hm2264  (Hycult Biotech)

Journal: Frontiers in Immunology

Article Title: Complement-Coagulation Cross-talk: Factor H-mediated regulation of the Complement Classical Pathway activation by fibrin clots

doi: 10.3389/fimmu.2024.1368852

Figure Lengend Snippet: Detection of the membrane attack complex (MAC) on fibrin-coated wells (A) and in the supernatants of human serum incubated with the fibrin-coated wells (B) . (A) Serial dilutions of fresh human serum (1/10, 1/50, 1/250, 1/1,250, 1/6,250, 1/31,250, 1/156,250, 1/781,250, and 1/3,906,250) were incubated with ovalbumin antigen–antibody complex-, ovalbumin-, fibrinogen-, and fibrin-coated wells. After incubation, the supernatant from each well was removed and the well washed three times in PBS–0.1% Tween 20. MAC was detected in ovalbumin antigen–antibody complex-, ovalbumin-, fibrinogen-, and fibrin-coated wells with mouse anti-neo C9 antibody using an ELISA system. (B) MAC was also detected in the supernatants in a capture ELISA system with rabbit anti-C9 monoclonal antibody. To measure the nonspecific formation of MAC, serial dilutions of fresh human serum were incubated with blocked wells alone, and the MAC from each well and the supernatant were assayed in the same ELISA and c apture ELISA systems as above. These values were subtracted from each sample as a background. The means of three experiments are presented with standard deviations.

Article Snippet: In order to capture MAC in the supernatants, a capture antibody (rabbit anti-human C9 IgG) was purified from rabbit anti-human C9 antiserum using a HiTrap Protein G column (Sigma-Aldrich).

Techniques: Membrane, Incubation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Cryo-EM structure of the human G-protein coupled receptor 1 (GPR1) – Gi protein complex bound to the chemerin C-terminal nonapeptide

doi: 10.1101/2023.06.03.543554

Figure Lengend Snippet: (A) cAMP inhibition response of GPR1 and CMKLR1 stimulated by different concentrations of C9. (B) NanoBiT G protein dissociation response of GPR1 and CMKLR1 treated by different concentrations of C9. (C) Accumulation of IP-one upon treatment of different concentrations of C9 on GPR1 and CMKLR1. (D) NanoBiT β-arrestin recruitment of GPR1 and CMKLR1 upon stimulation of different concentration of C9. Data are shown as means ± SEM from three independent experiments.

Article Snippet: In this study, we report a cryo-EM structure of human GPR1-C9-Gi protein complex, providing structural evidence that GPR1 is able to couple to the Gi proteins.

Techniques: Inhibition, Concentration Assay

Journal: bioRxiv

Article Title: Cryo-EM structure of the human G-protein coupled receptor 1 (GPR1) – Gi protein complex bound to the chemerin C-terminal nonapeptide

doi: 10.1101/2023.06.03.543554

Figure Lengend Snippet: (A) Cryo-EM density map (left) and the structural model (right) of the GPR1-Gi-scFv16 complex bound to C9. (B) Cryo-EM density map (left) and the peptide backbone (right) of C9. (C) Overall structure of GPR1-C9 complex from side view (left) and key interaction residues (right). In the overall structure, the receptor (marine blue) is shown in cartoon and surface representation. The C9 peptide is shown in sticks with carbon in salmon orange. The residues of GPR1 within 4 Å from the C9 peptide (salmon orange licorice and ribbon) are shown in marine blue licorice. The hydrogen bonds are displayed as dashed lines. (D) Extracellular view of the overall structure (left) and polar interactions (right) of the GPR1-C9 complex. The residue numbering of GPR1 follows the Ballesteros–Weinstein nomenclature. (E) Schematic representation of interactions between GPR1 and C9 analyzed by LigPlot+ program. The stick representations of GPR1 and C9 are shown as orange and purple sticks, respectively.

Article Snippet: In this study, we report a cryo-EM structure of human GPR1-C9-Gi protein complex, providing structural evidence that GPR1 is able to couple to the Gi proteins.

Techniques: Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Cryo-EM structure of the human G-protein coupled receptor 1 (GPR1) – Gi protein complex bound to the chemerin C-terminal nonapeptide

doi: 10.1101/2023.06.03.543554

Figure Lengend Snippet: (A - D) cAMP response in HeLa cells transfected to express WT or mutant GPR1. Different concentrations of C9 are applied. (E - H) G protein dissociation in HEK293T cells co-transfected to express WT or mutant GPR1, Gαi1-LgBiT, Gβ1, and SmBiT-Gγ2. Different concentrations of C9 are applied. All data shown are means ± SEM from three independent experiments.

Article Snippet: In this study, we report a cryo-EM structure of human GPR1-C9-Gi protein complex, providing structural evidence that GPR1 is able to couple to the Gi proteins.

Techniques: Transfection, Mutagenesis

Journal: bioRxiv

Article Title: Cryo-EM structure of the human G-protein coupled receptor 1 (GPR1) – Gi protein complex bound to the chemerin C-terminal nonapeptide

doi: 10.1101/2023.06.03.543554

Figure Lengend Snippet: (A) The occupancy of all H-bonds between C9 and GPR1 observed in MD simulations. (B) Side view (upper panel) and top view (lower panel) of the distributions of functionally related residues around the C9 peptide.

Article Snippet: In this study, we report a cryo-EM structure of human GPR1-C9-Gi protein complex, providing structural evidence that GPR1 is able to couple to the Gi proteins.

Techniques:

Journal: bioRxiv

Article Title: Cryo-EM structure of the human G-protein coupled receptor 1 (GPR1) – Gi protein complex bound to the chemerin C-terminal nonapeptide

doi: 10.1101/2023.06.03.543554

Figure Lengend Snippet: (A) Intracellular view of the movement of GPR1 transmembrane helix 5, 6, and 7 (shown in marine blue) in comparison with inactive C5aR (PDB ID: 6C1R, shown in lime green). (B) Side close-up view of the D 3.49 -R 3.50 -Y 3.51 motif. A downward movement of Y 3.51 of GPR1 is highlighted by a red arrow. (C) Side close-up view of the “toggle switch”, W 6.48 and F 6.44 , an anti-clockwise rotation is highlighted for GPR1. (D) Rotamer conformational changes at the P 5.50 -I/V 3.40 -F 6.44 motif of GPR1 and C5aR, respectively. (E) Intracellular view of the movement of GPR1 transmembrane helix 5, 6, and 7 (shown in marine blue) in comparison with active CMKLR1 (PDB ID: 7YKD, shown in cyan). (F) Side close-up view of the D 3.49 -R 3.50 -Y 3.51 motif. A downward movement of H 3.50 of GPR1 is highlighted by a red arrow. (G) Side close-up view of the “toggle switch”, W 6.48 and F 6.44 , a clockwise rotation is highlighted for GPR1. (H) No significant conformational change at P 5.50 -I/V 3.40 -F 6.44 motif of GPR1 and CMKLR1, respectively.

Article Snippet: In this study, we report a cryo-EM structure of human GPR1-C9-Gi protein complex, providing structural evidence that GPR1 is able to couple to the Gi proteins.

Techniques:

Journal: bioRxiv

Article Title: Cryo-EM structure of the human G-protein coupled receptor 1 (GPR1) – Gi protein complex bound to the chemerin C-terminal nonapeptide

doi: 10.1101/2023.06.03.543554

Figure Lengend Snippet: (A) The interactions between the α5 helix of Gαi (pink) and GPR1 (marine blue) in the cavity at ICL3, TM5, TM6, and TM7 regions. (B) The interactions between Gβ subunit (yellow) and H8 of the receptor (marine blue). (C) Comparisons of the interactions between the α5 helix of Gαi and TM5, TM6, and ICL3 of several Gi-coupled receptors including GPR1 (marine blue), CMKLR1 (cyan, PDB ID: 7YKD), CCR5 (gray, PDB ID: 7F1R), C5aR (lime green, PDB ID: 6C1R) and CXCR4 (yellow, PDB ID: 3ODU). (D) 90°orientation of (C) for intracellular view showing the locations of ICL2, ICL1, and H8. (E) Same as (C) and (D) yet the interactions of the αN helix of Gαi with these receptors are compared.

Article Snippet: In this study, we report a cryo-EM structure of human GPR1-C9-Gi protein complex, providing structural evidence that GPR1 is able to couple to the Gi proteins.

Techniques:

Journal: bioRxiv

Article Title: Cryo-EM structure of the human G-protein coupled receptor 1 (GPR1) – Gi protein complex bound to the chemerin C-terminal nonapeptide

doi: 10.1101/2023.06.03.543554

Figure Lengend Snippet: (A - C) cAMP response in HeLa cells transfected to express WT or mutant GPR1. Different concentrations of C9 are applied. (D - F) G protein dissociation in HEK293T cells co-transfected to express WT or mutant GPR1, Gαi1-LgBiT, Gβ1, and SmBiT-Gγ2. Different concentrations of C9 are applied. All data shown are means ± SEM from three independent experiments.

Article Snippet: In this study, we report a cryo-EM structure of human GPR1-C9-Gi protein complex, providing structural evidence that GPR1 is able to couple to the Gi proteins.

Techniques: Transfection, Mutagenesis

Journal: bioRxiv

Article Title: In vivo evolution of a Klebsiella pneumoniae capsule defect promotes complement-mediated opsono-phagocytosis and persistence during recurrent infection

doi: 10.1101/2023.05.31.542722

Figure Lengend Snippet: (A) Mixing study of KPC-Kp clinical isolates KLP1 (genetic reference) and KLP7 (IS 5 insertions in mgrB and wcaJ ) after incubation for 4 hours in pooled serum from healthy volunteers and serum depleted of complement component C3. Groups include isolates grown in 85% vol/vol solution of sterile phosphate buffered saline (control; n=8 trials), healthy serum (NHS; n=8 trials), or sera depleted of complement component C3 (C3 depleted; n=7 trials). In select experiments, C3-depleted serum was also mixed 1:1 with healthy serum (n = 4 trials). Each point represents the median colony-forming units (CFU) per mL result from a single trial. (B-C) Direct ELISA of five serial ST258 KPC-Kp isolates to quantify binding of complement components C3 and C5b-C9 (membrane attack complex) after incubation with diluted pooled healthy serum for 30 minutes. The result of three separate trials quantifying fold change to the KLP1 reference isolate with median result are displayed. *p<0.05 by Kruskal-Wallis Test with Dunn’s post-hoc. (D-E) Flow cytometry histograms of D) C3b and E) MAC binding for KLP1, KLP6 ( mgrB insertional inactivation), and KLP7 ( mgrB and wcaJ insertional inactivation) after incubation for 30 minutes with diluted pooled healthy serum. (F) Immunogold transmission electron microscopy of KLP1 and KLP7 incubated for 30 minutes with diluted pooled healthy serum and primary antibody for C3 followed by colloidal gold secondary antibody. Representative images at 100,000X magnification are displayed.

Article Snippet: Membrane attack complex (MAC) binding was assessed by staining with rabbit anti-human C5b-C9 antibody (Catalog #204903, Sigma) at 1:1000 dilution at room temperature for 1 hour followed by washes × 3 then staining with goat anti-rabbit HRP-conjugated antibody (Catalog #204903, Sigma) at 1:5,000 dilution at room temperature for 1 hour.

Techniques: Incubation, Sterility, Saline, Control, Direct ELISA, Binding Assay, Membrane, Flow Cytometry, Transmission Assay, Electron Microscopy