Structural and functional analysis of bba03, borrelia burgdorferi competitive advantage promoting outer surface lipoprotein

Publisher Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. Copyright: Copyright 2020 Elsevier B.V., All rights reserved.BBA03 is a Borrelia burgdorferi outer surface lipoprotein encoded on one of the most conserved plasmids in Borrelia genome, linear plasmid 54 (lp54). Although many of its genes have been identified as contributing or essential for spirochete fitness in vivo, the majority of the proteins encoded on this plasmid have no known function and lack homologs in other organisms. In this paper, we report the solution NMR structure of the B. burgdorferi outer surface lipoprotein BBA03, which is known to provide a competitive advantage to the bacteria during the transmission from tick vector to mammalian host. BBA03 shows structural homology to other outer surface lipoproteins reflecting their genetic and evolutionary relatedness. Analysis of the structure reveals a pore in BBA03, which could potentially bind lipids.publishersversionPeer reviewe

Evaluation of Outer Surface Protein Vaccine Candidates of Borrelia burgdorferi for Lyme Disease

Publisher Copyright: © 2023 by the authors.Lyme disease affects several hundred thousand people worldwide annually, yet there is no registered vaccine for the disease available for human use. The disease is caused by Borrelia burgdorferi sensu lato complex bacteria, which harbor numerous outer surface proteins, and many of which have been targeted for vaccine development. However, to effectively combat various Borrelia species, the target protein should ideally be conserved and located in the chromosome. In this study, we evaluated the potential of seven conservative, chromosome-encoded outer surface proteins as vaccine candidates. Unfortunately, four of the initial candidates could not be produced in E. coli. The remaining BB0028, BB0158, and BB0689 proteins were administered to mice in both the free form and as conjugates with virus-like particles (VLPs). In most cases, high antibody titers were obtained, confirming the good immunogenicity of the selected proteins. However, for BB0158 and BB0689 proteins, adverse effects were observed following the injection of free proteins, which were not observed when they were coupled to VLPs. Bactericidity tests of the obtained antibodies suggested that none of the vaccine candidates could induce the production of bactericidal antibodies.Peer reviewe

Structural basis of epitope recognition by anti-alpha-synuclein antibodies MJFR14-6-4-2

Alpha-synuclein (α-syn) inclusions in the brain are hallmarks of so-called Lewy body diseases. Lewy bodies contain mainly aggregated α-syn together with some other proteins. Monomeric α-syn lacks a well-defined three-dimensional structure, but it can aggregate into oligomeric and fibrillar amyloid species, which can be detected using specific antibodies. Here we investigate the aggregate specificity of monoclonal MJFR14-6-4-2 antibodies. We conclude that partial masking of epitope in unstructured monomer in combination with a high local concentration of epitopes is the main reason for MJFR14-6-4-2 selectivity towards aggregates. Based on the structural insight, we produced mutant α-syn that when fibrillated is unable to bind MJFR14-6-4-2. Using these fibrils as a tool for seeding cellular α-syn aggregation, provides superior signal/noise ratio for detection of cellular α-syn aggregates by MJFR14-6-4-2. Our data provide a molecular level understanding of specific recognition of toxic amyloid oligomers, which is critical for the development of inhibitors against synucleinopathies

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

Production and characterization of novel ssRNA bacteriophage virus-like particles from metagenomic sequencing data

Abstract Background Protein shells assembled from viral coat proteins are an attractive platform for development of new vaccines and other tools such as targeted bioimaging and drug delivery agents. Virus-like particles (VLPs) derived from the single-stranded RNA (ssRNA) bacteriophage coat proteins (CPs) have been important and successful contenders in the area due to their simplicity and robustness. However, only a few different VLP types are available that put certain limitations on continued developments and expanded adaptation of ssRNA phage VLP technology. Metagenomic studies have been a rich source for discovering novel viral sequences, and in recent years have unraveled numerous ssRNA phage genomes significantly different from those known before. Here, we describe the use of ssRNA CP sequences found in metagenomic data to experimentally produce and characterize novel VLPs. Results Approximately 150 ssRNA phage CP sequences were sourced from metagenomic sequence data and grouped into 14 different clusters based on CP sequence similarity analysis. 110 CP-encoding sequences were obtained by gene synthesis and expressed in bacteria which in 80 cases resulted in VLP assembly. Production and purification of the VLPs was straightforward and compatible with established protocols, with the only exception that a considerable proportion of the CPs had to be produced at a lower temperature to ensure VLP assembly. The VLP morphology was similar to that of the previously studied phages, although a few deviations such as elongated or smaller particles were noted in certain cases. In addition, stabilizing inter-subunit disulfide bonds were detected in six VLPs and several possible candidate RNA structures in the phage genomes were identified that might bind to the coat protein and ensure specific RNA packaging. Conclusions Compared to the few types of ssRNA phage VLPs that were used before, several dozens of new particles representing ten distinct similarity groups are now available with a notable potential for biotechnological applications. It is believed that the novel VLPs described in this paper will provide the groundwork for future development of new vaccines and other applications based on ssRNA bacteriophage VLPs

Plasmepsin Inhibitory Activity and Structure-Guided Optimization of a Potent Hydroxyethylamine-Based Antimalarial Hit

Antimalarial hit 1SR (TCMDC-134674) identified in a GlaxoSmithKline cell based screening campaign was evaluated for inhibitory activity against the digestive vacuole plasmepsins (Plm I, II, and IV). It was found to be a potent Plm IV inhibitor with no selectivity over Cathepsin D. A cocrystal structure of 1SR bound to Plm II was solved, providing structural insight for the design of more potent and selective analogues. Structure-guided optimization led to the identification of structurally simplified analogues 17 and 18 as low nanomolar inhibitors of both, plasmepsin Plm IV activity and P. falciparum growth in erythrocytes

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.This work was supported by Goethe University (Corona funds), the DFG-funded CRC: “Molecular Principles of RNA-Based Regulation,” DFG infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611), the state of Hesse (BMRZ), the Fondazione CR Firenze (CERM), and the IWB-EFRE-program 20007375. This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant Agreement No. 871037. AS is supported by DFG Grant SCHL 2062/2-1 and by the JQYA at Goethe through project number 2019/AS01. Work in the lab of KV was supported by a CoRE grant from the University of New Hampshire. The FLI is a member of the Leibniz Association (WGL) and financially supported by the Federal Government of Germany and the State of Thuringia. Work in the lab of RM was supported by NIH (2R01EY021514) and NSF (DMR-2002837). BN-B was supported by theNSF GRFP.MCwas supported byNIH (R25 GM055246 MBRS IMSD), and MS-P was supported by the HHMI Gilliam Fellowship. Work in the labs of KJ and KT was supported by Latvian Council of Science Grant No. VPP-COVID 2020/1-0014. Work in the UPAT’s lab was supported by the INSPIRED (MIS 5002550) project, which is implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and cofinanced by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011- 285950–“SEE-DRUG” project (purchase of UPAT’s 700MHz NMR equipment). Work in the CM-G lab was supported by the Helmholtz society. Work in the lab of ABö was supported by the CNRS, the French National Research Agency (ANR, NMRSCoV2- ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), and the IR-RMN-THC Fr3050 CNRS. Work in the lab of BM was supported by the Swiss National Science Foundation (Grant number 200020_188711), the Günthard Stiftung für Physikalische Chemie, and the ETH Zurich. Work in the labs of ABö and BM was supported by a common grant from SNF (grant 31CA30_196256). This work was supported by the ETHZurich, the grant ETH40 18 1, and the grant Krebsliga KFS 4903 08 2019. Work in the lab of the IBS Grenoble was supported by the Agence Nationale de Recherche (France) RA-COVID SARS2NUCLEOPROTEIN and European Research Council Advanced Grant DynamicAssemblies. Work in the CA lab was supported by Patto per il Sud della Regione Siciliana–CheMISt grant (CUP G77B17000110001). Part of this work used the platforms of the Grenoble Instruct-ERIC center (ISBG; UMS 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-05-02) and GRAL, financed within the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE- 0003). Work at the UW-Madison was supported by grant numbers NSF MCB2031269 and NIH/NIAID AI123498. MM is a Ramón y Cajal Fellow of the Spanish AEI-Ministry of Science and Innovation (RYC2019-026574-I), and a “La Caixa” Foundation (ID 100010434) Junior Leader Fellow (LCR/BQ/PR19/11700003). Funded by project COV20/00764 fromthe Carlos III Institute of Health and the SpanishMinistry of Science and Innovation to MMand DVL. VDJ was supported by the Boehringer Ingelheim Fonds. Part of this work used the resources of the Italian Center of Instruct-ERIC at the CERM/ CIRMMP infrastructure, supported by the Italian Ministry for University and Research (FOE funding). CF was supported by the Stiftung Polytechnische Gesellschaft. Work in the lab of JH was supported by NSF (RAPID 2030601) and NIH (R01GM123249).Peer reviewe

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form