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| Thesis topic proposal |
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Description of the research topic:
The vacuolar proton-ATPase (V-ATPase) belongs to the family of membrane-attached biological macromolecules whose functioning involves true, full cycle rotation of some parts, called rotor, relative to other parts, called stator. The normal function of V-ATPase is to pump protons across certain biomembranes and it is a key rotary enzyme in all eukaryotic cells, acidifying intracellular compartments and the extracellular space in some tissues. According to their localisation in a multitude of eukaryotic endomembranes and plasma membranes, V-ATPases energise many different transport processes. V-ATPase is also a potential therapeutic target in several diseases. Proton transport by V-ATPase is energised from the chemical energy stored in ATP via binding and hydrolysis, which is converted into mechanical force rotating a group of certain subunits relative to the rest of the protein complex. The Vo domain is very important for the proton translocation, and the unique Glu residue is located on the 4th helix of subunit c of Vo. This intra-membrane region is important also because it contains binding sites for specific inhibitors. The Bafilomycins and Concanamycins, which inhibit all known eukaryotic V-ATPases, are the most specific and potent inhibitors. Concanamycin A (ConcA) binds to the intramembranous domain of V-ATPase and blocks rotation, hence proton transport. V-ATPase is Nature’s most versatile proton pump. Our main objective is to explore new molecular details of the effect of ConcA on V-ATPase. We have already determined the stoichiometry of binding of ConcA to native V-ATPase in yeast vacuolar vesicles. In those experiments binding was indirectly evidenced by inhibition of the enzyme. We have the following specific tasks:
• Since the linear region is still rather limited in our NADH-driven ATPase assay we will fit the decay curves according to the enzymatic reactions to eliminate this limitation. If this kinetic assay does not work because, e.g., disturbing interaction with components of the coupling enzyme system, we will develop and electron paramagnetic resonance method based on spin-labeled ATP or Mn-ATP.
• Next, we will test our hypothesis that ConcA is not able to stop the running engine by varying the sequence of adding the substrate and ConcA. In addition, if the substrate concentration is low, V-ATPase assumes a stepped rotation because the engine has to wait until an ATP binds to an open ATP pocket. Hence, lowering ATP concentration (down from excess) will make these standby periods longer, thereby making the probability of ConcA binding higher and inhibition stronger. We expect a sharp dependence of V-ATPase inhibition by excess ConcA on substrate concentration.
• Since there is nothing in the literature about the effect of light on ConcA, we want to explore this side of it in relation to its effect on V-ATPase. Using the selected ATPase assay we will test if ConcA can be inactivated, and then test if activity of V-ATPase (blocked by ConcA) re-gained by intense light in yeast vacuolar vesicles. If yes, we will test the dependence of the effect on light intensity and wavelength.
• This study has strong medical relevance. Therefore our tests will also be made on vacuoles, yeast cells and finally on rotifers (smallest animals) in collaboration with the Zs. Datki group (Psychiatry Department, Szeged University).
Selected references:
Farina, C., and Gagliardi, S. (1999) Selective inhibitors of the osteoclast vacuolar proton ATPase as novel bone antiresorptive agents [Review]. Drug Discovery Today 4, 163-172.
Nishi, T. and Forgac, M. (2002) The vacuolar (H+)-atpases - Nature's most versatile proton pumps. Nature Reviews Molecular Cell Biology 3(2), 94-103.
Pali, T., Whyteside, G., Dixon, N., Kee, T.P., Ball, S., Harrison, M.A., Findlay, J.B.C., Finbow, M. and Marsh, D. (2004) Interaction of inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector. Biochemistry 43(38), 12297-12305.
Pali, T., Dixon, N., Kee, T.P., and Marsh, D. (2004) Incorporation of the V-ATPase inhibitors concanamycin and indole pentadiene in lipid membranes. Spin-label EPR studies. Biochimica et Biophyisica Acta - Biomembranes 1663(1-2), 14-18.
Nakanishi-Matsui, M., Sekiya, M., Nakamoto, R.K., Futai, M. (2010) The mechanism of rotating proton pumping ATPases. Biochim Biophys Acta 1797, 1343-1352.
Ferencz, C., Petrovszki, P., Kota, Z., Fodor-Ayaydin, E., Haracska, L., Bota, A. et al. (2013). Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes. European Biophysics Journal, 42(2-3), 147-158.
Michel, V., Licon-Munoz, Y., Trujillo, K., Bisoffi, M., & Parra, K. J. (2013). Inhibitors of vacuolar ATPase proton pumps inhibit human prostate cancer cell invasion and prostate-specific antigen expression and secretion. Int J Cancer, 132(2), E1-10.
Vavassori, S., Mayer, A. (2014) A new life for an old pump: V-ATPase and neurotransmitter release. J Cell Biol 205, 7-9.
von Schwarzenberg, K., Lajtos, T., Simon, L., Muller, R., Vereb, G., and Vollmar, A. M. (2014). V-ATPase inhibition overcomes trastuzumab resistance in breast cancer. Mol Oncol, 8(1), 9-19.
Cotter, K., Stransky, L., McGuire, C., Forgac, M. (2015) Recent Insights into the Structure, Regulation, and Function of the V-ATPases. Trends Biochem Sci 40, 611-622.
Zhao, J., Benlekbir, S., Rubinstein, J.L. (2015) Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241-245.
Kitazawa, S., Nishizawa, S., Nakagawa, H., Funata, M., Nishimura, K., Soga, T. et al. (2017). Cancer with low cathepsin D levels is susceptible to vacuolar (H+)-ATPase inhibition. Cancer Sci, 108(6), 1185-1193.
Datki, Z. et al. (2018) Exceptional in vivo catabolism of neurodegeneration-related aggregates. Acta Neuropathol Commun 6, 6.
Datki, Z. et al. (2021) Exogenic production of bioactive filamentous biopolymer by monogonant rotifers. Ecotoxicol Environ Saf 208, 111666.
Required language skills: English
Number of students who can be accepted: 2
Deadline for application: 2022-04-06 |
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