The overall goal of the proposed research is to determine the three-dimensional structures of the FXYD family of small, homologous, ion transport regulators or channels, in phospholipid bilayer membranes, using NMR spectroscopy. The proteins of this family are implicated in various diseases, including breast cancer and heart disease, and include the Mammary tumor (Mat8) membrane protein which is expressed both in primary human breast tumors and breast tumor cell lines, and is believed to be a marker of specific oncogenic cell transformation. Mat8, like the other members of the FXYD family, functions as an ion channel or channel modulator, involved in the regulation of transmembrane ion flux and transport across breast epithelial tissue. The ion channel activities of Mat8, and of the other FXYD proteins, provide the initial insight into their mechanisms of action and potential as a drug receptors: since inhibitors of the Mat8 channel may kill breast cancer cells, this protein is an attractive candidate for the development of anti-cancer drugs. The structure of Mat8 is the starting point for the rational screening and design of molecules that interfere with its function. The structures of the FXYD membrane proteins will be determined using solid state and solution NMR spectroscopy of isotopically labeled samples of the proteins in lipid bilayers or in lipid micelles. The procedure for structure determination by NMR spectroscopy involves the following steps: (1) the expression and purification of milligram quantities of isotopically labeled recombinant proteins; (2) the preparation of samples of isotopically labeled protein in oriented lipid bilayers for solid state NMR spectroscopy, and in isotropic lipid micelles for solution NMR spectroscopy; (3) the execution of multi-dimensional solid state and solution NMR experiments needed for structure determination; and (4) the analysis of experimental data to determine the protein structure in the membrane. An important goal of this research will be to correlate the structures with the ion channel activities of the FXYD proteins, determined through concentrative ion uptake assays of the proteins reconstituted in liposomes, and by measuring channel recordings of the proteins reconstituted in planar lipid bilayers. The three-dimensional structures of the FXYD proteins, correlated with their ion channel activities, are an important step towards understanding their functions. They are also the starting point for the development of inhibitor molecules targeted to breast cancer and other diseases.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA082864-03
Application #
6603993
Study Section
Biophysical Chemistry Study Section (BBCB)
Program Officer
Lees, Robert G
Project Start
2001-07-01
Project End
2006-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
3
Fiscal Year
2003
Total Cost
$297,000
Indirect Cost
Name
Sanford-Burnham Medical Research Institute
Department
Type
DUNS #
020520466
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Gong, Xiao-Min; Ding, Yi; Yu, Jinghua et al. (2015) Structure of the Na,K-ATPase regulatory protein FXYD2b in micelles: implications for membrane-water interfacial arginines. Biochim Biophys Acta 1848:299-306
Bibert, St├ęphanie; Liu, Chia-Chi; Figtree, Gemma A et al. (2011) FXYD proteins reverse inhibition of the Na+-K+ pump mediated by glutathionylation of its beta1 subunit. J Biol Chem 286:18562-72
Teriete, Peter; Thai, Khang; Choi, Jungyuen et al. (2009) Effects of PKA phosphorylation on the conformation of the Na,K-ATPase regulatory protein FXYD1. Biochim Biophys Acta 1788:2462-70
Franzin, Carla M; Gong, Xiao-Min; Teriete, Peter et al. (2007) Structures of the FXYD regulatory proteins in lipid micelles and membranes. J Bioenerg Biomembr 39:379-83
Franzin, Carla M; Teriete, Peter; Marassi, Francesca M (2007) Membrane orientation of the Na,K-ATPase regulatory membrane protein CHIF determined by solid-state NMR. Magn Reson Chem 45 Suppl 1:S192-7
Franzin, Carla M; Gong, Xiao-Min; Thai, Khang et al. (2007) NMR of membrane proteins in micelles and bilayers: the FXYD family proteins. Methods 41:398-408
Teriete, Peter; Franzin, Carla M; Choi, Jungyuen et al. (2007) Structure of the Na,K-ATPase regulatory protein FXYD1 in micelles. Biochemistry 46:6774-83
Franzin, Carla M; Teriete, Peter; Marassi, Francesca M (2007) Structural similarity of a membrane protein in micelles and membranes. J Am Chem Soc 129:8078-9
Gong, Xiao-Min; Franzin, Carla M; Thai, Khang et al. (2007) Nuclear magnetic resonance structural studies of membrane proteins in micelles and bilayers. Methods Mol Biol 400:515-29
Pilch, Jan; Franzin, Carla M; Knowles, Lynn M et al. (2006) The anti-angiogenic peptide anginex disrupts the cell membrane. J Mol Biol 356:876-85

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