Journal Article Epublish
PubMed: 40565292 DOI: 10.3390/ijms26125829
Identification of SARS-CoV-2 Main Protease Cleavage Sites in Bovine β-Casein.
Int J Mol Sci 26(12)
Authors: János András Mótyán, Tibor Nagy, Ágota Nagyné Veres, Mária Golda, Mohamed Mahdi, József Tőzsér
Affiliations
- Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
- Department of Applied Chemistry, Faculty of Sciences and Technology, University of Debrecen, 4032 Debrecen, Hungary.
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease of 2019 (COVID-19) and has persistently caused infections since its emergence in late 2019. The main protease (Mpro) of SARS-CoV-2 plays a crucial role in its life-cycle; thus, it is an important target for drug development. One of the first virus-specific drugs that has been approved for the treatment of COVID-19 patients is Paxlovid, which contains nirmatrelvir, a covalent inhibitor of Mpro. Screening of inhibitor candidates and specificity studies also rely on efficient substrates and activity assays. Casein is one of the most commonly applied universal substrates that can be used to study a wide range of proteases, including SARS-CoV-2 Mpro. Casein is a known substrate for Mpro in vitro, but the specific casein isoform cleaved by Mpro remained unidentified, and the cleavage sites have yet to be determined. This work studied cleavage of α-, β- and κ-isoforms of bovine casein by SARS-CoV-2 Mpro, using in vitro and in silico approaches. The candidate cleavage sites were predicted in silico based on the protein sequences, and the cleavage positions were identified based on mass spectrometric analysis of cleavage fragments. Based on our results, only β-casein contains cleavage sites for Mpro and thus can be used as its substrate in vitro. The newly identified cleavage site sequences further widen the knowledge about the specificity of SARS-CoV-2 Mpro.
MeSH
Coronavirus, Sars-Cov-2, Pharmaceutical Preparations, Protein Isoforms, Viruses, Peptide Hydrolases, Severe Acute Respiratory Syndrome-Related Coronavirus, Caseins