Mechanistic Insights into COVID-19 by Global Analysis of the SARS-CoV-2 3CLpro Substrate Degradome. Pablos et al.

Published: 8 October 2021| Version 2 | DOI: 10.17632/b97d5nrb72.2
Contributor:
Christopher Overall

Description

This data comprises all the supplementary tables from the paper published in Cell Reports titled: "Mechanistic Insights into COVID-19 by Global Analysis of the SARS-CoV-2 3CLpro Substrate Degradome", by Isabel Pablos, I., Machado, Y., Ramos de Jesus, H.C., Mohamud, Y., Kappelhoff, R., Lindskog, C., Vlok, M., Bell, P.A., Butler, G.S., Grin, P.M., Cao, Q.T., Nguyen, J.P., Solis, N., Abbina, S., Rut, W., Vederas, J.C., Szekely, L., Szakos, A., Drag, M., Kizhakkedathu, J.N., Mossman, K., Hirota, J.A., Jan, E., Luo, H., Banerjee, A., Overall, C.M. Each table contains a legend and a brief experimental explanation of the proteomics and bioinformatics methods used to generate the data published in the Cell Reports paper. In this paper, Pablos et al delineate the human protein substrate landscape of SARS-CoV-2 main protease, 3CLpro, by TAILS substrate-targeted N-terminomics. We identify >100 substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLpro engaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. Cleaving the interactors of essential effector proteins, effectively stranding them from their binding partners, amplifies the consequences of proteolysis. We show that 3CLpro targets the Hippo pathway, including inactivation of MAP4K5, and key effectors of transcription, mRNA processing, and translation. We demonstrate that Spike glycoprotein directly binds galectin-8, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple antiviral autophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike healthy lung cells. The 3CLpro substrate degradome establishes a foundational substrate atlas to accelerate exploration of SARS-CoV-2 pathology and drug design. TAILS and MS/MS To induce interferon-stimulated gene proteins that may be 3CLpro substrates, HEK-293 and BEAS-2B cells were cultured in DMEM/F12 with 10% (v/v) FBS. BEAS-2B cells were treated with 10,000 U/ml carrier-free IFN-α2a or IFN-β1a or medium (control) for 18 h. Cells were harvested, and lysates were prepared under native conditions taking the necessary steps to reduce any cellular proteolytic activity. All steps were performed on ice. Protease inhibitor cocktail and 5 mM N-ethylmaleimide were added to the cell pellet in hypotonic lysis buffer and cells then pushed through a 27-gauge needle for 10 cycles and rested on ice for 1 h with agitations every 10 min. Cell lysates were flash-frozen in liquid N2 and stored at –80°C. 3CLpro or inactive 3CLpro-C145A (control) at 2.5 µM were incubated with 500 µg native cell protein, 37°C for 18 h (see paper: Figure 1A, panel a). The incubated samples were then analyzed by Terminal Amine Isotopic Labeling of Substrates (TAILS) and preTAILS shotgun proteomics (see paper: Figure 1 and Methods) using a modified protocol from (Kleifeld et al., 2011).

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All the primary data obtained in this study is well described in the STAR Methods section of the paper.

Institutions

The University of British Columbia

Categories

Proteomics, Western Blot, Protein-Protein Interaction Networks

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