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. First, they transport copper into the secretory pathway to metallate newly-synthesized cuproenzymes. They also efflux 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). This project focuses on cellular proteins that modulate the Cu-ATPases'trafficking and export functions.
In Aim 1 A, we will determine the dynamics of ATP7B in the polarized hepatic WIF-B cells, after over-expressing or knocking down the dynactin subunit, p62, which binds the N-terminal domain of ATP7B (not ATP7A) when copper is elevated. p62 is proposed to direct the apical movement of ATP7B-positive vesicles along microtubules. Titration of cellular copper with immunofluorescence and copper-64 efflux determinations will be performed.
Aim 1 B focuses on a 9 amino acid apical targeting motif we discovered in the N-terminus of only ATP7B. We will collaborate with Projects 3 + 4 to determine if the targeting motif interacts with other domains of ATP7B and if a chimera containing this motif fused to a basolateral protein will target to the apical membrane. To identify the protein (X) proposed to bind the targeting motif, we will use 2 approaches: a membrane-based yeast-two hybrid system;and a screen of putative binding domains of candidate proteins.
In Aim 2, we will determine the mechanism by which a PDZ protein, AIPP, targets/retains endogenous ATP7A to/in the basolateral region of polarized intestinal Caco-2 cells. We will over-express and knock down AIPP1 using approaches similar to those in Aim 1A and collaborate with Project 3 to determine if/how AIPP1 levels affect Cu export.
In Aim 3, we will study the role of ATOX1, the copper chaperone for both Cu-ATPases, and its proposed modifier, FKBP52, in copper homeostasis in vivo in compound heterozygote mice (ATOX1+/-; FKBP+/-). We will collaborate with Projects 2 and 3 to study the liver, intestine and kidney in these animals.
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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|
|Lutsenko, Svetlana (2014) Modifying factors and phenotypic diversity in Wilson's disease. Ann N Y Acad Sci 1315:56-63|
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|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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