The major goal of this project is to generate a comprehensive picture of cellular mechanisms regulating intracellular copper levels through copper delivery to the secretory pathway and copper export from the cell. The project focuses on the analysis of copper-transporting ATPases ATP7A and ATP7B, which play an essential role in this regulation. In humans, mutations in these transporters cause severe metabolic disorders, Menkes disease and Wilson's disease, respectively.
Specific Aim 1 outlines experiments dissecting the mechanism of copper release from Cu-ATPases. The hypothesis will be tested that the first luminal loop of the Cu-ATPases stimulates copper release from the transporters by sequestering copper at the previously unidentified metalbinding sites. The metal-binding properties of the loop region will be characterized, its structure will be determined by NMR, and the effect of mutations within the first loop on the function of Cu-ATPases will be examined.
Specific Aim 2 will determine the role of luminal copper acceptor proteins in transport activity and trafficking of Cu-ATPases from the secretory pathway. The experiments under Specific Aim 3 will investigate specific functions of ATP7A and ATP7B in balancing copper concentration in kidney. Both Wilson's disease and Menkes disease are associated with copper accumulation in renal proximal tubules suggesting distinct and nonoverlapping roles of ATP7A and ATP7B in renal copper homeostasis. This hypothesis will be tested by determining the effect of Cu-ATPases inactivation on total cellular copper content and on copper efflux from the basolateral and apical membranes of polarized renal cells. The effect of small molecular weight compound, which is highly elevated in the serum and urine of Atp7b-/- mice, on copper uptake and localization of ATP7A will be determined. The studies will produce novel mechanistic information necessary for better understanding of human copper homeostasis and better treatment of disorders associated with copper misbalance.

Public Health Relevance

The studies involve characterization of two copper transporters, ATP7A and ATP7B, that are associated with severe human disorders of copper metabolism. The results of the proposed experiments are expected to help better understanding of Menkes disease and Wilson disease phenotypes as well as to identify cellular factors that regulate human copper homeostasis in health and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM067166-10
Application #
8530245
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
$303,463
Indirect Cost
$107,912
Name
Johns Hopkins University
Department
Type
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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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
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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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