Journal: International Journal of Molecular Sciences

Article Title: Structural and Functional Analysis of Female Sex Hormones against SARS-CoV-2 Cell Entry

doi: 10.3390/ijms222111508

Figure Lengend Snippet: Estrogen effects on ACE2 structural energy. ( A ) 3D representation of the human ACE2 glycosylated residues and key regions used by the SARS-CoV-2 S protein to mediate entry into cells. S-RBD-binding sites are colored in dark blue and glycans in purple. ( B ) 3D molecular interactions between ACE2 and 17β-diol (magenta) or S-equol (orange) molecules obtained by 100 ns of molecular dynamics simulations (MDS). ( C ) A plain representation of solvated ACE2-helix α1 and α2 substructures by estrogen molecules. ( D ) FEL maps represent the conformational energy of helix α1 and α2 substructures with estrogen molecules during MDS (last 20 ns of MDS). The energy scale ranges from 12 kJ/mol (red) to 0 kJ/mol (blue). ( E ) Immunofluorescence microscopy analysis on HUVECs cells treated with or without conjugated 17β-diol (E2-Glow), shows colocalization (yellow) between ACE2 (green) and 17β-diol (red).

Article Snippet: Estrogen treatment : HUVECs at 60–70% confluence were supplemented with opti-MEM 24 h before treatment with complete growing media containing 17β-diol (or E2-Glow from Jena Bioscience cat# PR-958S) at a concentration of 3 nM or S-equol at a concentration of 10 nM or Tunicamycin at 0.05 μM for 24 h. Fresh media containing rS-RBD (10 μg) was supplied the next day.

Techniques: Binding Assay, Immunofluorescence, Microscopy, Analysis, Docking Assay

Journal: International Journal of Molecular Sciences

Article Title: Structural and Functional Analysis of Female Sex Hormones against SARS-CoV-2 Cell Entry

doi: 10.3390/ijms222111508

Figure Lengend Snippet: Estrogens bind to ACE2 glycans to promote its internalization. ( A ) Glycan–estrogen interactions stabilize ACE2 Glycan-residues at E57, N53, K341, and V339 (red color). ( B ) MEP maps show the electrostatic impact of estrogen molecules on the surface of ACE2 glycans. The energy scale ranges from −0.075 μa (red) to 0.075 μa (blue). ( C ) Immunofluorescence staining of human ACE2 (magenta) and the lysosome marker LAMP1 (green), shows loss of ACE2 membrane levels in HUVECs treated with 17β-diol or S-equol compared with the control group (DMSO). ( D ) Immunoblot shows decreased levels of total ACE2 protein which associates with increased endocytosis activity as evidenced by immunoblot for LC3b and LAMP1. ( E ) Histologic analysis of wild-type mouse lungs after 48 h of intratracheal instillation with 17β-diol or S-equol shows loss of Ace2 signal (red) on the membrane of alveoli cells. Estrogen-treated lungs show greater Ace2-Lamp1 colocalization (white arrows) indicating internalization of the receptor. ( F ) Immunoblot shows decreased levels of total and glycosylated Ace2 proteins in estrogen-treated lungs from male mice. Quantification of protein levels of three replicate experiments is shown. Student’s t -test, 2 tails. Bar graphs are presented as mean with error bars (±SD).

Article Snippet: Estrogen treatment : HUVECs at 60–70% confluence were supplemented with opti-MEM 24 h before treatment with complete growing media containing 17β-diol (or E2-Glow from Jena Bioscience cat# PR-958S) at a concentration of 3 nM or S-equol at a concentration of 10 nM or Tunicamycin at 0.05 μM for 24 h. Fresh media containing rS-RBD (10 μg) was supplied the next day.

Techniques: Glycoproteomics, Immunofluorescence, Staining, Marker, Membrane, Control, Western Blot, Activity Assay, Analysis, Docking Assay

Journal: International Journal of Molecular Sciences

Article Title: Structural and Functional Analysis of Female Sex Hormones against SARS-CoV-2 Cell Entry

doi: 10.3390/ijms222111508

Figure Lengend Snippet: Estrogen’s impact on ACE2 and S-RBD interactions. ( A ) Top view of the 3D ACE2 surface interacting with 5 top-scored S-RBDs (top 1—blue, 2—red, 3—orange, 4—purple, and top 5—yellow). S-RBDs were scored based on shape complementarity principles. ( B ) Heatmap of atomic contact energy between ACE2 and 57 S-RBDs shows spontaneous energy structures from most favorable (green) to less favorable S-RBD structures (red). Energy scale ranging from 500 Kcal/mol to −500 Kcal/mol. ( C ) Immunoblot of isolated proteins from cultured HUVECs shows a 90% inhibition of S-RBD entry into cells in estrogen-treated cells. Quantification of protein levels of three replicate experiments is shown. Student’s T -test, 2 tails. Bar graphs are presented as mean with error bars (±SD).

Article Snippet: Estrogen treatment : HUVECs at 60–70% confluence were supplemented with opti-MEM 24 h before treatment with complete growing media containing 17β-diol (or E2-Glow from Jena Bioscience cat# PR-958S) at a concentration of 3 nM or S-equol at a concentration of 10 nM or Tunicamycin at 0.05 μM for 24 h. Fresh media containing rS-RBD (10 μg) was supplied the next day.

Techniques: Western Blot, Isolation, Cell Culture, Inhibition

Journal: International Journal of Molecular Sciences

Article Title: Structural and Functional Analysis of Female Sex Hormones against SARS-CoV-2 Cell Entry

doi: 10.3390/ijms222111508

Figure Lengend Snippet: Estrogens block SARS-CoV-2 S protein uptake in the respiratory tract in vivo. ( A , B ) Immunofluorescence analysis of S-RBD entry into HUVECs pretreated estrogens (17β-diol or S-equol) or Tunicamycin (glycosylation inhibitor) followed by treatment with 10μg/mL of recombinant S-RBD (red) demonstrate that all 3 treatments cells reduced entry of S-RBD entry into cells via a reduction in Ace2 internalization as shown by colocalization with LAMP1 (green) and immunoblot. ( C ) ELISA-based binding assay using lung protein lysates from male mice (K18-ACE2) treated with 17β-diol (0.3 μM) or S-equol (1μM) or Tunicamycin (1 mg/Kg) shows reduced SARS-CoV-2 S protein affinity for the Ace2 receptor. ( D ) Immunoblot shows the glycosylation levels of Ace2 from total lung lysate in mice treated with estrogens or Tunicamycin. ( E ) Immunofluorescence of Ace2 (green) and RBD (magenta) in lungs from lysates from male mice (K18-ACE2) treated with estrogens or Tunicamycin.

Article Snippet: Estrogen treatment : HUVECs at 60–70% confluence were supplemented with opti-MEM 24 h before treatment with complete growing media containing 17β-diol (or E2-Glow from Jena Bioscience cat# PR-958S) at a concentration of 3 nM or S-equol at a concentration of 10 nM or Tunicamycin at 0.05 μM for 24 h. Fresh media containing rS-RBD (10 μg) was supplied the next day.

Techniques: Blocking Assay, In Vivo, Immunofluorescence, Analysis, Glycoproteomics, Recombinant, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay