Protein-Peptide Interactions in Endothelial Cell Von Willebrand Factor Secretion and Exocytosis

Background: Previous results (Bae et al., FASEB J, 2017) and those of others (Andreeva et al., JBC 2005) showed compelling evidence that the interaction between G protein Gα12 and α-SNAP (α-soluble N-ethylmaleimide-sensitive factor attachment protein) is required in membrane trafficking and exocytosis in many biological processes and important human diseases. The peptide derived from Gα12 (Gα12 N10-15) inhibited the secretion of von Willebrand factor from human pulmonary artery endothelial cells and in a septic mouse model (Bae et al., FASEB J, 2019). The peptides (Gα12 N10-15 and N6-20) directly bind to α-SNAP (Bae et al., ATVB 2020). However, the peptide substrate basis underlying the Gα12/α-SNAP interaction in a specific cellular response has not been investigated yet. Our recent work reported analyses of potential protein-protein interaction networks in exocytosis and von Willebrand factor secretion using the STRING database (Bae and Salinas, JPET 2024). Here, we report the heterogeneous expression and release of von Willebrand factor from human endothelial cells, peptide substrates in Gα12 that interact with α-SNAP, and peptide properties that characterize protein-peptide interactions using bioinformatics tools.

Methods: Human pulmonary artery endothelial cells from the same passage number were confluent, serum-starved, and challenged by thrombin. Von Willebrand factor expression from the cells were observed by confocal laser scanning microscopy. The protein data bank structures of Gα12 and α-SNAP proteins were obtained from the AlphaFold database (Jumper et al., Nature 2021). The HPEPDOCK 2.0 Server (Zhou et al., Nucleic Acids Res 2018) was employed for protein-peptide docking with α-SNAP as the receptor and Gα12 N1-30 peptide as the ligand. The HDOCK server (Yan et al., Nat Protoc 2020) was then used to search for other potential binding sites between α-SNAP (the input receptor) and Gα12 (the input ligand). The ligand interface residues of the top 10 models were ranked according to the distance between receptor and ligand. These peptide sequences were again docked to the α-SNAP using the HPEPDOCK 2.0 server. The docking scores and protein data bank files of the top 10 models were then obtained for each α-SNAP/Gα12 peptide pair. Based on the results, a sequence logo was constructed for the ten peptide sequences using the WebLogo server to show the frequency of an amino acid at each position (Crooks et al., Genome Res 2004).

Results: The expression and release of von Willebrand factor from human pulmonary artery endothelial cells were heterogeneous. From the HPEPDOCK 2.0 Server, the protein data bank files of top 10 clusters of α-SNAP and Gα12 N1-30 peptide were obtained according to the docking scores and used as the positive control. From the HDOCK server, the top 10 models were obtained according to docking scores. The amino acid residue with the least distance was taken as the starting amino acid of the peptide candidate. A 30-amino acid string was taken downstream from the starting amino acid to create peptide sequences of the Gα12 protein. Analysis of Gα12 N1-30 peptide binding to the α-SNAP protein resulted in docking scores from -187.8 to -164.7. Searching for other potential binding sites between Gα12 and α-SNAP proteins using HDOCK presented a range of docking scores from -244.9 to -222.8. The sequence logo of the Gα12 peptide sequences was prepared to show a preference for amino acids with basic side chains at neutral pH at the starting position. Arginine was reported as the most frequent amino acid at the N terminus, and leucine was the most frequent at the C terminus.

Conclusion: Our results demonstrate that human pulmonary artery endothelial cells show heterogenous expression and release of von Willebrand factor. The more negative docking score connotes a more possible receptor-ligand binding model. The docking scores of the top 10 predictions are all more negative than that of experimentally-verified dataset, indicating tighter bindings than a positive control. Hence, these predicted models provide a set of peptide substrates to investigate cellular responses from Gα12/α-SNAP interactions and may serve as a platform to investigate specific cellular responses including von Willebrand factor secretion.

No relevant conflicts of interest to declare.