Protein Structure and Dynamics - Lukáš Žídek
CEITEC MU CEITEC MU
Protein Structure and Dynamics - Lukáš Žídek

PhD Topics

1.     Are Tau fibrils induced by phosphorylation and the interaction with 14-3-3 proteins relevant for Alzheimer disease?

Supervisor: Jozef Hritz, Ph.D.                                                

Consultant: Jiří Nováček, Ph.D.; Jan Přibyl, Ph.D.

Annotation:
Several neurodegenerative diseases are associated with the formation of fibrous protein aggregates. The fibrillization of amyloid beta peptide into amyloid plaques and the agregation of hyperphosphorylated tau protein into neurofibrillar tangles are main neuropatological signs of Alzheimer disease. Studying of how different factors influence the formation of protein fibrils is the key for understanding this neurodegerative processes. The main aim of this PhD project will be preparation and analysis of tau fibrils prepared by variety of approaches. Major focus will be given on phosphorylation and interaction with 14-3-3 proteins. Interdisciplinary approach combining molecular biology and structural biology (mainly cryoEM tomography and AFM) methods will be applied. The described activities are part of international research projects allowing to spend the part of PhD study at the collaborative groups in Europe or North and South America and to learn specific research techniques, there. 

Literuture:

  1. Fitzpatrick, A. W. P., Falcon, B., He, S., Murzin, A. G., Murshudov, G., Garringer, H. J., Crowther, R. A., Ghetti, B., Goedert, M., and Scheres, S. H. W. Cryo-EM structures of tau filaments from Alzheimer’s disease. Nature 2017, 547, 185–190
  2. Falcon, B., Zhang, W., Murzin, A. G., Murshudov, G., Garringer, H. J., Vidal, R., Crowther, R. A., Ghetti, B., Scheres, S. H. W., and Goedert, M. Structures of filaments from Pick’s disease reveal a novel tau protein fold. Nature 2018, 561, 137–140
  3. Zhang, W., Falcon, B., Murzin, A. G., Fan, J., Crowther, R. A., Goedert, M., and Scheres, S. H. Heparin-induced tau filaments are polymorphic and differ from those in Alzheimer’s and Pick’s diseases. eLife 2019, 8, e43584
  4. Jansen, S.; Melková, K.; Trošanová, Z.; Hanáková, K.; Zachrdla, M.; Nováček, J.; Župa,E.;Zdráhal, Z.; Hritz, J.; Žídek, L.: Quantitative Mapping of MAP2c Phosphorylation and 14-3-3ζ Binding Sites Reveals Key Differences Between MAP2c and Tau. J. Biol. Chem. 2017, 292, 6715-6727
  5. Melková, K.; Zapletal, V.; Narasimhan, S.; Jansen, S.; Hritz, J.; Škrabana, R.; Zweckstetter, M.; Ringkjøbing Jensen, M.; Blackledge, M.; Žídek, L. Structure and Functions of Microtubule Associated Proteins Tau and MAP2c: Similarities and Differences. Biomolecules 20199, 105.

Keywords: neurodegenerative diseases, Alzheimer disease, protein fibrils, phosphorylation state, 14-3-3 proteins, cryoEM tomography

2.     Interactions underlying physiological function of Microtubule Associated Protein 2c

Supervisor: prof. Lukáš Žídek

Consultants: Jozef Hritz, Ph.D.

Annotation:
Microtubule associated protein 2c (MAP2c) is a key factor regulating microtubule dynamics in developing brain neurons, and an example of an intrinsically disordered proteins with an important physiological function and detectable structure-function relationship. The first goal is to study MAP2c in a natural complexity and by methods providing atomic resolution. Such methods include paramagnetic relaxation interference, to detect and describe transient local structures of MAP2c important for its function, and real-time NMR, to monitor kinetics of MAP2c phosphorylation by relevant kinases of different signalling pathways. The second goal is to characterize interactions of MAP2c with biologically important binding partners, especially with isoforms and a monomeric form of regulatory protein 14-3-3. The third goal is to test the effect of cellular environment on MAP2c by recording NMR spectra at near-to-native conditions (in cells and/or cell lysates) and/or by performing cryo-electron tomography on monolayered neurons.

Keywords: Microtubule Associated Protein 2c, MAP2c, NMR spectroscopy, cryo-electron tomography, 14-3-3 protein

3.     Structure, dynamics and interactions of bacterial RNA polymerase subunits and sigma factors

Supervisor: prof. Lukáš Žídek                                                            

Annotation:
Bacterial RNA polymerase (RNAP) is a key enzyme involved in gene expression and proved target of antibiotics. Although model RNAP of Gram-negative bacteria is well characterized, RNAP of other bacterial species differs substantially in subunit composition and regulation. In collaboration with Libor Krasny (Microbiology Institute of the Czech Academy of Sciences), we have recently characterized delta subunit and sigma-A factor of Bacillus subtilis. Topic of the thesis will be continuation of structural studies, namely investigation of dynamics and localization of the delta subunit in the holoenzyme, and structural characterization of proteins involved in non-canonical transcription initiation of RNAP of Mycobacterium smegmatis. The experimental approach will reflect the size and properties of the studied proteins. Cryo-electron microscopy will be the key technique of the holoenzyme studies, NMR spectroscopy will be applied to small a dynamic subunits. In particular, advanced NMR techniques developed in the group will facilitate studies of intrinsically disordered regions of the proteins.

Literature:

1.    Zachrdla et al. Solution structure of domain 1.1 of the sigma(A) factor from Bacillus subtilis is preformed for binding to the RNA polymerase core. Journal of Biological Chemistry, 292 (2017) 11610-11617.

2. Kuban et al. Quantitative Conformational Analysis of Functionally Important Electrostatic Interactions in the Intrinsically Disordered Region of Delta Subunit of Bacterial RNA Polymerase. J Am Chem Soc. 2019 Oct 10. doi: 10.1021/jacs.9b07837. [Epub ahead of print]

Keywords: RNA polymerase, transcription, bacteria, nuclear magnetic resonance

4.     Tau conformational changes induced by phosphorylation and 14-3-3 proteins relevant in neurodegenerative diseases

Supervisor: Jozef Hritz, Ph.D.                                                

Consultant: prof. Lukas Žídek, Ph.D.

Annotation:
The main neuropatological signs of Alzheimer disease are associated with the fibrillization of tau protein into neurofibrillar tangles. Studying of how different factors influence the formation of protein fibrils is the key for understanding these neurodegerative processes. The main aim of this PhD project will be characterization of conformational changes in the formation of tau fibrils due to their truncations, phosphorylation and the interaction with 14-3-3 proteins. Interdisciplinary approach combining biomolecular NMR, biophysical interaction techniques and computational methods will be applied. The described activities are part of international research projects allowing to spend the part of PhD study at the groups of our collaborators in Europe or North and South America and to learn specific research techniques, there. 

Literature:

  1. Lippens, G., and Gigant, B. Elucidating Tau function and dysfunction in the era of cryo-EM. J. Biol. Chem. 2019, 294, 9316–9325.
  2. Přecechtělová, J.; Mládek, A.; Zapletal, V.; Hritz, J.: Quantum Chemical Calculations of NMR Chemical Shifts in Phosphorylated Intrinsically Disordered Proteins, JCTC 2019, 15, 5642-5658.
  3. Melková, K.; Zapletal, V.; Jansen, S.; Nomilner, E.; Zachrdla, M.; Hritz, J.; Novácek, J.; Zweckstetter, M.; Ringkjøbing-Jensen, M.; Blackledge, M. and Žídek, L.: Functionally specific binding regions of microtubule-associated protein 2c exhibit distinct conformations and dynamics.J. Biol. Chem. 2018, 293, 13297-13309.
  4. Nagy, G., Oostenbrink, C., and Hritz, J. Exploring the binding pathways of the 14-3-3ζ protein: Structural and free-energy profiles revealed by Hamiltonian replica exchange molecular dynamics with distancefield distance restraints. PLoS One 2017, 12, 1–30.
  5. Motáčková, V., Nováček, J., Zawadzka-Kazimierczuk, A., Kazimierczuk, K., Žídek, L., Šanderová, H., Krásný, L., Koźmiński, W., and Sklenář, V. Strategy for complete NMR assignment of disordered proteins with highly repetitive sequences based on resolution-enhanced 5D experiments. J. Biomol. NMR 2010, 48, 169–177.

Keywords: Molecular Biophysics, tau protein, phosphorylation, 14-3-3 proteins, neurodegenerative diseases, NMR, computational simulations

5.      Role of phosphorylated 14-3-3 proteins 

Supervisor: Jozef Hritz, Ph.D.                                                  

Consultant:  prof. Vítězslav Bryja

Annotation:
14-3-3 proteins play key role in the neurobiochemistry of brain and are also associated with neurodegenerative amyloid diseases such as Alzheimer and Parkinson disease. 14-3-3 proteins bind more than 850 diverse target phosphoproteins, thereby forcing conformational changes or/and stabilizing active conformations in their target proteins. The main aim of this PhD project is to quantify extent of the phosphorylation of different isoforms of 14-3-3 protein family in different cell types. Subsequently, the potency of homo and heterodimerization involving the phosphorylated 14-3-3 proteins will be quantified by fluorescence, ITC and NMR biophysical assays. Finally, the impact on the binding of phosphorylated client proteins will be analyzed.

Literature

  1. Sluchanko, N.N.Association of Multiple Phosphorylated Proteins with the 14-3-3 Regulatory Hubs: Problems and Perspectives. J. Mol. Biol. 2018, 430, 20–26.
  2. Jandova, Z.; Trosanova, Z.; Weisova, V.; Oostenbrink, C.; Hritz, J. Free energy calculations on the stability of the 14-3-3z protein. BBA - Proteins and Proteomics, 2018, 1866, 442-450
  3. Nagy, G., Oostenbrink, C., and Hritz, J. Exploring the binding pathways of the 14-3-3ζ protein: Structural and free-energy profiles revealed by Hamiltonian replica exchange molecular dynamics with distancefield distance restraints. PLoS One 2017, 12, 1–30.
  4. Petr Louša, Hana Nedozrálová, Erik Župa, Jiří Nováček, Jozef Hritz:Phosphorylation of the regulatory domain of human tyrosine hydroxylase 1 monitored using non-uniformly sampled NMR. Biophysical Chemistry 2017, 223, 25-29
  5. Hritz J.; Byeon I-J.; T.; Martinez A.; Sklenář V.; Gronenborn A.M. Dissection of binding between a phosphorylated tyrosine hydroxylase peptide and 14-3-3ζ: a complex story elucidated by NMR. Biophys. J. 2014, 107, 2185-2194

Keywords: Biophysical Chemistry, neurodegenerative diseases, phosphorylation state, 14-3-3 proteins, NMR, ITC, fluorescence