Journal: International Journal of Biological Macromolecules

Article Title: Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

doi: 10.1016/j.ijbiomac.2022.12.251

Figure Lengend Snippet: Physicochemical properties of royal jelly (RJ)-PF 50 protein composites and genomic organization of the SARS-CoV-2 S protein. (a, b) 3D structures of MRJP2 (orange) and MRJP2 isoform X1 (yellow), respectively, were generated with the Iterative Threading ASSEmbly Refinement (I-TASSER) protein-modelling online server ( http://zhanglab.ccmb.med.umich.edu/I-TASSER/ ). (c) SDS-PAGE (12 %) chromatogram profile of RJ proteins stained with silver nitrate. Lane M: protein markers (M) with various molecular weights, Lane 1: PF 50 (RJ protein fraction precipitated at 40–50 % ammonium sulphate saturation), Lane 2: MRJP2 isoform X1, and Lane 3: MRJP2. MRJP2 and its isoform X1 were purified using CM-Sephadex ion-exchange column chromatography. (d) Predicted exposed and buried amino acid residues in MRJP2 and its isoform X1 using the DeepREx-WS webserver ( https://deeprex.biocomp.unibo.it ). The light orange highlighted regions represent the polar amino acid content of the two tested MRJPs. (e) Schematic representation of SARS-CoV-2 S glycoprotein, illustrating S1 and S2 subunits and showing the S1-receptor binding mechanism. The SARS-CoV-2 trimeric S glycoprotein crystallographic structure (PDB ID: 6VXX ) shows the fusion protein (FP), N-terminal domain (NTD), intracellular tail (I.T.), and transmembrane anchor (T.A.) protein domains, as well as the receptor-binding domain (RBD) that represents the human ACE2 receptor binding site. The box displays the genomic architecture of the S gene, highlighting the coding regions of the S1 and S2 subunits. (f) Schematic representation of the key mutations in the SARS-CoV-2 S protein of the UK (Alpha, B.1.1.7) and South African (Beta, B.1.351) variants of concern. These mutation sites were retrieved from the PDBsum database ( http://www.ebi.ac.uk/thornton-srv/databases/pdbsum ).

Article Snippet: In addition, Thaw Medium 1 (#60187, growth medium without puromycin), anti-S antibody (#101024; clone No. C-A11), luciferase (Luc) reagent (#60690), and SARS-CoV-2 RdRp homogeneous assay kit (#78109) were supplied from BPS Bioscience.

Techniques: Generated, SDS Page, Staining, Purification, Column Chromatography, Binding Assay, Mutagenesis

Journal: International Journal of Biological Macromolecules

Article Title: Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

doi: 10.1016/j.ijbiomac.2022.12.251

Figure Lengend Snippet: PF 50 (MRJP2 with its isoform X1) neutralizes SARS-CoV-2 UK S protein (S UK )-mediated pseudovirus entry and structures models. (a, c) Surface and cartoon representations of trimeric SARS CoV-2 S UK (PDB ID: 7LWT , S1 “green “, RBD “dark red”, S2 “pale purple”) with multivalent interactions of three MRJP2 and MRJP2 X1 molecules (orange and yellow colored cartoons, respectively), followed by the human ACE2 (PDB ID: 1R42 “purple”). The two views of the docking model (a, c) are rotated by 180°. (b, d) The interacting amino acid residues (black) between SARS-CoV-2 S UK -RBD (only the matching residues with S UK RBD-ACE2 complex's interface pocket were presented) and the three molecules of (b) MRJP2 (orange) and one molecule of (d) MRJP2 X1 (yellow). The yellow-highlighted residues are those responsible for the high virulence and transmissibility of the UK SARS-CoV-2. The docked complex structure (a) was given by the PatchDock server ( https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php ) and was visualized by Discovery Studio 2020 Client software. (e) The binding affinity (ΔG) and other PDBePISA ( https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver ) results (hydrogen bonds “HB”, area, and residues number “ i N res ”) for the interfaces of the docked complexes obtained. The pseudotyped SARS-CoV-2 S UK was preincubated for 1 h with a three-fold serially diluted (f) PF 50 or (g) S1 neutralizing antibody (clone No. C-A11), and then the S UK /PF 50 mixture was incubated for 48 h with ACE2-HEK 293 T cells. A luciferase assay system was utilized to evaluate the neutralization potencies of tested proteins. The half-maximal inhibitory concentration (IC 50 ) values were determined using GraphPad Prism version 9.2.0 by fitting the luciferase activity from serially diluted PF 50 or antibody to a sigmoidal dose-response curve. (f) The cytotoxicity (red line) of PF 50 on the ACE2-HEK 293 T cells was done using the cell titer-Glo luminescent cell viability assay. Data ( n = 3) is represented as the mean ± S.E.

Article Snippet: In addition, Thaw Medium 1 (#60187, growth medium without puromycin), anti-S antibody (#101024; clone No. C-A11), luciferase (Luc) reagent (#60690), and SARS-CoV-2 RdRp homogeneous assay kit (#78109) were supplied from BPS Bioscience.

Techniques: Software, Binding Assay, Incubation, Luciferase, Neutralization, Concentration Assay, Activity Assay, Cell Viability Assay

Journal: International Journal of Biological Macromolecules

Article Title: Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

doi: 10.1016/j.ijbiomac.2022.12.251

Figure Lengend Snippet: PF 50 (MRJP2 with its isoform X1) neutralizes SARS-CoV-2 South African S protein (S Afr )-mediated pseudovirus entry and structures models. (a, c) Surface and cartoon representations of trimeric SARS CoV-2 S Afr (PDB ID: 7LYN , S1 “white”, RBD “brown”, S2 “turquoise”) with multivalent interactions of three PF 50 molecules (orange and yellow colored cartoons), followed by the human ACE2 (PDB ID: 1R42 “purple”). The two views of the docking model (a, c) are rotated by 180°. (b, d) The interacting amino acid residues (black) between SARS-CoV-2 S Afr -RBD (only the matching residues with S Afr RBD-ACE2 complex's interface pocket were presented) and the two molecules of (c) MRJP2 X1 (yellow) and one molecule of (b) MRJP2 (orange). The yellow-highlighted residue is one of the residues that are responsible for the high virulence and transmissibility of the South African SARS-CoV-2. The docked complex structure (a) was given by the PatchDock server ( https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php ) and was visualized by Discovery Studio 2020 Client software. (e) The binding affinity (ΔG) and other PDBePISA ( https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver ) results (hydrogen bonds “HB”, area, and residues number “ i N res ”) for the interfaces of the docked complexes obtained. (f) The heatmap clustered the number of RBD residues of S UK and S Afr that were bound to one, two, or three molecules of MRJP or three molecules of PF 50 . The pseudotyped SARS-CoV-2 S Afr was preincubated for 1 h with a three-fold serially diluted (g) PF 50 or (h) S1 neutralizing antibody (clone No. C-A11), and then the S Afr /PF 50 mixture was incubated for 48 h with ACE2-HEK 293 T cells. A luciferase assay system was utilized to evaluate the neutralization potencies of tested proteins. The half-maximal inhibitory concentration (IC 50 ) values were determined using GraphPad Prism version 9.2.0 by fitting the luciferase activity from serially diluted PF 50 or antibody to a sigmoidal dose-response curve. The cytotoxicity (red line) of PF 50 on the ACE2-HEK 293 T cells (g) was done using the cell titer-Glo luminescent cell viability assay. Data ( n = 3) is represented as the mean ± S.E.

Article Snippet: In addition, Thaw Medium 1 (#60187, growth medium without puromycin), anti-S antibody (#101024; clone No. C-A11), luciferase (Luc) reagent (#60690), and SARS-CoV-2 RdRp homogeneous assay kit (#78109) were supplied from BPS Bioscience.

Techniques: Software, Binding Assay, Incubation, Luciferase, Neutralization, Concentration Assay, Activity Assay, Cell Viability Assay

Journal: International Journal of Biological Macromolecules

Article Title: Major royal jelly proteins elicited suppression of SARS-CoV-2 entry and replication with halting lung injury

doi: 10.1016/j.ijbiomac.2022.12.251

Figure Lengend Snippet: PF 50 (MRJP2 with its isoform X1) potently inhibits the activity of SARS-CoV-2 RNA dependent RNA polymerase (RdRp) in vitro and structures models. (a, b) Dose-dependent inhibition of RdRp activity by PF 50 compared to 6-Chloropurine-ribose-5′-triphosphate (CPR—P), respectively. PF 50 was incubated with the purified RdRp (67 ng/μl) for 1 h, then the Alpha counts (the signal-to-noise ratio) that reflect the generation of the digoxigenin-labeled RNA duplex were measured. The half-maximal inhibitory concentration (IC 50 ) values were computed using GraphPad Prism version 9.2.0 by fitting the Alpha-screen intensity data from serially diluted PF 50 or CPR-P to a sigmoidal dose-response curve. Results are the mean ± S.E. of two independent experiments. (c) The complex structure of RdRp (green) and the NSP7 “deep blue”-NSP8 “light blue” dimeric protein (PDB ID: 7BV2 ). (d) Docking model of RdRp (PDB ID: 7BV2 ) with PF 50 (orange and yellow colored cartoons), followed by NSP7-NSP8 dimeric protein. The interacting residues in the docked complex are magnified and elucidate the binding of MRJP2 to the NSP8-binding sites on the RdRp (red residues). The docked complex of RdRp with PF 50 was given by the GRAMM-X ProteinProtein Docking Web Server ( http://vakser.compbio.ku.edu/resources/gramm/grammx/ ), while the docked complexes with NSP7-NSP8 dimeric complex were obtained by the PatchDock server ( https://bioinfo3d.cs.tau.ac.il/PatchDock/php.php ). All the docked complexes were visualized by Discovery Studio 2020 Client software. (e) The binding affinity (ΔG) and other PDBePISA ( https://www.ebi.ac.uk/msd-srv/prot_int/cgi-bin/piserver ) results (hydrogen bonds “HB”, area, and residues number “ i N res ”) for the interfaces of the docked complexes obtained.

Article Snippet: In addition, Thaw Medium 1 (#60187, growth medium without puromycin), anti-S antibody (#101024; clone No. C-A11), luciferase (Luc) reagent (#60690), and SARS-CoV-2 RdRp homogeneous assay kit (#78109) were supplied from BPS Bioscience.

Techniques: Activity Assay, In Vitro, Inhibition, Incubation, Purification, Labeling, Concentration Assay, Binding Assay, Software