Protein interactome of the deamidase phosphoribosylformylglycinamidine synthetase (PFAS) by LC-MS/MS.

Phosphoribosylformylglycinamidine synthase (PFAS) is an essential enzyme in de novo synthesis of purine. Previously, PFAS has been reported to modulate RIG-I activation during viral infection via deamidation. In this study, we sought to identify potential substrates that PFAS can deamidate. Flag-PFAS was transfected into HEK-293T cells and PFAS associated proteins were purified with anti-Flag M2 magnetic beads. PFAS associated proteins were identified using mass spectrometry and were analyzed using bioinformatics tools including KEGG pathway analysis, gene ontology annotation, and protein interaction network analysis. A total of 441 proteins is suggested to potentially interact with PFAS. Of this number, 12 were previously identified and 429 are newly identified. The interactions of PFAS with CAD, CCT2, PRDX1, and PHGDH were confirmed by co-immunoprecipitation and western blotting. This study is first to report the interaction of PFAS with several proteins which play physiological roles in tumor development including CAD, CCT2, PRDX1, and PHGDH. Furthermore, we show here that PFAS is able to deamidate PHGDH, and induce other posttranslational modification into CAD, CCT2 and PRDX1. The present data provide insight on the biological function of PFAS. Further study to explore the role of these protein interactions in tumorigenesis and other diseases is recommended.

Epigallocatechin-3-gallate, an active ingredient of Traditional Chinese Medicines, inhibits the 3CLpro activity of SARS-CoV-2.

SARS-CoV-2 is the etiological agent responsible for the ongoing pandemic of coronavirus disease 2019 (COVID-19). The main protease of SARS-CoV-2, 3CLpro, is an attractive target for antiviral inhibitors due to its indispensable role in viral replication and gene expression of viral proteins. The search of compounds that can effectively inhibit the crucial activity of 3CLpro, which results to interference of the virus life cycle, is now widely pursued. Here, we report that epigallocatechin-3-gallate (EGCG), an active ingredient of Chinese herbal medicine (CHM), is a potent inhibitor of 3CLpro with half-maximum inhibitory concentration (IC50) of 0.874 ± 0.005 µM. In the study, we retrospectively analyzed the clinical data of 123 cases of COVID-19 patients, and found three effective Traditional Chinese Medicines (TCM) prescriptions. Multiple strategies were performed to screen potent inhibitors of SARS-CoV-2 3CLpro from the active ingredients of TCMs, including network pharmacology, molecular docking, surface plasmon resonance (SPR) binding assay and fluorescence resonance energy transfer (FRET)-based inhibition assay. The SPR assay showed good interaction between EGCG and 3CLpro with KD ~6.17 µM, suggesting a relatively high affinity of EGCG with SARS-CoV-2 3CLpro. Our results provide critical insights into the mechanism of action of EGCG as a potential therapeutic agent against COVID-19.