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1.
Elife ; 112022 07 04.
Article in English | MEDLINE | ID: covidwho-1924603

ABSTRACT

T cells play a critical role in the adaptive immune response, recognizing peptide antigens presented on the cell surface by major histocompatibility complex (MHC) proteins. While assessing peptides for MHC binding is an important component of probing these interactions, traditional assays for testing peptides of interest for MHC binding are limited in throughput. Here, we present a yeast display-based platform for assessing the binding of tens of thousands of user-defined peptides in a high-throughput manner. We apply this approach to assess a tiled library covering the SARS-CoV-2 proteome and four dengue virus serotypes for binding to human class II MHCs, including HLA-DR401, -DR402, and -DR404. While the peptide datasets show broad agreement with previously described MHC-binding motifs, they additionally reveal experimentally validated computational false positives and false negatives. We therefore present this approach as able to complement current experimental datasets and computational predictions. Further, our yeast display approach underlines design considerations for epitope identification experiments and serves as a framework for examining relationships between viral conservation and MHC binding, which can be used to identify potentially high-interest peptide binders from viral proteins. These results demonstrate the utility of our approach to determine peptide-MHC binding interactions in a manner that can supplement and potentially enhance current algorithm-based approaches.


Subject(s)
COVID-19 , Saccharomyces cerevisiae , Humans , Peptides/metabolism , Protein Binding , Proteome/metabolism , SARS-CoV-2 , Saccharomyces cerevisiae/metabolism
2.
Elife ; 112022 Jun 20.
Article in English | MEDLINE | ID: covidwho-1903839

ABSTRACT

With the continual evolution of new strains of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that are more virulent, transmissible, and able to evade current vaccines, there is an urgent need for effective anti-viral drugs. The SARS-CoV-2 main protease (Mpro) is a leading target for drug design due to its conserved and indispensable role in the viral life cycle. Drugs targeting Mpro appear promising but will elicit selection pressure for resistance. To understand resistance potential in Mpro, we performed a comprehensive mutational scan of the protease that analyzed the function of all possible single amino acid changes. We developed three separate high throughput assays of Mpro function in yeast, based on either the ability of Mpro variants to cleave at a defined cut-site or on the toxicity of their expression to yeast. We used deep sequencing to quantify the functional effects of each variant in each screen. The protein fitness landscapes from all three screens were strongly correlated, indicating that they captured the biophysical properties critical to Mpro function. The fitness landscapes revealed a non-active site location on the surface that is extremely sensitive to mutation, making it a favorable location to target with inhibitors. In addition, we found a network of critical amino acids that physically bridge the two active sites of the Mpro dimer. The clinical variants of Mpro were predominantly functional in our screens, indicating that Mpro is under strong selection pressure in the human population. Our results provide predictions of mutations that will be readily accessible to Mpro evolution and that are likely to contribute to drug resistance. This complete mutational guide of Mpro can be used in the design of inhibitors with reduced potential of evolving viral resistance.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , COVID-19/drug therapy , Cysteine Endopeptidases/metabolism , Humans , Protease Inhibitors , SARS-CoV-2/genetics , Saccharomyces cerevisiae/metabolism , Viral Nonstructural Proteins/metabolism
3.
Eur J Cell Biol ; 101(2): 151222, 2022 Apr.
Article in English | MEDLINE | ID: covidwho-1881962

ABSTRACT

Clathrin-mediated endocytosis (CME) is the major route through which cells internalise various substances and recycle membrane components. Via the coordinated action of many proteins, the membrane bends and invaginates to form a vesicle that buds off-along with its contents-into the cell. The contribution of the actin cytoskeleton to this highly dynamic process in mammalian cells is not well understood. Unlike in yeast, where there is a strict requirement for actin in CME, the significance of the actin cytoskeleton to mammalian CME is variable. However, a growing number of studies have established the actin cytoskeleton as a core component of mammalian CME, and our understanding of its contribution has been increasing at a rapid pace. In this review, we summarise the state-of-the-art regarding our understanding of the endocytic cytoskeleton, its physiological significance, and the questions that remain to be answered.


Subject(s)
Actin Cytoskeleton , Clathrin , Actin Cytoskeleton/metabolism , Actins/metabolism , Animals , Cell Membrane/metabolism , Clathrin/metabolism , Cytoskeleton/metabolism , Endocytosis/physiology , Mammals/metabolism , Saccharomyces cerevisiae/metabolism
4.
Front Biosci (Landmark Ed) ; 27(3): 93, 2022 03 09.
Article in English | MEDLINE | ID: covidwho-1766334

ABSTRACT

BACKGROUND: Inhibition of human topoisomerase I (TOP1) by camptothecin and topotecan has been shown to reduce excessive transcription of PAMP (Pathogen-Associated Molecular Pattern)-induced genes in prior studies, preventing death from sepsis in animal models of bacterial and SARS-CoV-2 infections. The TOP1 catalytic activity likely resolves the topological constraints on DNA that encodes these genes to facilitate the transcription induction that leads to excess inflammation. The increased accumulation of TOP1-DNA covalent complex (TOP1cc) following DNA cleavage is the basis for the anticancer efficacy of the TOP1 poisons developed for anticancer treatment. The potential cytotoxicity and mutagenicity of TOP1 targeting cancer drugs pose serious concerns for employing them as therapies in sepsis prevention. METHODS: In this study we set up a novel yeast-based screening system that employs yeast strains expressing wild-type or a dominant lethal mutant recombinant human TOP1. The effect of test compounds on growth is monitored with and without overexpression of the recombinant human TOP1. RESULTS: This yeast-based screening system can identify human TOP1 poisons for anticancer efficacy as well as TOP1 suppressors that can inhibit TOP1 DNA binding or cleavage activity in steps prior to the formation of the TOP1cc. CONCLUSIONS: This yeast-based screening system can distinguish between TOP1 suppressors and TOP1 poisons. The assay can also identify compounds that are likely to be cytotoxic based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.


Subject(s)
COVID-19 , Poisons , Animals , DNA Topoisomerases, Type I/chemistry , DNA Topoisomerases, Type I/genetics , DNA Topoisomerases, Type I/metabolism , Humans , SARS-CoV-2 , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
5.
Protein Eng Des Sel ; 352022 02 17.
Article in English | MEDLINE | ID: covidwho-1692165

ABSTRACT

Understanding how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with different mammalian angiotensin-converting enzyme II (ACE2) cell entry receptors elucidates determinants of virus transmission and facilitates development of vaccines for humans and animals. Yeast display-based directed evolution identified conserved ACE2 mutations that increase spike binding across multiple species. Gln42Leu increased ACE2-spike binding for human and four of four other mammalian ACE2s; Leu79Ile had an effect for human and three of three mammalian ACE2s. These residues are highly represented, 83% for Gln42 and 56% for Leu79, among mammalian ACE2s. The above findings can be important in protecting humans and animals from existing and future SARS-CoV-2 variants.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Animals , Humans , Mutation , Protein Binding , Saccharomyces cerevisiae/metabolism , Spike Glycoprotein, Coronavirus/genetics
6.
Protein Expr Purif ; 190: 106003, 2022 02.
Article in English | MEDLINE | ID: covidwho-1474960

ABSTRACT

SARS-CoV-2 protein subunit vaccines are currently being evaluated by multiple manufacturers to address the global vaccine equity gap, and need for low-cost, easy to scale, safe, and effective COVID-19 vaccines. In this paper, we report on the generation of the receptor-binding domain RBD203-N1 yeast expression construct, which produces a recombinant protein capable of eliciting a robust immune response and protection in mice against SARS-CoV-2 challenge infections. The RBD203-N1 antigen was expressed in the yeast Pichia pastoris X33. After fermentation at the 5 L scale, the protein was purified by hydrophobic interaction chromatography followed by anion exchange chromatography. The purified protein was characterized biophysically and biochemically, and after its formulation, the immunogenicity was evaluated in mice. Sera were evaluated for their efficacy using a SARS-CoV-2 pseudovirus assay. The RBD203-N1 protein was expressed with a yield of 492.9 ± 3.0 mg/L of fermentation supernatant. A two-step purification process produced a >96% pure protein with a recovery rate of 55 ± 3% (total yield of purified protein: 270.5 ± 13.2 mg/L fermentation supernatant). The protein was characterized to be a homogeneous monomer that showed a well-defined secondary structure, was thermally stable, antigenic, and when adjuvanted on Alhydrogel in the presence of CpG it was immunogenic and induced high levels of neutralizing antibodies against SARS-CoV-2 pseudovirus. The characteristics of the RBD203-N1 protein-based vaccine show that this candidate is another well suited RBD-based construct for technology transfer to manufacturing entities and feasibility of transition into the clinic to evaluate its immunogenicity and safety in humans.


Subject(s)
COVID-19 Vaccines , Gene Expression , SARS-CoV-2 , Spike Glycoprotein, Coronavirus , Animals , COVID-19 Vaccines/chemistry , COVID-19 Vaccines/genetics , COVID-19 Vaccines/pharmacology , Humans , Mice , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/pharmacology
7.
STAR Protoc ; 2(4): 100869, 2021 12 17.
Article in English | MEDLINE | ID: covidwho-1433914

ABSTRACT

Here, we describe a protocol to identify escape mutants on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) receptor-binding domain (RBD) using a yeast screen combined with deep mutational scanning. Over 90% of all potential single S RBD escape mutants can be identified for monoclonal antibodies that directly compete with angiotensin-converting enzyme 2 for binding. Six to 10 antibodies can be assessed in parallel. This approach has been shown to determine escape mutants that are consistent with more laborious SARS-CoV-2 pseudoneutralization assays. For complete details on the use and execution of this protocol, please refer to Francino-Urdaniz et al. (2021).


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , DNA Mutational Analysis/methods , Mutation , SARS-CoV-2/genetics , Saccharomyces cerevisiae/metabolism , Spike Glycoprotein, Coronavirus/genetics , Angiotensin-Converting Enzyme 2/metabolism , Binding Sites , COVID-19/metabolism , COVID-19/virology , Humans , Saccharomyces cerevisiae/genetics , Spike Glycoprotein, Coronavirus/metabolism
8.
Nat Commun ; 12(1): 4918, 2021 08 13.
Article in English | MEDLINE | ID: covidwho-1397870

ABSTRACT

Ribosomal RNA genes (rDNA) are highly unstable and susceptible to rearrangement due to their repetitive nature and active transcriptional status. Sequestration of rDNA in the nucleolus suppresses uncontrolled recombination. However, broken repeats must be first released to the nucleoplasm to allow repair by homologous recombination. Nucleolar release of broken rDNA repeats is conserved from yeast to humans, but the underlying molecular mechanisms are currently unknown. Here we show that DNA damage induces phosphorylation of the CLIP-cohibin complex, releasing membrane-tethered rDNA from the nucleolus in Saccharomyces cerevisiae. Downstream of phosphorylation, SUMOylation of CLIP-cohibin is recognized by Ufd1 via its SUMO-interacting motif, which targets the complex for disassembly through the Cdc48/p97 chaperone. Consistent with a conserved mechanism, UFD1L depletion in human cells impairs rDNA release. The dynamic and regulated assembly and disassembly of the rDNA-tethering complex is therefore a key determinant of nucleolar rDNA release and genome integrity.


Subject(s)
Cell Nucleolus/genetics , DNA Repair , DNA, Ribosomal/genetics , Saccharomyces cerevisiae Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/genetics , Valosin Containing Protein/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Nucleolus/metabolism , DNA Damage , DNA, Ribosomal/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Phosphorylation , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Small Ubiquitin-Related Modifier Proteins/metabolism , Sumoylation , Two-Hybrid System Techniques , Valosin Containing Protein/metabolism
9.
Cell Mol Immunol ; 18(8): 1847-1860, 2021 08.
Article in English | MEDLINE | ID: covidwho-1387308

ABSTRACT

CD4+ T cells orchestrate adaptive immune responses via binding of antigens to their receptors through specific peptide/MHC-II complexes. To study these responses, it is essential to identify protein-derived MHC-II peptide ligands that constitute epitopes for T cell recognition. However, generating cells expressing single MHC-II alleles and isolating these proteins for use in peptide elution or binding studies is time consuming. Here, we express human MHC alleles (HLA-DR4 and HLA-DQ6) as native, noncovalent αß dimers on yeast cells for direct flow cytometry-based screening of peptide ligands from selected antigens. We demonstrate rapid, accurate identification of DQ6 ligands from pre-pro-hypocretin, a narcolepsy-related immunogenic target. We also identify 20 DR4-binding SARS-CoV-2 spike peptides homologous to SARS-CoV-1 epitopes, and one spike peptide overlapping with the reported SARS-CoV-2 epitope recognized by CD4+ T cells from unexposed individuals carrying DR4 subtypes. Our method is optimized for immediate application upon the emergence of novel pathogens.


Subject(s)
CD4-Positive T-Lymphocytes/metabolism , COVID-19/metabolism , Epitopes, T-Lymphocyte/metabolism , HLA-DQ Antigens/metabolism , HLA-DR4 Antigen/metabolism , Saccharomyces cerevisiae/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Two-Hybrid System Techniques , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/virology , COVID-19/genetics , COVID-19/immunology , Epitopes, T-Lymphocyte/genetics , Epitopes, T-Lymphocyte/immunology , Flow Cytometry , HLA-DQ Antigens/genetics , HLA-DQ Antigens/immunology , HLA-DR4 Antigen/genetics , HLA-DR4 Antigen/immunology , Ligands , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/immunology
10.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1294968

ABSTRACT

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Subject(s)
COVID-19/virology , Peptide Library , SARS-CoV-2/metabolism , Viral Proteins/metabolism , A549 Cells , Cell Line , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Host Microbial Interactions/physiology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Time-Lapse Imaging , Viral Proteins/genetics
11.
PLoS One ; 16(1): e0244885, 2021.
Article in English | MEDLINE | ID: covidwho-1251754

ABSTRACT

Human influenza virus infections occur annually worldwide and are associated with high morbidity and mortality. Hence, development of novel anti-influenza drugs is urgently required. Rice Power® extract developed by the Yushin Brewer Co. Ltd. is a novel aqueous extract of rice obtained via saccharization and fermentation with various microorganisms, such as Aspergillus oryzae, yeast [such as Saccharomyces cerevisiae], and lactic acid bacteria, possessing various biological and pharmacological properties. In our previous experimental screening with thirty types of Rice Power® extracts, we observed that the 30th Rice Power® (Y30) extract promoted the survival of influenza A virus-infected Madin-Darby canine kidney (MDCK) cells. Therefore, to identify compounds for the development of novel anti-influenza drugs, we aimed to investigate whether the Y30 extract exhibits anti-influenza A virus activity. In the present study, we demonstrated that the Y30 extract strongly promoted the survival of influenza A H1N1 Puerto Rico 8/34 (A/PR/8/34), California 7/09, or H3N2 Aichi 2/68 (A/Aichi/2/68) viruses-infected MDCK cells and inhibited A/PR/8/34 or A/Aichi/2/68 viruses infection and growth in the co-treatment and pre-infection experiments. The pre-treatment of Y30 extract on MDCK cells did not induce anti-influenza activity in the cell. The Y30 extract did not significantly affect influenza A virus hemagglutination, and neuraminidase and RNA-dependent RNA polymerase activities. Interestingly, the electron microscopy experiment revealed that the Y30 extract disrupts the integrity of influenza A virus particles by permeabilizing the viral membrane envelope, suggesting that Y30 extract has a direct virucidal effect against influenza A virus. Furthermore, we observed that compared to the ethyl acetate (EtOAc) extract, the water extract of Y30 extract considerably promoted the survival of cells infected with A/PR/8/34 virus. These results indicated that more anti-influenza components were present in the water extract of Y30 extract than in the EtOAc extract. Our results highlight the potential of a rice extract fermented with A. oryzae and S. cerevisiae as an anti-influenza medicine and a drug source for the development of anti-influenza compounds.


Subject(s)
Aspergillus oryzae/metabolism , Influenza A virus/drug effects , Oryza/chemistry , Oryza/microbiology , Plant Extracts/pharmacology , Saccharomyces cerevisiae/metabolism , Water/chemistry , Acetates/chemistry , Animals , Antiviral Agents/pharmacology , Dogs , Fermentation , Influenza A virus/growth & development , Influenza A virus/physiology , Madin Darby Canine Kidney Cells , Microbial Viability/drug effects
12.
Nat Chem Biol ; 17(10): 1057-1064, 2021 10.
Article in English | MEDLINE | ID: covidwho-1281726

ABSTRACT

The predominant approach for antibody generation remains animal immunization, which can yield exceptionally selective and potent antibody clones owing to the powerful evolutionary process of somatic hypermutation. However, animal immunization is inherently slow, not always accessible and poorly compatible with many antigens. Here, we describe 'autonomous hypermutation yeast surface display' (AHEAD), a synthetic recombinant antibody generation technology that imitates somatic hypermutation inside engineered yeast. By encoding antibody fragments on an error-prone orthogonal DNA replication system, surface-displayed antibody repertoires continuously mutate through simple cycles of yeast culturing and enrichment for antigen binding to produce high-affinity clones in as little as two weeks. We applied AHEAD to generate potent nanobodies against the SARS-CoV-2 S glycoprotein, a G-protein-coupled receptor and other targets, offering a template for streamlined antibody generation at large.


Subject(s)
Antibody Formation/immunology , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Antibodies/immunology , Antigens , COVID-19/immunology , Humans , Peptide Library , Recombinant Proteins/metabolism , SARS-CoV-2/immunology , SARS-CoV-2/pathogenicity , Saccharomyces cerevisiae/metabolism , Single-Domain Antibodies/genetics , Single-Domain Antibodies/metabolism , Spike Glycoprotein, Coronavirus/immunology
13.
Curr Genet ; 67(5): 755-759, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1216214

ABSTRACT

With the current COVID-19 pandemic, we all realized how important interactions are. Interactions are everywhere. At the cellular level, protein interactions play a key role and their ensemble, also called interactome, is often referred as the basic building blocks of life. Given its importance, the maintenance of the integrity of the interactome is a real challenge in the cell. Many events during evolution can disrupt interactomes and potentially result in different characteristics for the organisms. However, the molecular underpinnings of changes in interactions at the cellular level are still largely unexplored. Among the perturbations, hybridization puts in contact two different interactomes, which may lead to many changes in the protein interaction network of the hybrid, including gains and losses of interactions. We recently investigated the fate of the interactomes after hybridization between yeast species using a comparative proteomics approach. A large-scale conservation of the interactions was observed in hybrids, but we also noticed the presence of proteostasis-related changes. This suggests that, despite a general robustness, small differences may accumulate in hybrids and perturb their protein physiology. Here, we summarize our work with a broader perspective on the importance of interactions.


Subject(s)
Fungal Proteins/metabolism , Hybridization, Genetic , Protein Interaction Maps , Saccharomyces cerevisiae/metabolism , Saccharomyces/metabolism , Animals , Fungal Proteins/chemistry , Fungal Proteins/genetics , Proteomics , Saccharomyces/chemistry , Saccharomyces/classification , Saccharomyces/genetics , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/genetics
14.
Nat Biotechnol ; 39(7): 846-854, 2021 07.
Article in English | MEDLINE | ID: covidwho-1152861

ABSTRACT

Accurate quantification of the proteome remains challenging for large sample series and longitudinal experiments. We report a data-independent acquisition method, Scanning SWATH, that accelerates mass spectrometric (MS) duty cycles, yielding quantitative proteomes in combination with short gradients and high-flow (800 µl min-1) chromatography. Exploiting a continuous movement of the precursor isolation window to assign precursor masses to tandem mass spectrometry (MS/MS) fragment traces, Scanning SWATH increases precursor identifications by ~70% compared to conventional data-independent acquisition (DIA) methods on 0.5-5-min chromatographic gradients. We demonstrate the application of ultra-fast proteomics in drug mode-of-action screening and plasma proteomics. Scanning SWATH proteomes capture the mode of action of fungistatic azoles and statins. Moreover, we confirm 43 and identify 11 new plasma proteome biomarkers of COVID-19 severity, advancing patient classification and biomarker discovery. Thus, our results demonstrate a substantial acceleration and increased depth in fast proteomic experiments that facilitate proteomic drug screens and clinical studies.


Subject(s)
Proteomics/methods , Tandem Mass Spectrometry , Arabidopsis/metabolism , Biomarkers/metabolism , COVID-19/blood , COVID-19/diagnosis , Cell Line , Humans , Peptides/analysis , Proteome/analysis , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Severity of Illness Index
15.
Biol Cell ; 113(7): 311-328, 2021 Jul.
Article in English | MEDLINE | ID: covidwho-1119222

ABSTRACT

BACKGROUND INFORMATION: Comprehensive libraries of plasmids for SARS-CoV-2 proteins with various tags (e.g., Strep, HA, Turbo) are now available. They enable the identification of numerous potential protein-protein interactions between the SARS-CoV-2 virus and host proteins. RESULTS: We present here a large library of SARS CoV-2 protein constructs fused with green and red fluorescent proteins and their initial characterisation in various human cell lines including lung epithelial cell models (A549, BEAS-2B), as well as in budding yeast. The localisation of a few SARS-CoV-2 proteins matches their proposed interactions with host proteins. These include the localisation of Nsp13 to the centrosome, Orf3a to late endosomes and Orf9b to mitochondria. CONCLUSIONS AND SIGNIFICANCE: This library should facilitate further cellular investigations, notably by imaging techniques.


Subject(s)
COVID-19/virology , Peptide Library , SARS-CoV-2/metabolism , Viral Proteins/metabolism , A549 Cells , Cell Line , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Host Microbial Interactions/physiology , Humans , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Protein Interaction Domains and Motifs , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Time-Lapse Imaging , Viral Proteins/genetics
16.
Int J Mol Sci ; 21(19)2020 Sep 28.
Article in English | MEDLINE | ID: covidwho-963280

ABSTRACT

Some years inspire more hindsight reflection and future-gazing than others. This is even more so in 2020 with its evocation of perfect vision and the landmark ring to it. However, no futurist can reliably predict what the world will look like the next time that a year's first two digits will match the second two digits-a numerical pattern that only occurs once in a century. As we leap into a new decade, amid uncertainties triggered by unforeseen global events-such as the outbreak of a worldwide pandemic, the accompanying economic hardship, and intensifying geopolitical tensions-it is important to note the blistering pace of 21st century technological developments indicate that while hindsight might be 20/20, foresight is 50/50. The history of science shows us that imaginative ideas, research excellence, and collaborative innovation can, for example, significantly contribute to the economic, cultural, social, and environmental recovery of a post-COVID-19 world. This article reflects on a history of yeast research to indicate the potential that arises from advances in science, and how this can contribute to the ongoing recovery and development of human society. Future breakthroughs in synthetic genomics are likely to unlock new avenues of impactful discoveries and solutions to some of the world's greatest challenges.


Subject(s)
Disease Outbreaks/prevention & control , Genetic Engineering/methods , Genome, Fungal , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Synthetic Biology/methods , Saccharomyces cerevisiae/classification
17.
Sci Rep ; 10(1): 17090, 2020 10 13.
Article in English | MEDLINE | ID: covidwho-867590

ABSTRACT

The triterpene oil squalene is an essential component of nanoemulsion vaccine adjuvants. It is most notably in the MF59 adjuvant, a component in some seasonal influenza vaccines, in stockpiled, emulsion-based adjuvanted pandemic influenza vaccines, and with demonstrated efficacy for vaccines to other pandemic viruses, such as SARS-CoV-2. Squalene has historically been harvested from shark liver oil, which is undesirable for a variety of reasons. In this study, we have demonstrated the use of a Synthetic Biology (yeast) production platform to generate squalene and novel triterpene oils, all of which are equally as efficacious as vaccine adjuvants based on physiochemical properties and immunomodulating activities in a mouse model. These Synthetic Biology adjuvants also elicited similar IgG1, IgG2a, and total IgG levels compared to marine and commercial controls when formulated with common quadrivalent influenza antigens. Injection site morphology and serum cytokine levels did not suggest any reactogenic effects of the yeast-derived squalene or novel triterpenes, suggesting their safety in adjuvant formulations. These results support the advantages of yeast produced triterpene oils to include completely controlled growth conditions, just-in-time and scalable production, and the capacity to produce novel triterpenes beyond squalene.


Subject(s)
Adjuvants, Immunologic/chemistry , Influenza Vaccines/immunology , Triterpenes/chemistry , Animals , Antibodies, Viral/blood , Betacoronavirus/isolation & purification , COVID-19 , Coronavirus Infections/prevention & control , Coronavirus Infections/virology , Cytokines/blood , Immunoglobulin G/blood , Influenza Vaccines/chemistry , Mice , Mice, Inbred BALB C , Nanoparticles/chemistry , Orthomyxoviridae Infections/pathology , Orthomyxoviridae Infections/prevention & control , Orthomyxoviridae Infections/virology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Pneumonia, Viral/virology , SARS-CoV-2 , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae/metabolism , Synthetic Biology/methods
18.
Nature ; 585(7826): 614-619, 2020 09.
Article in English | MEDLINE | ID: covidwho-744380

ABSTRACT

Tropane alkaloids from nightshade plants are neurotransmitter inhibitors that are used for treating neuromuscular disorders and are classified as essential medicines by the World Health Organization1,2. Challenges in global supplies have resulted in frequent shortages of these drugs3,4. Further vulnerabilities in supply chains have been revealed by events such as the Australian wildfires5 and the COVID-19 pandemic6. Rapidly deployable production strategies that are robust to environmental and socioeconomic upheaval7,8 are needed. Here we engineered baker's yeast to produce the medicinal alkaloids hyoscyamine and scopolamine, starting from simple sugars and amino acids. We combined functional genomics to identify a missing pathway enzyme, protein engineering to enable the functional expression of an acyltransferase via trafficking to the vacuole, heterologous transporters to facilitate intracellular routing, and strain optimization to improve titres. Our integrated system positions more than twenty proteins adapted from yeast, bacteria, plants and animals across six sub-cellular locations to recapitulate the spatial organization of tropane alkaloid biosynthesis in plants. Microbial biosynthesis platforms can facilitate the discovery of tropane alkaloid derivatives as new therapeutic agents for neurological disease and, once scaled, enable robust and agile supply of these essential medicines.


Subject(s)
Alkaloids/biosynthesis , Alkaloids/supply & distribution , Hyoscyamine/biosynthesis , Saccharomyces cerevisiae/metabolism , Scopolamine/metabolism , Acyltransferases/genetics , Acyltransferases/metabolism , Animals , Atropa belladonna/enzymology , Atropine Derivatives/metabolism , Biological Transport , Datura/enzymology , Glucosides/biosynthesis , Glucosides/metabolism , Hyoscyamine/supply & distribution , Lactates/metabolism , Ligases/genetics , Ligases/metabolism , Models, Molecular , Nervous System Diseases/drug therapy , Oxidoreductases/genetics , Oxidoreductases/metabolism , Protein Engineering , Saccharomyces cerevisiae/genetics , Scopolamine/supply & distribution , Vacuoles/metabolism
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