25–30 May 2020, Spetses Island, Greece
Two main projects are pursued in my lab.
The Membrane biology group works on ABC transporters and multidrug resistance. We are interested in the mechanistic and molecular relationships between catalytic activity, conformational changes and microenvironment of ABC transporters. P-glycoprotein (ABCB1, Pgp) is in the focus of our interest; we have currently extended our work to ABCG2 (BCRP) and plan to do similar studies on MRP1 (ABCC1). The members of the ABC superfamily of membrane transporters are involved in the regulation of the uptake into and distribution within our body of physiological substrates as well as various xenobiotics, drugs. Due to their wide substrate spectrum, a consequence of their preference for lipophylic compounds, they also play a critical role in the multidrug resistance phenomenon severely limiting therapeutical success in cancer. Our ambition is to understand the molecular details of their catalytic cycle and the intimate molecular interactions with their microenvironment, as well as to apply the knowledge obtained at the cell/molecule level in the context of the whole organism, in vivo.
The Nuclear organization and chromatin structure group is interested in the regulatory signficance of chromatin being divided into superhelical and relaxed domains. Due to the repressive nature of nucleosomes, the main underlying scheme of gene regulation in eukaryotic cells is de-repression. On the other hand, nucleosome stability is highly sensitive to the degree of superhelicity, a transcription dependent feature of the matrix anchored DNA loops. However, at their anchorage points, genomic DNA is relaxed due to strand discontinuities arising in conjunction with transcription, likely introduced by topoisomerase enzymes. We are investigating the molecular determinants and dynamic features of these two distinct chromatin domains in the context of gene activition and global nuclear compartmentalization.
In conjunction with the first project, our experimental approaches include: (a) sensitive flow-cytometric methods to detect substrate efflux; (b) ATPase assays to measure the interaction of drugs with ABC transporters in membrane fractions; (c) intracellular ATP concentration measurements in live cells using a FRET-based intracellular ATP sensor; (d) a flow-cytometric platform for the measurement of raft- and cytoskeleton-association of cell-surface proteins, applicable also for rapid, simple, serial analysis of intermolecular associations between different cell surface proteins; (e) immunoprecipitation and co-immunprecipitation to study protein-protein interactions by Western-blot and MALDI analysis; (f) a sensitive flow cytometric assay to measure ATP dependent conformational changes of Pgp and ABCG2 in Staphylococcus toxin permeabilized cells; (g) a confocal microscopic assay to measure substrate binding to transporters in live as well as permeabilized cells; (h) xenotransplantation system in SCID mice to study the effect of ABC transporter modulators on multidrug resistant tumors by PET.
Regarding the second, our experimental systems include mammalian cells, mouse embryonic stem cells and S. cerevisiae, studied by (a) confocal microscopic analysis of the localisation patterns of free 3’OHs detected by in situ nick translation or terminal transferase reaction relative to those of transcription related R-loops (RNA/DNA-hybrids), topoisomerase enzymes and epigenetic marks (PTMs); (b) a laser scanning cytometric (LSC) in situ assay of nucleosome stability, measured in a PTM and cell cycle dependent manner; (c) an LSC assay of superhelical density and size distribution of loops; (d) molecular combing of DNA; (e,f) various gelelectrophoretic techniques (denaturing urea-agarose, CHEF), including single-cell electrophoresis of large DNA molecules; (g) a reverse South-Western blotting technique to map strand breaks and R-loops.
Superresolution microscopy, electronmicroscopy of the cell nucleus, mass spectrometry of immunoprecipitates, bioinformatics knowhow for chipseq analyses
1: Bársony O, Szalóki G, Türk D, Tarapcsák S, Gutay-Tóth Z, Bacsó Z, Holb IJ,
Székvölgyi L, Szabó G, Csanády L, Szakács G, Goda K. A single active catalytic
site is sufficient to promote transport in P-glycoprotein. Sci Rep. 2016 Apr 27;6:24810
2: Goda K, Bacsó Z, Szabó G. Multidrug resistance through the spectacle of
P-glycoprotein. Curr Cancer Drug Targets. 2009 May;9(3):281-97. Review.
3: Krasznai ZT, Tóth A, Mikecz P, Fodor Z, Szabó G, Galuska L, Hernádi Z, Goda K.
Pgp inhibition by UIC2 antibody can be followed in vitro by using
tumor-diagnostic radiotracers, 99mTc-MIBI and 18FDG. Eur J Pharm Sci. 2010 Dec
4: Szekvolgyi L, Imre L, Minh DX, Hegedus E, Bacso Z, Szabo G. Flow cytometric
and laser scanning microscopic approaches in epigenetics research. Methods Mol
5: Székvölgyi L, Rákosy Z, Bálint BL, Kókai E, Imre L, Vereb G, Bacsó Z, Goda K,
Varga S, Balázs M, Dombrádi V, Nagy L, Szabó G. Ribonucleoprotein-masked nicks at
50-kbp intervals in the eukaryotic genomic DNA. Proc Natl Acad Sci U S A. 2007
25–30 May 2020, Spetses Island, Greece