Presented by

Dr. Christopher Overall

Transcriptomics identifies mRNA levels, but not protein levels—yet whereas proteomics identifies protein levels, these do not necessarily reflect the biological activity of the protein. Protein N and C terminal peptides provide critical information on protein function and stability. Our terminomics methods (TAILS, PICS) enrich and annotate N- and C-terminomes, and our N- and C-termini database TopFIND v 4.1 (https://topfind.clip.msl.ubc.ca) reveal widespread truncation and generation of termini in normal and diseased tissues and the associated change in protein functions. In the pandemic, we pivoted to understanding the pathogenesis of COVID-19 by identifying the SARS-CoV-2 3CLpro main protease human protein substrate repertoire. 3CLpro, the main viral protease, is indispensable for SARS-CoV-2 replication.

We delineate the interconnected human protein substrate landscape of 3CLpro using TAILS analysis of human lung and kidney cells treated or not with interferons (N = 12), supported by analyses of SARS-CoV-2-infected human lung cells. Over 100 substrate discoveries were identified and validated by MALDI-TOF analysis of synthetic peptide cleavage kinetics for each cleavage site in >100 substrates. Molecular docking simulations of 3CLpro engaging substrates confirmed that ~10% of validated sites were noncanonical. PPI analysis shows that cleavage by 3CLpro of the interactors of essential effector proteins effectively stranding these from their binding partners, and so amplifies the consequences of proteolysis throughout the cell by protein complex disassembly. Using recombinant protein digestion, Edman degradation, and digestion of normal human bronchial epithelial cell from 5 subjects, we further confirmed substrate cleavages. We show that 3CLpro targets multiple proteins in the Hippo pathway, including the inactivation of MAP4K5, as well as key effectors of transcription, mRNA processing and translation. We discovered that the SARS-CoV-2 spike protein directly binds galectin-8, the cleavage of which disengages CALCOCO2/NDP52 and decouples protective anti-viral-autophagy. Indeed, unlike healthy lung, in post-mortem COVID-19 human lung samples obtained from the Human Protein Atlas, NDP-52 rarely colocalizes with galectin-8. Thus, great mechanistic insight into novel mechanisms of evasion of antiviral host cell immunity, hijacking of the host mRNA binding and translation apparatus, and disruptions in cell shape and syncytium formation was enabled by rationale start points identified in the 3CLpro substrate degradome. The Atlas of CoV-2 substrates establishes a foundational resource to accelerate further exploration of SARS-CoV-2 pathology in the COVID-19 cellular coup d’etat.