Targeted protein S-nitrosylation of ACE2 inhibits SARS-CoV-2 infection

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1. Version published to 10.1038/s41589-022-01149-6

   Sep 29, 2022
2. 

   ScreenIT

   Apr 8, 2022

   SciScore for 10.1101/2022.04.05.487060: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	Aminoadamantane and aminoadamantane nitrate drugs: Aminoadamantane nitrate compounds (blindly coded NMT2, NMT3, NMT5-NMT9, and NMT5-Met (metabolite, sans nitro group) were synthesized by and obtained from EuMentis Therapeutics, Inc. (Newton, MA), and have been described previously6–9, 33.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After incubation with secondary antibodies (IR-dye 680LT-conjugated goat anti-mouse [1:20,000; Li-Cor, 926-68020] or IR-dye 800CW-conjugated goat anti-rabbit [1:15,000; Li-Cor, 926-32211]), membranes were scanned …

   SciScore for 10.1101/2022.04.05.487060: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	not detected.
   Sex as a biological variable	not detected.
   Randomization	not detected.
   Blinding	Aminoadamantane and aminoadamantane nitrate drugs: Aminoadamantane nitrate compounds (blindly coded NMT2, NMT3, NMT5-NMT9, and NMT5-Met (metabolite, sans nitro group) were synthesized by and obtained from EuMentis Therapeutics, Inc. (Newton, MA), and have been described previously6–9, 33.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   After incubation with secondary antibodies (IR-dye 680LT-conjugated goat anti-mouse [1:20,000; Li-Cor, 926-68020] or IR-dye 800CW-conjugated goat anti-rabbit [1:15,000; Li-Cor, 926-32211]), membranes were scanned with an Odyssey infrared imaging system (Li-Cor)	anti-mouse suggested: (LI-COR Biosciences Cat# 926-68020, RRID:AB_10706161) anti-rabbit suggested: (Santa Cruz Biotechnology Cat# sc-53804, RRID:AB_783976)
   Immunoprecipitants were eluted and subjected to immunoblotting with anti-ACE2 antibody (1:1000, Cell Signaling, #15983) and anti-SARS-CoV-2 Spike protein antibody (1:2000, Abcam, ab275759)	anti-SARS-CoV-2 Spike protein suggested: None
   Primary antibodies and dilutions were as follows: Mouse anti-TNFα (5 µg/ml, Abcam, #ab1793) and rabbit anti-macrophage inflammatory protein 1α (MIP-1α)/CCL3+CCL3L1 (1:250	anti-TNFα suggested: None anti-macrophage inflammatory protein 1α ( MIP-1α)/CCL3+CCL3L1 suggested: None
   After 30 min, all cells were collected and subjected to biotin switch-assay and immunoblotting with anti-ACE2 antibody to assess the levels of SNO-ACE2 and total input ACE2.	anti-ACE2 suggested: None total input ACE2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Cell lines: HEK293T (System Biosciences, LV900A-1) and HEK293-Spike cells (SARS-CoV-2 Spike (D614)-expressing 293 cells [293-SARS2-S cells, InvivoGen]) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with GlutaMAX™ (DMEM, high glucose, GlutaMAX™ Supplement, Life Technologies, 10566016) supplemented with 10% fetal bovine serum (FBS; Sigma, F7524), 100 IU/ml, and 100 µg/ml penicillin-streptomycin (Thermo Fisher Scientific, 10378016) at 37 °C in a 5% CO2 incubator.	HEK293T suggested: None HEK293-Spike suggested: None 293 suggested: None
   SARS-CoV-2 virus generation: Monkey Vero E6 cells were plated in a T225 flask with complete DMEM containing 10% FBS, 1×PenStrep, 2 mM L-glutamine and incubated for overnight at 37 °C in a humified atmosphere of 5% CO2.	Vero E6 suggested: None
   HeLa-ACE2 cells were seeded in the assay-ready plates at 1.6×103 cells/well in assay medium, and plates were incubated for 24 h at 37 ℃ with 5% CO2.	HeLa-ACE2 suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)
   Homogenized lungs were titrated 1:10 over 6 steps and layered over Vero cells.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Recombinant DNA
   Sentences	Resources
   . Monkey Vero E6 cells (ATCC CRL-1586) were maintained in complete DMEM (Corning, 15-013-CV) containing 10% FBS, 1×PenStrep (Corning 20-002-CL), 2 mM L-glutamine (Corning, 25-005-CL) at 37 °C in a 5% CO2 incubator. Plasmids: hACE2 was a gift from Hyeryun Choe (Addgene plasmid #1786; http://n2t.net/addgene:1786 ; RRID:Addgene_1786)46.	detected: RRID:Addgene_1786)
   The C262A, C498A, C261/498A mutant ACE2 constructs were generated using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies, 210514) according to the manufacturer’s protocol. pGBW-m4252984 (SARS-CoV-2 E [envelope]) was a gift from Ginkgo Bioworks (Addgene plasmid #153898; http://n2t.net/addgene:153898; RRID:Addgene_153898).	detected: RRID:Addgene_153898)
   MLV-gag/pol, MLV-CMV-Luciferase, SARS-CoV-2, and VSV-G plasmids were a gift from David Nemazee	VSV-G suggested: RRID:Addgene_138479)
   Expression and purification of human ACE2 protein: The N-terminal peptidase domain of human ACE2 (residues 19 to 615, GenBank: BAB40370.1) was cloned into phCMV3 vector and fused with C-terminal His-tag.	phCMV3 suggested: RRID:Addgene_173431)
   In brief, HEK293T cells were transiently co-transfected with MLV-gag/pol, MLV-CMV-Luciferase plasmid, and SARS- CoV-2 Spike (D614) or VSV-G plasmid.	MLV-CMV-Luciferase suggested: None
   For transient expression in HEK293T cells, we used a transfection reagent (Fugene® HD, Promega) to co-transfect plasmids containing cDNAs for SARS-CoV-2 E protein (pGBW-m4133502, Addgene) and green fluorescent protein (GFP) at a ratio of 1:0.1 (0.5:0.05 µg/well, respectively).	pGBW-m4133502 suggested: RRID:Addgene_153565)
   Software and Algorithms
   Sentences	Resources
   Maximum intensity projection of images was generated, and fluorescence intensity was analyzed with ImageJ software (https://imagej.nih.gov/ij/download.html) as previously described50.	ImageJ suggested: (ImageJ, RRID:SCR_003070)
   Molecular dynamics (MD) simulations were performed on the Frontera supercomputer at the Texas Advanced Supercomputing Center (TACC) using NAMD 2.1461 and CHARMM36m all-atom additive force fields62–64.	NAMD suggested: (NAMD, RRID:SCR_014894)
   Molecular Devices) with a 10× objective, and total live cells per well quantified in the acquired images using the Live Dead Application Module (MetaXpress).	MetaXpress suggested: (MetaXpress, RRID:SCR_016654)
   Statistical analyses were performed using GraphPad Prism software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your data.

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2022.04.05.487060 on bioRxiv

   Apr 5, 2022