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Article

Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction

by
Noémi Miltner
1,
Gergő Kalló
1,2,
Éva Csősz
1,2,
Márió Miczi
1,
Tibor Nagy
3,
Mohamed Mahdi
1,
János András Mótyán
1,* and
József Tőzsér
1,*
1
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary
2
Proteomics Core Facility, Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary
3
Department of Applied Chemistry, Faculty of Science and Technology, University of Debrecen, 4032 Debrecen, Hungary
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2023, 24(4), 3236; https://doi.org/10.3390/ijms24043236
Submission received: 23 January 2023 / Revised: 3 February 2023 / Accepted: 3 February 2023 / Published: 6 February 2023
(This article belongs to the Special Issue Newly Emerging or Re-emerging Viruses as a Potential Threat to Humans)
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Abstract

The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.
Keywords: coronavirus; SARS-CoV-2; main protease; Mpro; NetCorona; cleavage site identification; cleavage site prediction; host protein cleavage; specificity; two-dimensional gel electrophoresis coronavirus; SARS-CoV-2; main protease; Mpro; NetCorona; cleavage site identification; cleavage site prediction; host protein cleavage; specificity; two-dimensional gel electrophoresis
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MDPI and ACS Style

Miltner, N.; Kalló, G.; Csősz, É.; Miczi, M.; Nagy, T.; Mahdi, M.; Mótyán, J.A.; Tőzsér, J. Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction. Int. J. Mol. Sci. 2023, 24, 3236. https://doi.org/10.3390/ijms24043236

AMA Style

Miltner N, Kalló G, Csősz É, Miczi M, Nagy T, Mahdi M, Mótyán JA, Tőzsér J. Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction. International Journal of Molecular Sciences. 2023; 24(4):3236. https://doi.org/10.3390/ijms24043236

Chicago/Turabian Style

Miltner, Noémi, Gergő Kalló, Éva Csősz, Márió Miczi, Tibor Nagy, Mohamed Mahdi, János András Mótyán, and József Tőzsér. 2023. "Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction" International Journal of Molecular Sciences 24, no. 4: 3236. https://doi.org/10.3390/ijms24043236

APA Style

Miltner, N., Kalló, G., Csősz, É., Miczi, M., Nagy, T., Mahdi, M., Mótyán, J. A., & Tőzsér, J. (2023). Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction. International Journal of Molecular Sciences, 24(4), 3236. https://doi.org/10.3390/ijms24043236

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