Our long-term goal is to elucidate the molecular mechanisms by which two mammalian Cu-ATPases regulate copper homeostasis in polarized epithelial cells. These Cu-ATPases have two functions that necessitate their intracellular redistribution, or trafficking, In a copper-sensitive and reversible manner. 1) They transport copper Into the secretory pathway to metallate newly-synthesized cuproenzymes. 2) They also export copper. ATP7A in intestinal epithelial cells delivers dietary Cu to the circulation (basolateral environment), and ATP7B in hepatic cells delivers excess Cu to the bile (apical environment). In this PPG, we will focus on the hepatic Cu-ATPase, ATP7B. Like the other membrane transport proteins being studied in this PPG, ATP7B trafficks In a regulated manner to the apical region of a polarized epithelial cell, the hepatocyte. Once there, it exports the metal ion Cu(l) Into bile. But the signals/mechanism(s) mediating the copper-sensitive targeting of ATP7B, the functional nature ofthe vesicles carrying the protein and the Cu(l) export mechanisms it uses at the apical membrane are currently unknown. Based on new results from two years of PPG funding, we will continue our studies in WIF-B cells and in vivo.
In Aim 1, we will identify sequences and domains in ATP7B itself that regulate its copper-dependent dynamics (TGN-retention, apical targeting and Cu(l) export). We will generate adenoviruses encoding C- and new N-terminal Wilson Disease :(WD)-causing GFP-ATP7B mutations, express them in WlF-B for protein expression and trafficking +/- Cu, and In MNK y/- cells (no ATP7A or ATP7B) for their Cu(l)-loading activity. We will determine Intramolecular interactions of ATP7B through analysis of 7B/7A chimera expression/trafficking phenotypes. We will perform FRAP studies on selected mutants.
In Aim 2, we will continue Identification, isolation and functional characterization of vesicles carrying endogenous ATP7B (and Cu(l)?) to the apical region of hepatocytes of copper-loaded mice. We will also identify ATP7B protein interactors +/- Cu. We will determine if ATP7B trafficks via the well-studied Rablla apical recycling compartment. Finally, we will express selected WD-ATP7B mutants in ATP7B-/- mice to determine the mutant protein's Cu(l) export phenotype in vivo.

Public Health Relevance

Copper-transporting ATPases, ATP7A and ATP7B, are important In health and disease. Hepatic ATP7B;normally exports excess copper from the liver. In the absence of ATP7B function, affected people develop j Wilson Disease, which is due to copper overload and is characterized by liver damage and/or abnormal;neurological symptoms. Thus, understanding the dynamics of hepatic ATP7B is important. ?:

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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