The purpose of the Metal Ion Core is to provide the instrumentation, technical support, and to generate reagents necessary to fulfill the goals of this application. The core will provide atomic absorption analysis of metals; equipment and expertise to perform two-dimensional electrophoresis analysis of proteins, a mass spectrometry facility for the analysis of proteins and peptides;and a confocal microscope facility for the immunolocalization of proteins in cells and tissues. The atomic absorption instrument will allow sensitive quantitative analysis of copper and selenium ions necessary to study copper transport and transfer (Projects 1,2, and 4). The mass spectrometer will be used to identify protein interacting with the copper transporters and regulating their intracellular localization and trafficking behavior (Projects 3,4, and 5), as well as to confirm the efficiency of incorporation of selenocysteine into proteins prepared for small angle x-ray diffraction experiments. The confocal microscope will allow direct visualization of the subcellular localization of ion transport proteins so that the mechanisms controlling their trafficking in normal and diseased tissue can be understood (Project 4). The important charge of the core will be to express and purify recombinant proteins for production of antibodies against proteins involved in copper homeostasis and well as to generate proteins for collaborative research between all five projects. These latter category of proteins includes various mutants of ATP7B (Project 5), NBDdelta TMS1,2 (Project 2 and 4), and deltal-63 protein (Projects 4 and 5)

Public Health Relevance

The services provided by the Metal Ion Core and the shared reagents that will be generated in the course of the proposed experiments will facilitate studies by the Program Project. The major goal of the Program Project is to understand the metal ion regulation in human health and disease. The protocols and reagents produced by the Core will also be made available to the biomedical community.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM067166-10
Application #
8530247
Study Section
Special Emphasis Panel (ZRG1-CB-L)
Project Start
Project End
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
10
Fiscal Year
2013
Total Cost
$230,415
Indirect Cost
$89,918
Name
Johns Hopkins University
Department
Type
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Malinouski, Mikalai; Hasan, Nesrin M; Zhang, Yan et al. (2014) Genome-wide RNAi ionomics screen reveals new genes and regulation of human trace element metabolism. Nat Commun 5:3301
Braiterman, Lelita T; Murthy, Amrutha; Jayakanthan, Samuel et al. (2014) Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B. Proc Natl Acad Sci U S A 111:E1364-73
Nyasae, Lydia K; Schell, Michael J; Hubbard, Ann L (2014) Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes. Traffic 15:1344-65
Lutsenko, Svetlana (2014) Modifying factors and phenotypic diversity in Wilson's disease. Ann N Y Acad Sci 1315:56-63
Maryon, Edward B; Molloy, Shannon A; Kaplan, Jack H (2013) Cellular glutathione plays a key role in copper uptake mediated by human copper transporter 1. Am J Physiol Cell Physiol 304:C768-79
Maryon, Edward B; Molloy, Shannon A; Ivy, Kristin et al. (2013) Rate and regulation of copper transport by human copper transporter 1 (hCTR1). J Biol Chem 288:18035-46
Ivy, Kristin D; Kaplan, Jack H (2013) A re-evaluation of the role of hCTR1, the human high-affinity copper transporter, in platinum-drug entry into human cells. Mol Pharmacol 83:1237-46
Flores, Adrian G; Unger, Vinzenz M (2013) Atox1 contains positive residues that mediate membrane association and aid subsequent copper loading. J Membr Biol 246:903-13
Barry, Amanda N; Otoikhian, Adenike; Bhatt, Sujata et al. (2011) The lumenal loop Met672-Pro707 of copper-transporting ATPase ATP7A binds metals and facilitates copper release from the intramembrane sites. J Biol Chem 286:26585-94
Braiterman, L; Nyasae, L; Leves, F et al. (2011) Critical roles for the COOH terminus of the Cu-ATPase ATP7B in protein stability, trans-Golgi network retention, copper sensing, and retrograde trafficking. Am J Physiol Gastrointest Liver Physiol 301:G69-81

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