Author

Vesna Zuzel

Date

July 2010

Document Type

Thesis

Degree Name

M.S.

Department

Dept. of Biochemistry and Molecular Biology

Institution

Oregon Health & Science University

Abstract

All cells require copper as it is a co-factor for enzymes that catalyze essential reactions. A cell must balance its need for copper against the devastating oxidative damage caused by an excess of the metal. Cells express an array of copper-binding proteins that orchestrate its homeostasis and transport copper to various destinations within the cell. The consequences of copper misbalance can be observed in two diseases of copper metabolism known as Menkes and Wilson's disease; inactivating mutations in the copper ATPases ATP7A and ATP7B, respectively, causes these. Copper-ATPases are polytopic membrane proteins that catalyze the transfer of copper across biological membranes. ATP7B is expressed in the liver where it transports copper into the secretory pathway. It also traffics from the Trans-Golgi network towards the apical membrane of hepatocytes where it sequesters copper in exocytic vesicles for release into the bile. ATP7B is found in many other tissues, and in these cases it is coexpressed with ATP7A. While the role of ATP7B in the liver is understood, its function in other tissues remains elusive. Here, the expression and trafficking behavior of ATP7B is investigated in the large intestine and the ovary. The results suggest that ATP7B is expressed apically in epithelia lining the lumen and crypts of the human colon. It is shown to undergo copper-dependent trafficking in Caco-2 cells, a human cell line modeling the intestine. With this information we propose a novel model of copper excretion. ATP7B functions (in part) to sequester excess copper in vesicles near the apical/luminal membrane of colonic epithelial cells. Subsequently, the metal is released from the cells into the lumen of the large intestine where it is incorporated into the feces for removal from the body. This is hypothesized to be a physiological process secondary to biliary excretion. Expression of copper-ATPases has been linked to resistance to the chemotherapeutic agent cisplatin in tumor cells. The mechanism of resistance is highly debated, and there is evidence both for and against direct transport of cisplatin by ATP7B. A2780-CP20 cells over-express ATP7B and are resistant to cisplatin. We show that ATP7B does not traffic when these cells are treated with either copper or cisplatin. These findings suggest that ATP7B binds to, and sequesters copper in these cells for fine-tuning its release when required. The lack of trafficking argues against ATP7B-mediated efflux of cisplatin across plasma membrane in these cells, and therefore resistance is caused by an alternate mechanism such as binding and sequestration.

Identifier

doi:10.6083/M41834GZ

School

School of Medicine

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