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 targefing 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 confinue 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 mutafions, 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 interacfions 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 characterizafion 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.
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 overioad and is characterized by liver damage and/or abnormal ;neurological symptoms. Thus, understanding the dynamics of hepafic ATP7B is important. ?:
|Singh, Varsha; Yang, Jianbo; Yin, Jianyi et al. (2018) Cholera toxin inhibits SNX27-retromer-mediated delivery of cargo proteins to the plasma membrane. J Cell Sci 131:|
|Yin, Jianyi; Tse, Chung-Ming; Avula, Leela Rani et al. (2018) Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers. Cell Mol Gastroenterol Hepatol 5:591-609|
|Sarker, Rafiquel; Cha, Boyoung; Kovbasnjuk, Olga et al. (2017) Phosphorylation of NHE3-S719 regulates NHE3 activity through the formation of multiple signaling complexes. Mol Biol Cell 28:1754-1767|
|Qiang, Xiaoling; Liotta, Anthony S; Shiloach, Joseph et al. (2017) New melanocortin-like peptide of E. coli can suppress inflammation via the mammalian melanocortin-1 receptor (MC1R): possible endocrine-like function for microbes of the gut. NPJ Biofilms Microbiomes 3:31|
|Cha, Boyoung; Yang, Jianbo; Singh, Varsha et al. (2017) PDZ domain-dependent regulation of NHE3 protein by both internal Class II and C-terminal Class I PDZ-binding motifs. J Biol Chem 292:8279-8290|
|Yu, Huimin; Hasan, Nesrin M; In, Julie G et al. (2017) The Contributions of Human Mini-Intestines to the Study of Intestinal Physiology and Pathophysiology. Annu Rev Physiol 79:291-312|
|Foulke-Abel, Jennifer; In, Julie; Yin, Jianyi et al. (2016) Human Enteroids as a Model of Upper Small Intestinal Ion Transport Physiology and Pathophysiology. Gastroenterology 150:638-649.e8|
|Gupta, Arnab; Schell, Michael J; Bhattacharjee, Ashima et al. (2016) Myosin Vb mediates Cu+ export in polarized hepatocytes. J Cell Sci 129:1179-89|
|Zachos, Nicholas C; Kovbasnjuk, Olga; Foulke-Abel, Jennifer et al. (2016) Human Enteroids/Colonoids and Intestinal Organoids Functionally Recapitulate Normal Intestinal Physiology and Pathophysiology. J Biol Chem 291:3759-66|
|Cebotaru, Liudmila; Liu, Qiangni; Yanda, Murali K et al. (2016) Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease. Kidney Int 90:90-9|
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