Current work in the Berger-Schaffitzel lab focuses on the ribosome, translational control mechanisms, in particular nonsense-mediated mRNA decay (NMD), and ribosome display.

Nonsense-mediated mRNA decay (NMD)

Nonsense-mediated mRNA decay (NMD) represents a key surveillance mechanism to quality control the expression of normal and aberrant mRNAs in eukaryotes. NMD recognizes and degrades transcripts with premature termination codons (PTCs). These can be introduced into the mRNA by mutations, transcriptional errors, and aberrant splicing, but are also present in 5-20% of normal transcripts. By degrading PTC-containing mRNAs, NMD limits the production of potentially harmful, C-terminally truncated proteins. NMD thus represents one of the main quality control mechanisms of the eukaryotic cell. In addition, NMD is an important regulator of overall eukaryotic gene expression.

Translation is a prerequisite for the recognition of a PTC-containing mRNA. Despite years of intense research, the molecular mechanisms which allows discriminating a PTC from a normal termination codon, the key step in NMD, remains poorly understood. A set of conserved NMD effectors has been identified: the UP-Frameshift factors UPF1, UPF2, and UPF3B and the SMG proteins. Our team studies the function and structure of these NMD factors using biochemistry, biophysics and structural biology, supported by a Wellcome Trust Investigator award, a Marie Sklodowska-Curie Fellowship to Mirella Vivoli-Vega, (CSC) and SWBio DTP scholarships.

Selected recent publications:

1. Bufton, J.C., Powers, K.T., Szeto, J.A., Toelzer, C., Berger, I. & Schaffitzel, C.* (2022) Structures of nonsense-mediated mRNA decay factors UPF3B and UPF3A in complex with UPF2 reveal molecular basis for competitive binding and for neurodevelopmental disorder-causing mutation. Nucleic Acids Res. 50, 5934-5947. https://doi.org/10.1093/nar/gkac421.
2. Mailliot, J., Vivoli-Vega, M. & Schaffitzel, C.* (2022) No-nonsense: insights into the functional interplay of nonsense-mediated mRNA decay factors. J. 479, 973-993. doi: 10.1042/BCJ20210556
3. Powers, K.T., Stevenson-Jones, F.. Yadav, S.K.N., Amthor, B., Bufton, J., Borucu, U., Shen, D. , Becker, J., Larysh D., Hentze, M., Kulozik, A., Neu-Yilik, G. & Schaffitzel, C.* (2021) Blasticidin S inhibits peptide bond formation and release in mammalia and modulates translation dynamics at premature termination codons. Nucleic Acids Res. 49, 7665-7679. https://doi.org/10.1093/nar/gkab532
4. Powers, K.T., Szeto, J.A. & Schaffitzel C.* (2020) New insights into no-go, non-stop and nonsense-mediated mRNA decay complexes. Opin. Struct. Biol. 65, 110-118. doi: 10.1016/j.sbi.2020.06.011. https://sci-hub.se/10.1016/j.sbi.2020.06.011
5. Eliseev, B., Yeramala, L., Leitner, A., Karuppasamy, M., Raimondeau, E., Huard, K., Alkalaeva, E., Aebersold, & Schaffitzel, C.* (2018) Structure of human cap-dependent 48S translation pre-initiation complex. Nucleic Acids Res. 46, 2678–2689. https://doi.org/10.1093/nar/gky054
6. Neu-Yilik, G., Raimondeau, E., Eliseev, B., Yeramala, L., Amthor, B., Deniaud, A., Huard, K., Kerschgens, K., Hentze, M.W.*, Schaffitzel, C.* & Kulozik, A.E.* (2017) Dual function of UPF3B in early and late translation termination. EMBO J. 36, 2968-2986. https://doi.org/10.15252/embj.201797079

Ribosome Display and Novel Snakebite Therapy Platforms

The first-choice treatment of snakebite envenoming is antivenom, and yet this Neglected Tropical Disease still, annually, causes up to 138,000 deaths and 400,000 disabilities in surviving victims. Current anti-venoms (AVs) are based on antibodies from hyperimmunised horses and sheep and are weakly effective. This approach has many additional disadvantages, in particular it cannot rationally incorporate the distinct immunogenicity or toxicity of the venoms’ proteins into the design - venoms comprise between 20 to >100 proteins that vary in molecular mass, bioactivity and pathogenicity.

We work on an innovative solution to change snakebite treatment in two international consortia funded by the EC and the Wellcome Trust. We rationally design nanobodies and ADDobodies/ ADDomers (ADDovenom, https://addovenom.com) to bind and neutralise the pathogenic function of all toxins of the medically most important snakes in Sub-Saharan Africa, and without adverse effect risk. We will achieve this by applying cutting-edge mass spectroscopy techniques to snake venoms to enable informed target choices, by protein engineering, by ribosome display in vitro selection technology and high-throughput protein production methods.

Selected publications:

1. Sari-AK, D., Bufton, J., Garzoni, F., Fitzgerald, D., Schaffitzel, C. & Berger, I. (2021) VLP-factoryTM and ADDomer©: Self-assembling Virus-Like Particle (VLP) technologies for multiple protein and peptide epitope display. Protoc. 1, e55. doi: 10.1002/cpz1.55.
2. Sari-Ak, D., Bahrami, S., Laska, M.J., Drncova, P., Fitzgerald, D.J., Schaffitzel, C., Garzoni, F. & Berger, I. (2019) High-Throughput Production of Influenza Virus-Like Particle (VLP) Array by Using VLP-factory™, a MultiBac Baculoviral Genome Customized for Enveloped VLP Expression. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. doi: 10.1007/978-1-4939-9624-7_10. https://link.springer.com/protocol/10.1007/978-1-4939-9624-7_10.
3. Vragniau, C., Bufton, J.C., Garzoni, F., Stermann, E., Rabi, F., Terrat, C., Verneret, M., Josserand, V., Verrier, B., Chaperot, L., Schaffitzel, C.*, Berger, I.* & Fender, P.* (2019) Synthetic Self-assembling ADDomer Platform for Highly Efficient Vaccination by Genetically-encoded Multi-epitope Display. Advances 5, eaaw2853. DOI: 10.1126/sciadv.aaw2853.

Free Fatty Acid Binding to SARS-CoV-2 Spike protein

We discovered a novel Free Fatty Acid (FFA)-binding pocket in SARS-CoV-2 Spike (S) glycoprotein, with low nanomolar affinity for linoleic acid (LA), an essential FFA with key roles in lipid metabolism and inflammation modulation. Intriguingly, we found that LA binding is highly conserved in severely pathogenic human CoVs SARS-CoV, MERS-CoV, SARS-CoV-2 and all Variants of Concern (VOCs) including omicron. In marked contrast, endemic human CoVs causing only mild disease (common cold) do not appear to have a functional LA-binding pocket. We determined that LA treatment inhibits viral infectivity outside of cells, and viral replication inside of cells, revealing two modes of action that hold promise for potential future FFA-based treatments.

Selected Publications:

1. Toelzer, C., Gupta, K., Yadav, S.K.N., Hodgson, L., Williamson, M.K., Buzas, D., Borucu, U., Powers, K., Stenner, R., Vasileiou, K., Garzoni, F., Fitzgerald, D., Payré, C., Gautam, G., Lambeau, G., Davidson, A.D., Verkade, P., Frank, M., Berger, I. & Schaffitzel, C. (2022) The free fatty acid-binding pocket is a conserved hallmark in pathogenic β-coronavirus spike proteins from SARS-CoV to Omicron. Sci Adv. 8, eadc9179. https://doi.org/10.1126/sciadv.adc9179.
2. Buchanan, C.J., Gaunt, B., Harrison, P.J., Yang, Y., Liu, J., Khan, A., Giltrap, A.M., Le Bas, A., Ward, P.N., Gupta, K., Dumoux, M., Tan, T.K., Schimaski, L., Daga, S., Picchiotti, N., Baldassarri, M., Benetti, E., Fallerini, C., Fava, F., Giliberti, A., Koukos, P.I., Davy, M.J., Lakshminarayanan, A., Xue, X., Papadakis, G., Deimel, L.P., Casablancas-Antràs, V., Claridge, T.D.W., Bonvin, A.M.J.J., Sattentau, Q.J., Furini, S., Gori, M., Huo, J., Owens, R.J., Schaffitzel, C., Berger, I., Renieri, A.; GEN-COVID Multicenter Study, Naismith, J.H., Baldwin, A.J. & Davis, B.G. (2022) Pathogen-sugar interactions revealed by universal saturation transfer analysis. Science 377, eabm3125. https://doi.org/10.1126/science.abm3125
3. Gupta, K., Toelzer, C., Williamson, M.K., Shoemark, D.K., Oliveira, A.S.F., Matthews, D.A., Almuqrin, A., Staufer, O., Yadav, S.K.N., Borucu, U., Garzoni, F., Fitzgerald, D., Spatz, J., Mulholland, A.J., Davidson, A.D., Schaffitzel, C.* & Berger. I.* (2022) Structural insights in cell-type specific evolution of intra-host diversity by SARS-CoV-2. Commun. 13, 222. https://doi.org/10.1038/s41467-021-27881-6
4. Staufer, O., Gupta, K., Hernandez Bücher, J.E., Kohler, F., Sigl, C., Singh, G., Vasileiou, K., Yagüe Relimpio, A., Macher, M., Fabritz, S., Dietz, H., Cavalcanti Adam, E.A., Schaffitzel, C., Ruggieri, A., Platzman, I., Berger, I. & Spatz, J.P. (2022) Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein. Commun. 13, 868. https://doi.org/10.1038/s41467-022-28446-x
5. Shoemark, DK, Colenso, CK, Toelzer, C, Gupta, K, Sessions, RB, Davidson, AD, Berger, I, Schaffitzel, C, Spencer, J, Mulholland, AJ. (2021) Molecular Simulations suggest Vitamins, Retinoids and Steroids as Ligands of the Free Fatty Acid Pocket of the SARS-CoV-2 Spike Protein.  Chem. Int. Ed. Engl. 60, 7098-7110.DOI: 10.1002/anie.202015639.
6. Toelzer, C., Gupta, K., Yadav, S.K.N., Borucu, U., Davidson, A.D., Kavanagh Williamson, M,, Shoemark, D.K., Garzoni, F., Staufer, O., Milligan, R., Capin, J., Mulholland, A.J., Spatz, J., Fitzgerald, D., Berger, I. & Schaffitzel. * (2020) Free fatty acid binding pocket in the locked structure of SARS-CoV-2 spike protein. Science 370, 725-730. https://doi.org/10.1126/science.abd3255

In all our projects, we rely on biochemical methods, state-of-the-art protein production and in vitro translation systems. We use biophysics to study multi-protein complexes and structural biology, crystallography and cryo-EM. We have established a Wellcome Trust-funded GW4 Facility for high-resolution Cryo-EM with a 200kV Talos Arctica with energy filter and K2 Direct Electron Detector http://www.bristol.ac.uk/wolfson-bioimaging/equipment/cryo-em/. Image processing is supported by a BBSRC-funded BlueCryo high-performance computing cluster.