Finished grants:

  • Study of function of membrane proteins

    Grant agency: GAUK
    Identification number of grant: GAUK 456213
    Principal investigator: Mgr. Jakub Zahumenský, Ph.D.
    Co-Principal investigators: doc. RNDr. Dana Gášková, CSc.

    Grant annotation

    The main aim of the project is a detailed and systematic study of yeast S. cerevisiae membrane transporters, their function and reaction to chemical stress in various conditions. Main subjects of the study will be multidrug resistance (MDR) proteins Pdr10 and Pdr15 and plasma H+-ATPase (Pma1) – means of activation and inhibition.
    We will also focus on non-native S. cerevisiae transporters that can be expressed heterologously, above all Cdr1 and Cdr2 – MDR proteins native to yeast Candida albicans (resistance of yeast infections and contaminations) [1, 2].
    Chemical stress will be induced by medical and agricultural compounds, e.g. antibiotics, fungicides, immunosuppressants, cytostatics. The proteins will be studied in intact yeast cells using the staining curves method [3] with the redistribution potentiometric fluorescent probe diS-C3(3) that is a substrate of two important yeast MDR pumps (Pdr5, Snq2) [4].
    Yeast MDR proteins are responsible for high resistance of infections and contaminations. At the same time, they share a high homology with human MDR proteins which elevate resistance of cancer cells against chemotherapeutics. Yeast and higher eukaryotes share the same basic structural and functional organization of the cell [5]. The results of the project may therefore be significant for medicine.

  • The study of multidrug resistance of yeast Kluyveromyces lactis

    Grant agency: GAUK
    Identification number of grant: GAUK 1072313
    Principal investigator: Mgr. Iva Jančíková, Ph.D.
    Co-Principal investigators: doc. RNDr. Dana Gášková, CSc.

    Grant annotation

    During treatment of some diseases (e.g. cancer), lower efficiency of drugs was observed as a consequence of evolved cell resistance. It is among others due to specific transporters present in cell membranes and actively expelling wide range of drugs from the cell. Their overexpression leads to the multidrug resistance (MDR). These proteins (pumps) are present in all type of cells. Pdr5p of yeast Saccharomyces cerevisiae and human P-glycoprotein belong to the group of the better-studied eukaryotic MDR pumps.
    This project proposal focuses on less explored Pdr5p homolog from yeast Kluyveromyces lactis. We plan to use the fluorescent method developed for studying the activity of two major pumps of S. cerevisiae Pdr5p and Snq2p, to characterize the Pdr5p pump from Kluyveromyces lactis (termed Kl Pdr5p). The method is based on the monitoring of differences in the accumulation of the potentiometric probe diS-C3(3) in the cells with diverse activity of pumps, which extrude the probe from the cells (the probe is their substrate).
    The successful implementation of the fluorescent method to study pump Kl Pdr5p enable us to observe the factors affecting its activity, including searching for efficient inhibitors, and we could also compare the activity of Pdr5p for both yeast species.

  • Biophysical studies of functional characteristics of bacterial MntH protein (Proton-dependent Manganese Transporter) from SLC11 (SoLute Carrier 11) family and its selected transmembrane segments

    Grant agency: GAUK
    Identification number of grant: GAUK 701512
    Principal investigator: Mgr. Iva Jančíková, Ph.D.
    Co-Principal investigators: RNDr. Roman Chaloupka, Ph.D.

    Grant annotation

    The SLC11 family (or Nramp) includes secondary active transport proteins transporting divalent metal ions together with protons into a cell. These proteins are also connected with the response of immune system. The exact mechanism of this membrane transport is still unknown. The MntH protein is structural and functional homologous to the eukaryote proteins Nramp, so it represents suitable prototypic model for the functional study of the whole SLC11 family. Furthermore some synthetic peptides corresponding to the transmembrane™ segments of membrane protein MntH can associate and form together a channel. Fluorescence spectrometry is representing a suitable instrument for the transport study of the whole protein in vivo and also to observe the behavior of the peptides in the model membranes of liposomes. Fluorescence spectrometry combined with mass spectrometry will be used to find the transport stoichiometry, influence of the different conditions, interaction of MntH with other ions (e.g. Ca2+) and other functionally important characteristics. Finally two synthetic peptides will be studied (corresponding to TMS3 and TMS6), their channel specificity and their interactions with another ions. The special procedures will be developed and optimized for the experiments using varied suitable fluorescence probes.