A BioID-derived proximity interactome for SARS-CoV-2 proteins.
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ABSTRACT:
SUBMITTER: May DG
PROVIDER: S-EPMC8475972 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here, the cellular impact of expressing SARS-CoV-2 viral proteins was studied by global proteomic analysis, and proximity biotinylation (BioID) was used to map the SARS-CoV-2 virus-host interactome in human lung cancer-derived cells. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are associated with SARS-CoV-2 proteins. We have established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Furthermore, we identified 66 high-confidence interactions by comparing this study with previous reports, providing a strong foundation for future follow-up studies. Finally, we cross-referenced candidate interactors with the CLUE drug library to identify potential therapeutics for drug-repurposing efforts. Collectively, these studies provide a valuable resource to uncover novel SARS-CoV-2 biology and inform development of antivirals.
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Project description:The novel coronavirus SARS-CoV-2 is responsible for the ongoing COVID-19 pandemic and has caused a major health and economic burden worldwide. Understanding how SARS-CoV-2 viral proteins behave in host cells can reveal underlying mechanisms of pathogenesis and assist in development of antiviral therapies. Here the cellular impact of expressing SARS-CoV-2 viral proteins was studied by global proteomic analysis and proximity biotinylation (BioID) was used to map the SARS-CoV-2 virus-host interactome in human lung cancer derived cells. Functional enrichment analyses revealed previously reported and unreported cellular pathways that are associated with SARS-CoV-2 proteins. We have also established a website to host the proteomic data to allow for public access and continued analysis of host-viral protein associations and whole-cell proteomes of cells expressing the viral-BioID fusion proteins. Furthermore, we identified 154 high-confidence interactions by comparing this study with previous reports, providing a strong foundation for future follow-up studies. Finally, we cross-referenced candidate interactors with the CLUE drug library to identify potential therapeutics for drug-repurposing efforts. Collectively, these studies provide a valuable resource to uncover novel SARS-CoV-2 biology and inform development of antivirals.
2021-10-18 | MSV000088245 | MassIVE
Project description:Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1.
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Project description:Lysine specific demethylase 1 (LSD1) regulates gene expression as part of the CoREST complex, along with co-repressor of REST (CoREST) and histone deacetylase 1 (HDAC1). CoREST is recruited to specific genomic loci by core components and numerous transient interactions with chromatin-associated factors and transcription factors. We hypothesise that many of these weaker and transient associations may be difficult to identify using traditional co-immunoprecipitation methods. We have therefore employed proximity-dependent biotin-identification (BioID) with four different members of the CoREST complex, in three different cell types, to identify a comprehensive network of LSD1/CoREST associated proteins. In HEK293T cells, we identified 302 CoREST-associated proteins. Among this group were 16 of 18 known CoREST components and numerous novel associations, including readers (CHD3, 4, 6, 7 and 8), writers (KMT2B and KMT2D) and erasers (KDM2B) of histone methylation. However, components of other HDAC1 containing complexes (e.g. Sin3) were largely absent. To examine the dynamic nature of the CoREST interactome in a primary cell type, we replaced endogenous LSD1 with BirA*-LSD1 in embryonic stem (ES) cells and performed BioID in pluripotent, early- and late-differentiating environments. We identified 156 LSD1-associated proteins of which 67 were constitutively associated across all three time-points (43%), including novel associations with the MMB and ChAHP complexes, implying that the majority of interactors are both dynamic and cell type dependent. In total, we have performed 16 independent BioID experiments for LSD1 in three different cell types, producing a definitive network of LSD1-assoicated proteins that should provide a major resource for the field.
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Project description:Proximity dependent biotin identification (BioID) has emerged as a powerful tool for studies of proteome architecture, including insoluble or membrane-associated proteins. The technique has been well established in mammalian cells but has yet to be applied to whole plant systems. Here we demonstrate the application of BioID on leaf tissues of the model plant Arabidopsis thaliana, thereby expanding the versatility of this important technique and providing a powerful proteomics tool for plant biologists.
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Project description:World over, people are looking for solutions to tackle the pandemic coronavirus disease (COVID-19) caused by the virus SARS-CoV-2/nCoV-19. Notable contributions in biomedical field have been characterizing viral genomes, host transcriptomes and proteomes, repurposable drugs and vaccines. In one such study, 332 human proteins targeted by nCoV19 were identified. We expanded this set of host proteins by constructing their protein interactome, including in it not only the known protein-protein interactions (PPIs) but also novel, hitherto unknown PPIs predicted with our High-precision Protein-Protein Interaction Prediction (HiPPIP) model that was shown to be highly accurate. In fact, one of the earliest discoveries made possible by HiPPIP is related to activation of immunity upon viral infection. We found that several interactors of the host proteins are differentially expressed upon viral infection, are related to highly relevant pathways, and that the novel interaction of NUP98 with CHMP5 may activate an antiviral mechanism leading to disruption of viral budding. We are making the interactions available as downloadable files to facilitate future systems biology studies and also on a web-server at http://hagrid.dbmi.pitt.edu/corona that allows not only keyword search but also queries such as "PPIs where one protein is associated with 'virus' and the interactors with 'pulmonary'".
| S-EPMC7336710 | biostudies-literature
Project description:The BioID method uses a promiscuous biotin ligase to detect protein-protein associations as well as proximate proteins in living cells. Here we report improvements to the BioID method centered on BioID2, a substantially smaller promiscuous biotin ligase. BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, and exhibits enhanced labeling of proximate proteins. Thus BioID2 improves the efficiency of screening for protein-protein associations. We also demonstrate that the biotinylation range of BioID2 can be considerably modulated using flexible linkers, thus enabling application-specific adjustment of the biotin-labeling radius.
| S-EPMC4831873 | biostudies-literature
Project description:SARS-CoV-2 has a higher chance of progression in adults of any age with certain underlying health conditions or comorbidities like cancer, neurological diseases and in certain cases may even lead to death. Like other viruses, SARS-CoV-2 also interacts with host proteins to pave its entry into host cells. Therefore, to understand the behaviour of SARS-CoV-2 and design of effective antiviral drugs, host-virus protein-protein interactions (PPIs) can be very useful. In this regard, we have initially created a human-SARS-CoV-2 PPI database from existing works in the literature which has resulted in 7085 unique PPIs. Subsequently, we have identified at most 10 proteins with highest degrees viz. hub proteins from interacting human proteins for individual virus protein. The identification of these hub proteins is important as they are connected to most of the other human proteins. Consequently, when they get affected, the potential diseases are triggered in the corresponding pathways, thereby leading to comorbidities. Furthermore, the biological significance of the identified hub proteins is shown using KEGG pathway and GO enrichment analysis. KEGG pathway analysis is also essential for identifying the pathways leading to comorbidities. Among others, SARS-CoV-2 proteins viz. NSP2, NSP5, Envelope and ORF10 interacting with human hub proteins like COX4I1, COX5A, COX5B, NDUFS1, CANX, HSP90AA1 and TP53 lead to comorbidities. Such comorbidities are Alzheimer, Parkinson, Huntington, HTLV-1 infection, prostate cancer and viral carcinogenesis. Subsequently, using Enrichr tool possible repurposable drugs which target the human hub proteins are reported in this paper as well. Therefore, this work provides a consolidated study for human-SARS-CoV-2 protein interactions to understand the relationship between comorbidity and hub proteins so that it may pave the way for the development of anti-viral drugs.
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Project description:SARS-CoV-2 is an RNA virus whose success as a pathogen relies on its abilities to repurpose host RNA-binding proteins (RBPs) and to evade antiviral RBPs. To uncover the SARS-CoV-2 RNA interactome, we here develop a robust ribonucleoprotein (RNP) capture protocol and identify 109 host factors that directly bind to SARS-CoV-2 RNAs. Applying RNP capture on another coronavirus, HCoV-OC43, revealed evolutionarily conserved interactions between coronaviral RNAs and host proteins. Transcriptome analyses and knockdown experiments delineated 17 antiviral RBPs, including ZC3HAV1, TRIM25, PARP12, and SHFL, and 8 proviral RBPs, such as EIF3D and CSDE1, which are responsible for co-opting multiple steps of the mRNA life cycle. This also led to the identification of LARP1, a downstream target of the mTOR signaling pathway, as an antiviral host factor that interacts with the SARS-CoV-2 RNAs. Overall, this study provides a comprehensive list of RBPs regulating coronaviral replication and opens new avenues for therapeutic interventions.
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Project description:BioID is a well-established method for identifying protein-protein interactions and has been utilized within live cells and several animal models. However, the conventional labeling period requires 15-18 h for robust biotinylation which may not be ideal for some applications. Recently, two new ligases termed TurboID and miniTurbo were developed using directed evolution of the BioID ligase and were able to produce robust biotinylation following a 10 min incubation with excess biotin. However, there is reported concern about inducibility of biotinylation, cellular toxicity, and ligase stability. To further investigate the practical applications of TurboID and ascertain strengths and weaknesses compared to BioID, we developed several stable cell lines expressing BioID and TurboID fusion proteins and analyzed them via immunoblot, immunofluorescence, and biotin-affinity purification-based proteomics. For TurboID we observed signs of protein instability, persistent biotinylation in the absence of exogenous biotin, and an increase in the practical labeling radius. However, TurboID enabled robust biotinylation in the endoplasmic reticulum lumen compared to BioID. Induction of biotinylation could be achieved by combining doxycycline-inducible expression with growth in biotin depleted culture media. These studies should help inform investigators utilizing BioID-based methods as to the appropriate ligase and experimental protocol for their particular needs.
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