The long-term goal of this research is to elucidate the systemic and cellular processes that control copper homeostasis. Copper is an essential trace element. The misbalance of copper, either through poor absorption or overload results in severe disorders such as Menkes disease, progressive ataxic myelopathy, and Wilson's disease (WD). WD is caused by mutations in the ATP7B gene. ATP7B is a copper- transporting ATPase, that pumps copper out of the body and into the bile. ATP7B inactivation causes the accumulation of toxic levels of copper In the body, especially in the liver which can lead to cirrohosis and failure. WD patients also suffer from neurological and pyschiatric abnormalities. If WD is diagnosed early it can be treated with life-long treatment of copper-chelation therapy. Since, all the manifestations presented in Wilson's disease are not unique, diagnosing the disease is a challenge. Even though we now understand some general aspects of copper regulation, we have yet to explain some of the characteristic features of WD. Specifically, (1) why do WD patients have increased urinary copper? (2) What is the nature of the urinary copper? (3) How is the kidney able to balance copper so well? My recent discovery of a novel small copper-carrier (SCC) in the urine of WD patients can help fill these gaps.
Specific Aim I : To characterize SCC in the urine of the Wilson's disease mouse (Atp7b-/-). To do this, I will use a series of high-resolution chromatographic steps to purify SCC to homogeneity and characterize it by tandem mass-spectrometry.
Specific Aim II : To characterize the relationship between CTR1 levels in ATP7b-/- hepatoctyes and SCC in the urine. I hypothesize that CTR1 down-regulation leads to increased SCC in the urine. To test this I will measure CTR1 mRNA and protein (by real-Time PCR and Western blot) in the liver at different stages of the disease and compare it to amounts of SCC over the same time period Specific Aim III: To determine the molecular basis of ATP7A protein upregulation in Atp7b-/- kidney. I hypothesize that copper delivery by SCC, ATP7B inactivation, and decrease in miR-107 lead to upregulation of ATP7A. To test this, I will perform copper uptake, siRNA, and northern blot analyses. Relevance to public health: Understanding these questions would provide better insight into how the body balances copper levels. Additionally, SCC could be a unique indicator of WD.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1-DKUS-D (29))
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Agodoa, Lawrence Y
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Johns Hopkins University
Schools of Arts and Sciences
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Gray, Lawrence W; Peng, Fangyu; Molloy, Shannon A et al. (2012) Urinary copper elevation in a mouse model of Wilson's disease is a regulated process to specifically decrease the hepatic copper load. PLoS One 7:e38327
Hirayama, Tasuku; Van de Bittner, Genevieve C; Gray, Lawrence W et al. (2012) Near-infrared fluorescent sensor for in vivo copper imaging in a murine Wilson disease model. Proc Natl Acad Sci U S A 109:2228-33
Burkhead, Jason L; Gray, Lawrence W; Lutsenko, Svetlana (2011) Systems biology approach to Wilson's disease. Biometals 24:455-66