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The sudden global emergence of SARS-CoV-2 urgently requires an in-depth understanding of molecular functions of viral proteins and their interactions with the host proteome. Several omics studies have extended our knowledge of COVID-19 pathophysiology, including some focused on proteomic aspects 1-3 . To understand how SARS-CoV-2 and related coronaviruses manipulate the host we here characterized interactome, proteome and signaling processes in a systems-wide manner. This identified connections between the corresponding cellular events, revealed functional effects of the individual viral proteins and put these findings into the context of host signaling pathways. We investigated the closely related SARS-CoV-2 and SARS-CoV viruses as well as the influence of SARS-CoV-2 on transcriptome, proteome, ubiquitinome and phosphoproteome of a lung-derived human cell line. Projecting these data onto the global network of cellular interactions revealed relationships between the perturbations taking place upon SARS-CoV-2 infection at different layers and identified unique and common molecular mechanisms of SARS coronaviruses. The results highlight the functionality of individual proteins as well as vulnerability hotspots of SARS-CoV-2, which we targeted with clinically approved drugs. We exemplify this by identification of kinase inhibitors as well as MMPase inhibitors with significant antiviral effects against SARS-CoV-2. Main text:To identify interactions of SARS-CoV-2 and SARS-CoV with cellular proteins, we transduced A549 lung carcinoma cells with lentiviruses expressing individual HA-tagged viral proteins (Figure 1a;Extended data Fig. 1a; Supplementary Table 1). Affinity purification followed by mass spectrometry analysis (AP-MS) and statistical modelling of the MS1-level quantitative data allowed identification of 1484 interactions between 1086 cellular proteins and 24 SARS-CoV-2 and 27 SARS-CoV bait proteins (Figure 1b; Extended data Fig. 1b; Supplementary Table 2). The resulting virus-host interaction network revealed a wide range of cellular activities intercepted by SARS-CoV-2 and SARS-CoV (Figure 1b; Extended data Table 1; Supplementary Table 2). In particular, we discovered Extended data Figure 1 | Expression of viral proteins in transduced A549 cells induces changes to the host proteome. (a) Expression of HA-tagged viral proteins, in stably transduced A549 cells, used in AP-MS and proteome expression measurements. (b) The extended version of the virus-host protein-protein interaction network with 24 SARS-CoV-2 and 27 SARS-CoV proteins, as well as ORF3 of HCoV-NL63 and ORF4 and 4a of HCoV-229E, used as baits. Host targets regulated upon viral protein overexpression or SARS-CoV-2 infection (based on the analysis of all data of this study) are highlighted (see the in-plot legend). (c-f) Co-precipitation experiments in HEK 293T cells showing a specific enrichment of (c) endogenous MAVS co-precipitated with c-term HA-tagged ORF7b of SARS-CoV-2 and SARS-CoV (negative controls: SARS-CoV-2 ORF6-HA, ORF7a-HA), (d) ORF7b-H...

Highlights d Genetics, biochemistry, proteomics, and cryo-EM define GID E3 ligase regulation d Carbon stress induces assembly of an inactive anticipatory GID Ant complex d Environmental perturbations trigger substrate receptor assembly into active GID E3s d Structural model of N-degron substrate ubiquitylation by multisubunit RING-RING E3

Summary How are E3 ubiquitin ligases configured to match substrate quaternary structures? Here, by studying the yeast GID complex (mutation of which causes deficiency in glucose-induced degradation of gluconeogenic enzymes), we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryoelectron microscopy (cryo-EM) structures show that, to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two minimally functional GID E3s assemble into the 20-protein Chelator-GID SR4 , which resembles an organometallic supramolecular chelate. The Chelator-GID SR4 assembly avidly binds multiple Fbp1 degrons so that multiple Fbp1 protomers are simultaneously ubiquitylated at lysines near the allosteric and substrate binding sites. Importantly, key structural and biochemical features, including capacity for supramolecular assembly, are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical, and cell biological data, we propose that higher-order E3 ligase assembly generally enables multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. Here, we develop a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identifies 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells – double the number and quantitative accuracy of data dependent acquisition. Applied to TNF signaling, the workflow comprehensively captures known sites while adding many novel ones. An in-depth, systems-wide investigation of ubiquitination across the circadian cycle uncovers hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting new connections between metabolism and circadian regulation.