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| Thesis topic proposal |
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Description of the research topic:
THESIS TOPIC PROPOSAL
Institute: University of Szeged
theoretical medicine
The vacuolar proton-ATPase (V-ATPase) belongs to the family of membrane-attached biological macromolecules of the rotary enzymes. 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. Because of its size and complexity, understanding the details of structure-function relationship of this membrane-bound molecular machine is one of the major challenges in biophysics and molecular biology today. V-ATPase is also a potential therapeutic target in several diseases. It is known that lysosomes are highly acidified (by lysosomal V-ATPase) only when cells need their degradative function. For instance, starving animals for 3 hours produces a robust lysosomal acidification. According preliminary work by our partner (Momentum Drosophila Autophagy group, G. Juhász), control of V-ATPase in lysosome acidification can not be explained with known regulatory factors. It is hypothesised that incoming lipids might be new regulatory factors. Therefore lipid-protein interaction is of great functional relevance, but it has never been studied, in either direction, on native V-ATPase or on the Vo domain. Spin-label electron paramagnetic resonance (EPR) spectroscopy has several unique benefits in membrane-protein research: it can be used in native and reconstituted proteins and membranes; causes small or no perturbation; spectra are clean for interpretation and fitting; proteins, lipids, inhibitors, substrates all can be spin-labeled; it yields data on dynamics in an optimal time-scale for biomembranes; it can detect non-covalent interactions; and it yields structural data. The objecive of this proposal is to use vacuolar vesicles to study the effect of V-ATPase on lipid dynamics with EPR and study the effect of the lipid environment on native V-ATPase, also focusing on how the activity of the enzyme is affected by altering the native lipid environment. Our preliminary results already indicate that lipid dynamics is affected by the functioning of the enzyme. We can separate the spectral components of the first-shell and bulk lipids if both are significant. However, we know that ConcA has a membrane effect even alone, and the substrate (ATP) might have some effect too. The challenge here is to separate the inhibitor and substrate effects from that of the activity of the enzyme. We will address these questions with the following research programme:
• We will do experiments with other inhibitors, that are less specific to V-ATPase but are not expected to affect lipids significantly.
• We will use spin-labeled inhibitor analogues to quantitate their binding (based on changes in their rotational dynamics) and correlate it with changes in the spectra of spin-labeled lipids.
• We will turn on the enzyme by light, in the EPR resonator, uning light-sensitive substrates. This will allow timed control of the reactions, and also high resolution difference spectroscopy.
• We will study different positional isomers of lipid spin-labels such as stearic acid (SA), phosphocholine (PC) and TEMPO. TEMPO partitions between the membrane and the aqueous phase, yielding information on the rigidity and compactness of the membrane.
• Experiments will be done at different temperatures in order to better separate the spectra of first-shell and bulk lipids and to modulate membrane fluidity. Spectra will be analysed with our routine algorithms yielding orientational order parameter, rotational correlation time and relative polarity. These experiments will show how first-shell and bulk lipids react to different functional states of the enzyme.
• As concerns the reverse direction of lipid-protein interaction, our hypothesis is that V-ATPase is sensitive to its lipid environment because 2/3 of the surface of the rotor is always in contact with lipids (strong binding of lipids to the rotor could block it), and previously we have seen a clear selectivity towards negatively charged lipids of the lobster protein (a c-ring model of the rotor). We will study the effect of exogenously added lipids on ATPase activity.
Selected references:
Mosior, M., Mikołajczak, A. and Gomułkiewicz, J. (1990). The effect of ATP on the order and the mobility of lipids in the bovine erythrocyte membrane. Biochim Biophys Acta, 1022(3), 361-364.
Pali, T., Finbow, M.E., Holzenburg, A., Findlay, J.B.C., and Marsh, D. (1995) Lipid-Protein Interactions and Assembly of the 16-kDa Channel Polypeptide from Nephrops norvegicus. Studies with Spin-Label Electron Spin Resonance Spectroscopy and Electron Microscopy. Biochemistry 34(28), 9211-9218.
Kota, Z., Horvath, L. I., Droppa, M., Horvath, G., Farkas, T., & Pali, T. (2002). Protein assembly and heat stability in developing thylakoid membranes during greening. Proceedings of the National Academy of Sciences USA, 99(19), 12149-12154.
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.
Marsh, D. and Pali, T. (2004) The protein-lipid interface: perspectives from magnetic resonance and crystal structures (review article) Biochimica et Biophyisica Acta - Biomembranes 1666(1-2), 118-141.
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.
Forgac, M. (2007) Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat Rev Mol Cell Biol 8, 917-929.
Xu, J., Cheng, T., Feng, H.T., Pavlos, N.J. and Zheng, M.H. (2007) Structure and function of V-ATPases in osteoclasts: potential therapeutic target. Histol Histopathol 22, 443-54.
Juhasz, G. and Neufeld, T.P. (2008) Experimental control and characterization of autophagy in Drosophila. Methods Mol. Biol. 445, 125-133.
Perez-Sayansa, M., Somoza-Martina, J.M, Barros-Angueirab, F., Reya, J.M.G. and Garcia-Garcia, A. (2009) V-ATPase inhibitors and implication in cancer treatment. Cancer Treatment Reviews 35(8), 707-713.
Takats, S., Nagy, P., Varga, A., Pircs, K., Karpati, M., Varga, K., Kovacs, A.L., Hegedus, K. and Juhasz, G. (2013) Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila. J. Cell Biol. 201, 531-539.
Takats, S., Pircs, K., Nagy, P., Varga, A., Karpati, M., Hegedus, K., Kramer, H., Kovacs, A.L., Sass, M. and Juhasz, G. (2014) Interaction of the HOPS complex with Syntaxin 17 mediates autophagosome clearance in Drosophila. Mol. Biol. Cell 25, 1338-1354.
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.
Nagy, P., Varga, A., Kovacs, A.L., Takats, S. and Juhasz, G. (2015) How and why to study autophagy in Drosophila: It's more than just a garbage chute. Methods 75, 151-161.
Sun-Wada, G.H. and Wada, Y. (2015) Role of vacuolar-type proton ATPase in signal transduction. Biochim Biophys Acta 1847, 1166-1172.
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.
Lorincz, P., Mauvezin, C. and Juhasz, G. (2017) Exploring Autophagy in Drosophila. Cells 6.
Lorincz, P., Toth, S., Benko, P., Lakatos, Z., Boda, A., Glatz, G., Zobel, M., Bisi, S., Hegedus, K., Takats, S., Scita, G. and Juhasz, G. (2017) Rab2 promotes autophagic and endocytic lysosomal degradation. J. Cell Biol. 216, 1937-1947.
Pali, T. and Kota, Z. (2019). Studying Lipid-Protein Interactions with Electron Paramagnetic Resonance Spectroscopy of Spin-Labeled Lipids. Methods Mol Biol, 2019, 2013, 529-561.
Required language skills: English
Number of students who can be accepted: 2
Deadline for application: 2022-04-06 |
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