Copper (Cu) is an essential trace metal that is acquired from the diet and serves as a catalytic co-factor for a wide variety of enzymatic reactions that play critical roles in life. Cu deficiency leads to pathophysiological manifestations including impaired iron absorption, neutropenia, cognitive defects, peripheral neuropathy and hypertrophic cardiomyopathy. Understanding the mechanisms responsible for the accumulation of Cu in cells and tissues, the regulation of Cu accumulation, and the consequences due to dysregulated Cu acquisition are important to human health. Ctr1 is the only known Cu+ importer in mammals and while Ctr1 plays an essential role in dietary and peripheral Cu acquisition, embryonic development, cardiac function and normal growth, little is known about the mechanisms that regulate Ctr1 activity. Ctr1 exists both as a full-length protein and as a truncated form (tCtr1) lacking the extracellular Cu binding domain, which has reduced Cu uptake activity. We identified cathepsin as a protease that carries out the rate-limiting step in Ctr1 ecto-domain cleavage and demonstrated that this cleavage is stimulated by the Ctr2 integral membrane protein. Among patients in a large cardiac catheterization clinic cohort, we identified a single nucleotide polymorphism (SNP) in the human Ctr1 gene that occurs predominantly in African Americans, resulting in hyper-cleavage of the Ctr1 Cu-binding ecto-domain and decreased cellular Cu acquisition. Here we detail experiments to test the hypothesis that Ctr1 ecto-domain cleavage, through the cathepsin L/B proteases and Cu-responsive Ctr2 levels, is a critical regulatory mechanism for mammalian Cu acquisition. Our experiments will identify new components in mammalian Cu homeostasis, decipher a new mechanism for Cu- dependent proteolysis, generate a new animal model and validate a link between a defect in Ctr1 ecto-domain cleavage, Cu deficiency and hypertrophic cardiomyopathy in African Americans.

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Copper is an essential trace element for embryonic development, growth and metabolism in humans and copper deficiency leads to anemia, cognitive and neurological defects and cardiac disease. The research outlined in this application will enhance our understanding of how copper absorption is regulated and ascertain how defects in these regulatory mechanisms, which may disproportionately occur in African Americans, impacts copper metabolism and cardiac disease.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Macromolecular Structure and Function A Study Section (MSFA)
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Maruvada, Padma
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Duke University
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Logeman, Brandon L; Thiele, Dennis J (2018) Reconstitution of a thermophilic Cu+ importer in vitro reveals intrinsic high-affinity slow transport driving accumulation of an essential metal ion. J Biol Chem 293:15497-15512
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Öhrvik, Helena; Logeman, Brandon; Turk, Boris et al. (2016) Cathepsin Protease Controls Copper and Cisplatin Accumulation via Cleavage of the Ctr1 Metal-binding Ectodomain. J Biol Chem 291:13905-16
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