images were processed with LAS AF software (Leica Microsystems) and ImageJ software (National Institute of Mental Health, Bethesda, Maryland, ImageJsuggested: (ImageJ, RRID:SCR_003070)Software and statistics: Graphs were prepared with Origin 8.1 (http://www.originlab.com/), and GraphPad Prism 5 (http://www.graphpad.com/) was used for statistical purposes. 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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