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.
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.
|Jayakanthan, Samuel; Braiterman, Lelita T; Hasan, Nesrin M et al. (2017) HUMAN COPPER TRANSPORTER ATP7B (WILSON DISEASE PROTEIN) FORMS STABLE DIMERS IN VITRO AND IN CELLS. J Biol Chem :|
|Hamilton, James P; Koganti, Lahari; Muchenditsi, Abigael et al. (2016) Activation of liver X receptor/retinoid X receptor pathway ameliorates liver disease in Atp7B(-/-) (Wilson disease) mice. Hepatology 63:1828-41|
|Krishnamoorthy, Lakshmi; Cotruvo Jr, Joseph A; Chan, Jefferson et al. (2016) Copper regulates cyclic-AMP-dependent lipolysis. Nat Chem Biol 12:586-92|
|Gupta, Arnab; Schell, Michael J; Bhattacharjee, Ashima et al. (2016) Myosin Vb mediates Cu+ export in polarized hepatocytes. J Cell Sci 129:1179-89|
|Kline, Chelsey D; Gambill, Benjamin F; Mayfield, Mary et al. (2016) pH-regulated metal-ligand switching in the HM loop of ATP7A: a new paradigm for metal transfer chemistry. Metallomics 8:729-33|
|Clifford, Rebecca J; Maryon, Edward B; Kaplan, Jack H (2016) Dynamic internalization and recycling of a metal ion transporter: Cu homeostasis and CTR1, the human Cu? uptake system. J Cell Sci 129:1711-21|
|Dmitriev, Oleg Y; Lutsenko, Svetlana; Muyldermans, Serge (2016) Nanobodies as Probes for Protein Dynamics in Vitro and in Cells. J Biol Chem 291:3767-75|
|Braiterman, Lelita T; Gupta, Arnab; Chaerkady, Raghothama et al. (2015) Communication between the N and C termini is required for copper-stimulated Ser/Thr phosphorylation of Cu(I)-ATPase (ATP7B). J Biol Chem 290:8803-19|
|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|
|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|
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