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Abstract

Tobias Madl, Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Molecular Biology and Biochemistry, Medical University of Graz

Supervisor: Prof. Dr. Tobias Madl
Availability: This position is available.
Offered by: Medical University of Graz
Application deadline:Applications are accepted between August 01, 2018 00:00 and September 23, 2018 23:59 (CEST)

Description

Background: Motifs rich in arginine and glycine residues were recognized several decades ago to play functional roles in RNA-binding and were termed RG/RGG motifs1,2. More than 1000 proteins harbor the intrinsically disordered RG/RGG motif, and these proteins play essential roles in a plethora of physiological processes such as transcription, pre-mRNA splicing, DNA damage signaling and mRNA translation2, and very recently in neuroprotection3. We have shown that the RG/RGG-motif of FUS is involved in transportin-1 – mediated nuclear import, and that transportin-1 acts as a molecular chaperone regulating FUS phase separation4-6. Arginine methylation of the RG/RGG motif in combination with RNA-binding and mutations regulate phase separation of FUS and determine the formation of pathogenic inclusions in Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) patients. Very recently we have discovered a novel regulator of phase separation of not only FUS but many other proteins which will be the focus of this project (Madl lab, unpublished).

Hypothesis and Objectives: Based on our recent studies and supported by our preliminary data, we propose an alternative mechanism for chaperoning phase separation involving a novel regulator. We propose that this regulator acts in coordination with transportin-1 and that a code of post-translational modifications regulates import of the large class of RG/RGG proteins and a new class of proteins and that disease mutations found in cancer and neurodegeneration modulate these interactions. We propose to use newly discovered target proteins as model systems to reveal the structural and functional mechanisms of nuclear import and phase separation by:

  1. 1) studying interaction, structure and function of the novel protein complexes
  2. 2) studying regulation of the novel protein complexes by post-translational modifications, disease mutations, and co-factors

This might set the base for the discovery of new potential druggable targets in the future for the treatment of a plethora of diseases with different phenotypes, though caused by the same molecular disease mechanisms.

Methodology: The PhD candidate will make use of our recent methodological achievements for studying structure of large protein complexes by combining solution Nuclear Magnetic Resonance (NMR) spectroscopy, and molecular modeling7-12, and extend it with complementary approaches such as Mass Spectrometry (MS) and cell biology.

 

References:

  1. Kiledjian, M. & Dreyfuss, G. Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. The EMBO journal 11, 2655-2664 (1992).
  2. Thandapani, P., O'Connor, T. R., Bailey, T. L. & Richard, S. Defining the RGG/RG motif. Molecular cell 50, 613-623, doi:10.1016/j.molcel.2013.05.021 (2013).
  3. Peretti, D., Bastide, A., Radford, H., Verity, N., Molloy, C., Martin, M. G., Moreno, J. A., Steinert, J. R., Smith, T., Dinsdale, D., Willis, A. E. & Mallucci, G. R. RBM3 mediates structural plasticity and protective effects of cooling in neurodegeneration. Nature, doi:10.1038/nature14142 (2015).
  4. Dormann, D., Madl, T., Valori, C. F., Bentmann, E., Tahirovic, S., Abou-Ajram, C., Kremmer, E., Ansorge, O., Mackenzie, I. R., Neumann, M. & Haass, C. Arginine methylation next to the PY-NLS modulates Transportin binding and nuclear import of FUS. The EMBO journal 31, 4258-4275, doi:10.1038/emboj.2012.261 (2012).
  5. Suarez-Calvet, M., Neumann, M., Arzberger, T., Abou-Ajram, C., Funk, E., Hartmann, H., Edbauer, D., Kremmer, E., Gobl, C., Resch, M., Bourgeois, B., Madl, T., Reber, S., Jutzi, D., Ruepp, M. D., Mackenzie, I. R., Ansorge, O., Dormann, D. & Haass, C. Monomethylated and unmethylated FUS exhibit increased binding to Transportin and distinguish FTLD-FUS from ALS-FUS. Acta neuropathologica 131, 587-604, doi:10.1007/s00401-016-1544-2 (2016).
  6. Hofweber, M., Hutten, S., Bourgeois, B., Spreitzer, E., Niedner-Boblenz, A., Schifferer, M., Ruepp, M. D., Simons, M., Niessing, D., Madl, T. & Dormann, D. Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation. Cell 173, 706-719 e713, doi:10.1016/j.cell.2018.03.004 (2018).
  7. Gobl, C., Madl, T., Simon, B. & Sattler, M. NMR approaches for structural analysis of multidomain proteins and complexes in solution. Progress in nuclear magnetic resonance spectroscopy 80C, 26-63, doi:10.1016/j.pnmrs.2014.05.003 (2014).
  8. Huang, J. R., Warner, L. R., Sanchez, C., Gabel, F., Madl, T., Mackereth, C. D., Sattler, M. & Blackledge, M. Transient Electrostatic Interactions Dominate the Conformational Equilibrium Sampled by Multidomain Splicing Factor U2AF65: A Combined NMR and SAXS Study. Journal of the American Chemical Society 136, 7068-7076, doi:10.1021/ja502030n (2014).
  9. Karagoz, G. E., Duarte, A. M., Akoury, E., Ippel, H., Biernat, J., Moran Luengo, T., Radli, M., Didenko, T., Nordhues, B. A., Veprintsev, D. B., Dickey, C. A., Mandelkow, E., Zweckstetter, M., Boelens, R., Madl, T. & Rudiger, S. G. Hsp90-Tau complex reveals molecular basis for specificity in chaperone action. Cell 156, 963-974, doi:10.1016/j.cell.2014.01.037 (2014).
  10. Lorenz, O. R., Freiburger, L., Rutz, D. A., Krause, M., Zierer, B. K., Alvira, S., Cuellar, J., Valpuesta, J. M., Madl, T., Sattler, M. & Buchner, J. Modulation of the Hsp90 chaperone cycle by a stringent client protein. Molecular cell 53, 941-953, doi:10.1016/j.molcel.2014.02.003 (2014).
  11. Madl, T., Gabel, F. & Sattler, M. NMR and small-angle scattering-based structural analysis of protein complexes in solution. Journal of structural biology 173, 472-482, doi:10.1016/j.jsb.2010.11.004 (2011).
  12. Muller, R., Grawert, M. A., Kern, T., Madl, T., Peschek, J., Sattler, M., Groll, M. & Buchner, J. High-resolution structures of the IgM Fc domains reveal principles of its hexamer formation. Proceedings of the National Academy of Sciences of the United States of America 110, 10183-10188, doi:10.1073/pnas.1300547110 (2013).