Tamás Hegedűs

Semmelweis University, Budapest, Budapest fovaros, Hungary

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Publications (6)14.93 Total impact

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    ABSTRACT: Intrinsically disordered proteins (IDPs) lack a stable tertiary structure, but their short binding regions termed Pre-Structured Motifs (PreSMo) can form transient secondary structure elements in solution. Although disordered proteins are crucial in many biological processes and designing strategies to modulate their function is highly important, both experimental and computational tools to describe their conformational ensembles and the initial steps of folding are sparse. Here we report that discrete molecular dynamics (DMD) simulations combined with replica exchange (RX) method efficiently samples the conformational space and detects regions populating α-helical conformational states in disordered protein regions. While the available computational methods predict secondary structural propensities in IDPs based on the observation of protein-protein interactions, our ab initio method rests on physical principles of protein folding and dynamics. We show that RX-DMD predicts α-PreSMos with high confidence confirmed by comparison to experimental NMR data. Moreover, the method also can dissect α-PreSMos in close vicinity to each other and indicate helix stability. Importantly, simulations with disordered regions forming helices in X-ray structures of complexes indicate that a preformed helix is frequently the binding element itself, while in other cases it may have a role in initiating the binding process. Our results indicate that RX-DMD provides a breakthrough in the structural and dynamical characterization of disordered proteins by generating the structural ensembles of IDPs even when experimental data are not available.
    PLoS ONE 04/2014; 9(4):e95795. · 3.53 Impact Factor
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    ABSTRACT: ABCG2 is an important multidrug transporter involved also in urate transport, thus its mutations can lead to the development of gout and may also alter general drug absorption, distribution and excretion. The frequent ABCG2 polymorphism, Q141K, is associated with an elevated risk of gout and has been controversially reported to reduce the plasma membrane expression and/or the transport function of the protein. In the present work we examined the stability and cellular processing of the Q141K ABCG2 variant, as well as that of the ΔF142 ABCG2, corresponding to the ΔF508 mutation in the CFTR (ABCC7) protein, causing cystic fibrosis. The processing and localization of full length ABCG2 variants were investigated in mammalian cells, followed by Western blotting and confocal microscopy, respectively. Folding and stability were examined by limited proteolysis of Sf9 insect cell membranes expressing these ABCG2 constructs. Stability of isolated nucleotide binding domains, expressed in and purified from bacteria, was studied by CD spectroscopy. We find that the Q141K variant has a mild processing defect which can be rescued by low temperature, a slightly reduced activity, and a mild folding defect, especially affecting the NBD. In contrast, the ΔF142 mutant has major processing and folding defects, and no ATPase function. We suggest that although these mutations are both localized within the NBD, based on molecular modeling their contribution to the ABCG2 structure and function is different, thus rescue strategies may be devised accordingly.
    Biochemical and Biophysical Research Communications 06/2013; · 2.28 Impact Factor
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    ABSTRACT: To overcome the pathological phenomena caused by altered function of ABC (ATP Binding Cassette) proteins, their mechanisms of action are extensively investigated, often involving the design of mutant constructs for experiments. Designing mutagenetic constructs, interpreting the result of mutagenetic experiments, and finding individual genetic variants require an extensive knowledge of previously published mutations. To aid the recapitulation of mutations described in the literature, we set up a database of ABC protein mutations (ABCMdb) extracted from full-text papers using an automatic mining approach. We have also developed a Web application interface to compare mutations in different ABC proteins using sequence alignments and to interactively map the mutations to 3D structural models. Currently our database contains protein mutations published for ABCB1, ABCB11, ABCC1, ABCC6, ABCC7, and the proteins of the ABCG subfamily. The database will be extended to include other members and subfamilies, and to provide information on whether or not a mutation is disease causing, represents a high-incidence polymorphism, or was generated only in vitro. The ABCMdb database should already help to compare the effects of mutations at homologous positions in different ABC proteins, and its interactive tools aim to advance the design of experiments for a wider range of proteins. Hum Mutat 33:1547-1556, 2012. © 2012 Wiley Periodicals, Inc.
    Human Mutation 06/2012; 33(11):1547-56. · 5.05 Impact Factor
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    Biochimica et Biophysica Acta (BBA) - Biomembranes 05/2012; 1818(5):1435. · 3.43 Impact Factor
  • Tamás Hegedűs, John R. Riordan
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    ABSTRACT: The cystic fibrosis transmembrane conductance regulator (CFTR) comprises ATP binding and transmembrane domains, and a unique regulatory (R) domain not found in other ATP binding cassette proteins. Phosphorylation of the R domain at different sites by PKA and PKC is obligatory for the chloride channel function of CFTR. Sequence similarity searches on the R domain were uninformative. Furthermore, R domains from different species show low sequence similarity. Since these R domains resemble each other only in the location of the phosphorylation sites, we generated different R domain patterns masking amino acids between these sites. Because of the high number of the generated patterns we expected a large number of matches from the UniProt database. Therefore, a relational database management system (RDBMS) was set up to handle the results. During the software development our system grew into a general package which we term Modular BioSQL (mBioSQL). It has higher performance than other solutions and presents a generalized method for the storage of biological result-sets in RDBMS allowing convenient further analysis. Application of this approach revealed that the R domain phosphorylation pattern is most similar to those in nuclear proteins, including transcription and splicing factors.
    Central European Journal of Biology 02/2006; 1(1):29-42. · 0.63 Impact Factor
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    ABSTRACT: A kutatás célja az ABCA1 membránfehérje működésének és fehérje-kölcsönhatásainak jellemzése volt. Új modellrendszereket alakítottunk ki Sf9 rovarsejt-bakulovírus és retrovirális expressziós rendszerek segítségével, részletesen vizsgáltuk a vad-típusú és mutáns ABCA1 fehérjék sejten belüli lokalizációját, működését és PDZ fehérjékkel való kölcsönhatását. Bizonyítottuk, hogy az ABCA1 fehérje mind az ApoA1-függő koleszterin kiáramlás, mind a Ca2+-aktivált sejtfelszíni foszfatidilszerin expozíció folyamatában fontos szerepet játszik. Elsőként mutattunk ki összefüggést egy vérzékenységi betegség és az ABCA1 működése között. Elemeztük az ABCA1 mutációnak hatását a betegségre jellemző hibás foszfatidilszerin expozícióban. Vizsgáltuk a lipidanyagcserére ható vegyületek hatását az ABCA1-hez köthető funkciókra, azonosítottunk két új gátló vegyületet. Megállapítottuk, hogy egy speciális PDZ fehérje a vizsgált ABC fehérjék közül egyedül az ABCA1 fehérjével lép kölcsönhatásba, más ABC transzporterekhez kötő egyéb PDZ fehérjék nem kötődtek az ABCA1-hez. Kimutattuk polarizált sejtekben az ABCA1, a b2-syntrophin és az utrophin bazolaterális ko-lokalizációját. A kidolgozott mérési módszereket más ABC transzporterek működésének vizsgálatára is eredményesen alkalmaztuk. Megkezdtük a foszfolipid-transzportért felelős ABC fehérjék azonosítását trombocitákban. | The aims of this project were the functional characterization of the ABCA1 protein and identification of its potential interactions with intracellular proteins. We installed new assay systems to analyse the function, subcellular localization and protein interactions of the wild-type and mutant ABCA1 versions, by using two expression systems: the baculovirus-Sf9 insect cell system and retrovirus based expression system for mammalian cells. We proved that ABCA1 plays a key role both in cellular ApoAI-mediated cholesterol removal pathway, and in the exofacial translocation of phosphatidylserine. Our results provided the first link between a defect in a transbilayer phospholipid transport pathway, that of ABCA1, and the bleeding phenotype. We analysed the effects of various mutations of ABCA1 on the Ca2+-stimulated PS exposition. We screened the influence of potential inhibitors on the ABCA1-dependent processes and identified new inhibitors of the PS exposition. We demonstrated that among the examined ABC transporters only ABCA1 binds b2-syntrophin. A diverse group of PDZ proteins that interacts with other ABC proteins does not bind to ABCA1. We showed basolateral colocalization of ABCA1 protein with b2-syntrophin and utrophin. The assays for ABCA1 characterization were applied for studying other ABC proteins successfully. We started the identification of ABC proteins involved in phospholipid translocation in platelets.

Publication Stats

7 Citations
14.93 Total Impact Points

Institutions

  • 2013
    • Semmelweis University
      • Department of Biophysics and Radiation Biology
      Budapest, Budapest fovaros, Hungary
  • 2012
    • Hungarian Academy of Sciences
      • Membrane Research Group
      Budapeŝto, Budapest, Hungary
  • 2006
    • University of North Carolina at Chapel Hill
      • Department of Biochemistry and Biophysics
      North Carolina, United States