Current Projects

Membrane-associated proteins of the pH regulation and membrane fusion processes of organelles and transport vesicles of eukaryotic cells

The non-uniform distribution of ions in living organism is one most important driving forces of many life processes. The molecular mechanisms of pH regulation and membrane fusion of the eukaryotic cellular organelles are of high relevance to many normal and pathogenic physiological processes. We focus on 3 classes of key proteins in these processes: the vacuolar proton-ATPase (V-ATPase), the Ductin and Syntaxin proteins. The former two are our projects, and they are strongly related since part of the V-ATPase belongs to the Ductin family. Ductins and syntaxins play important roles in vesicle fusion mechanisms. Some Ductin proteins function as gap-junctions and synaptic mediatophores. Since our description of the Ductin family [Holzenburg et al., 1993], we have determined the membrane assembly and interaction of one of the gap-junction Ductin proteins with lipids [Pali et al., 1997; Pali et al., 1999; Kota et al., 2008]. We are now revisiting the Ductin family in order to identify new structural features and new factors regulating their membrane assembly.

The internal organelles of eukaryotic cells are more acidic than the cytoplasm. The protein complex that is responsible for their acidification is nature's most universal proton pump, the vacuolar proton-ATPase [Abuammar et al., 2021]. V-ATPase also takes part in membrane fusion processes by providing controlled local acidification in cells. It also plays crucial roles in a number diseases, and its tissue-specific inhibition is a key therapeutic objective. V-ATPase is a membrane-bound molecular rotary engine, which converts the chemical energy from ATP hydrolysis to the rotation of the rotor domain via a torque generated between specific subunits. This leads to trans-membrane proton pumping in the interface between the stator and rotor domains. We were the first to report the rotation rate in a V-ATPase that was not subjected to genetic or chemical modification and it is not fixed to a solid support via inhibitor titration [Ferencz et al., 2013] and the discovery that the activity of V-ATPase can be affected with an oscillating trans-membrane electric field [Ferencz et al., 2017; Petrovszki et al., 2021]. The electric field effects are being used to explore further details of the rotary mechanism. In reconstituted model membrane system we are to study the functional assembly of the c-ring, the ductin part of V-ATPase. Our current studies on V-ATPase also aim at subunit-subunit and subunit-lipid interactions, the effect of synthetic inhibitors and divalent cations on function and subunit assembly and the details of the rotary mechanism [Sebok-Nagy et al., 2023]. In collaboration with the Momentum Drosophila Autophagy group (G. Juhasz, Institute of Genetics) we are studying the binding of syntaxins to membranes [Laczkó-Dobos et al., 2024] and new factors regulating lysosome acidification by V-ATPase.

See our Research Topic at Frontiers in Biomolecular Sciences about Functions, Working Mechanisms, and Regulation of Rotary ATPases and Ductin Proteins [Pali et al., 2024].

Folding, insertion, structure and assembly of proteins in the membrane and at the membrane-water interface, in relation to their biological function

An experimentally and computationally challenging problem in biophysics is how membrane proteins are inserted, folded and assembled in the lipid bilayer, in some cases even pass it through, often in the absence of folding assistants and take up their native biologically functional conformation. In this membrane-protein folding problem specific interactions between the protein and lipids of the target membrane must clearly play crucial role yet to be explored in detail. In reconstituted lysozyme-lipid complexes, we are studying the influence of the lipid bilayer on activity, thermal (un)folding and stability of the antibacterial enzyme. Lysozyme is a soluble protein it is exposed to bacterial membranes during its function as hydrolase. Since even closely related soluble proteins unfold or refold at the membrane-water interface very differently, it is an interesting phenomenon to be understood. On the other hand, small membrane active antimicrobial peptides are able to insert and form channels or disrupt the bilayer integrity causing lysis without suffering major structural changes themselves. In the past, we have modelled the membran-embedded structure of several membrane proteins [Kostrzewa et al., 2000; Bashtovyy et al., 2003; Pali et al., 2006] and polypeptides [Bashtovyy et al., 2001; Kota et al., 2004; Kota et al., 2008] based on spectroscopic structural data. Currently we are focusing on a more systematic combination of experimental and theoretical techniques for structural biology of membrane proteins, including machine learning. Experimental structural biology of membrane proteins faces the challenges and limitations due to the need of isolating, solubilising or crystallising proteins (that are otherwise natively hosted by the lipid matrix) in a foreign environment. Theoretical approaches have therefore enormous importance. Since the native fold of a membrane protein assumes a native lipid annular shell, the sequence-to-fold coding is valid only in the native membranous environment. Therefore, our approach is to measure structural data on the target membrane proteins in their functional state and native environment, and combine the so-obtained data with theoretical methods in order to predict the 3-dimensional fold. To this aim, we are developing on a membrane protein structure prediction pipeline that uses spectroscopic data and predictions from bioinformatics and machine learning as restrains in molecular mechanics and -dynamics simulations.

Protein-lipid interactions and membrane reorganisations in photosynthetic membranes and lipid-based drug delivery formulas

Uncoupling or perturbing the selective structural and dynamic balance between membrane proteins and lipids may either force the organism to adapt to the new conditions, by e.g. modifying its lipid composition, or die when unable to do so. The protein-lipid interface takes several different forms, all of which are crucial to biology. For biological membranes the protein-lipid interface may be either polar in the case of surface-bound or absorbed peripheral proteins, or apolar in the case of integral transmembrane proteins. Membrane-active peptides of biological or synthetic origin may encompass either or both of these types. As the site of non-competitive inhibition by local anaesthetics and probably other hydrophobic drugs, the protein-lipid interface also has direct functional and pharmacological implications.

In collaborations with the Institute of Plant Biology, we are studying the role of non-bilayer lipid phases and free radicals [Kota et al., 2002; Lingvay et al., 2020; Rehman et al., 2021 ] in adaptive reorganisations and stress responses, respectively, of photosynthetic membranes, in which most of lipids are in contact with membrane proteins. In an international Visegrad grant (see below), we are studying the membrane dynamics and lipid-protein interaction in the native membrane of an innovative marine bioinspired algal drug carrier and the molecular details of its interaction with target membranes. This project is motivated by the observation that the major limitations in drug carrier production are protocol efficiency, bioincompatibility and cost. There is a great need to overcome these limitations and meet the requirements of sustainability and environmental friendliness, such as using algae. In national and international collaborations we are studying effect of waste-water TiO2-based filtration membranes on soluble proteins [Sebok-Nagy et al., 2023] and spectroscopically characterise nano metal-organic frameworks membranes for purification of waste water from organic contaminates, respectively.