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The lumenal loop Met672-Pro707 of copper-transporting ATPase ATP7A binds metals and facilitates copper release from the intramembrane sites.

ABSTRACT: The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met(672)-Pro(707) (HM-loop) that connects the first two transmembrane segments of ATP7A is important for copper release. Mutations within this loop do not prevent the ability of ATP7A to form a phosphorylated intermediate during ATP hydrolysis but inhibit subsequent dephosphorylation, a step associated with copper release. The HM-loop inserted into a scaffold protein forms two structurally distinct binding sites and coordinates copper in a mixed His-Met environment with an ?2:1 stoichiometry. Binding of either copper or silver, a Cu(I) analog, induces structural changes in the loop. Mutations of 4 Met residues to Ile or two His-His pairs to Ala-Gly decrease affinity for copper. Altogether, the data suggest a two-step process, where copper released from the transport sites binds to the first His(Met)(2) site, triggering a structural change and binding to a second 2-coordinate His-His or His-Met site. We also show that copper binding within the HM-loop stabilizes Cu(I) and protects it from oxidation, which may further aid the transfer of copper from ATP7A to acceptor proteins. The mechanism of copper entry into the secretory pathway is discussed.

SUBMITTER: Barry AN

PROVIDER: S-EPMC3143623 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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The lumenal loop Met672-Pro707 of copper-transporting ATPase ATP7A binds metals and facilitates copper release from the intramembrane sites.

Barry Amanda N AN Otoikhian Adenike A Bhatt Sujata S Shinde Ujwal U Tsivkovskii Ruslan R Blackburn Ninian J NJ Lutsenko Svetlana S

The Journal of biological chemistry 20110606 30

The copper-transporting ATPase ATP7A has an essential role in human physiology. ATP7A transfers the copper cofactor to metalloenzymes within the secretory pathway; inactivation of ATP7A results in an untreatable neurodegenerative disorder, Menkes disease. Presently, the mechanism of ATP7A-mediated copper release into the secretory pathway is not understood. We demonstrate that the characteristic His/Met-rich segment Met(672)-Pro(707) (HM-loop) that connects the first two transmembrane segments o ...[more]

PMID: 21646353

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Project description:OBJECTIVE:Copper (Cu) is essential micronutrient, and its dysregulation is implicated in aortic aneurysm (AA) development. The Cu exporter ATP7A (copper-transporting P-type ATPase/Menkes ATPase) delivers Cu via the Cu chaperone Atox1 (antioxidant 1) to secretory Cu enzymes, such as lysyl oxidase, and excludes excess Cu. Lysyl oxidase is shown to protect against AA formation. However, the role and mechanism of ATP7A in AA pathogenesis remain unknown. Approach and Results: Here, we show that Cu chelator markedly inhibited Ang II (angiotensin II)-induced abdominal AA (AAA) in which ATP7A expression was markedly downregulated. Transgenic ATP7A overexpression prevented Ang II-induced AAA formation. Conversely, Cu transport dysfunctional ATP7Amut/+/ApoE-/- mice exhibited robust AAA formation and dissection, excess aortic Cu accumulation as assessed by X-ray fluorescence microscopy, and reduced lysyl oxidase activity. In contrast, AAA formation was not observed in Atox1-/-/ApoE-/- mice, suggesting that decreased lysyl oxidase activity, which depends on both ATP7A and Atox1, was not sufficient to develop AAA. Bone marrow transplantation suggested importance of ATP7A in vascular cells, not bone marrow cells, in AAA development. MicroRNA (miR) array identified miR-125b as a highly upregulated miR in AAA from ATP7Amut/+/ApoE-/- mice. Furthermore, miR-125b target genes (histone methyltransferase Suv39h1 and the NF-?B negative regulator TNFAIP3 [tumor necrosis factor alpha induced protein 3]) were downregulated, which resulted in increased proinflammatory cytokine expression, aortic macrophage recruitment, MMP (matrix metalloproteinase)-2/9 activity, elastin fragmentation, and vascular smooth muscle cell loss in ATP7Amut/+/ApoE-/- mice and reversed by locked nucleic acid-anti-miR-125b infusion. CONCLUSIONS:ATP7A downregulation/dysfunction promotes AAA formation via upregulating miR-125b, which augments proinflammatory signaling in a Cu-dependent manner. Thus, ATP7A is a potential therapeutic target for inflammatory vascular disease.

Project description:During iron deficiency, perturbations in copper homeostasis have frequently been documented. Previous studies in iron-deprived rats demonstrated that enterocyte and hepatic copper levels increase and a copper transporter (the Menkes Copper ATPase; Atp7a) is induced in the duodenal epithelium in parallel to iron transport-related genes (e.g. Dmt1, Dcytb, Fpn1). Moreover, two ferroxidase proteins involved in iron homeostasis, hephaestin expressed in enterocytes and ceruloplasmin, produced and secreted into blood by the liver, are copper-dependent enzymes. We thus aimed to test the hypothesis that Atp7a function is important for the copper-related compensatory response of the intestinal epithelium to iron deficiency. Accordingly, iron homeostasis was studied for the first time in mice expressing a mutant Atp7a protein with minimal activity (Brindled [Mo (Br) (/y) ]). Mutant mice were rescued by perinatal copper injections, and, after a 7-8 week recovery period, were deprived of dietary iron for 3 weeks (along with WT littermates). Adult Mo (Br) (/y) mice displayed copper-deficiency anemia but had normal iron status; in contrast, iron-deprived Mo (Br) (/y) mice were iron deficient and more severely anemic with partial amelioration of the copper-deficient phenotype. Intestinal iron absorption in both genotypes (WT and Mo (Br) (/y) ) increased ∼3-fold when mice consumed a low-iron diet and ∼6-fold when mice were concurrently bled. WT mice exhibited no alterations in copper homeostasis in response to iron deprivation or phlebotomy. Conversely, upregulation of iron absorption was associated with increased enterocyte and liver copper levels and serum ferroxidase (ceruloplasmin) activity in Mo (Br) (/y) mice, typifying the response to iron deprivation in many mammalian species. We thus speculate that a copper threshold exists that is necessary to allow appropriate regulate of iron absorption. In summary, Mo (Br) (/y) mice were able to adequately regulate iron absorption, but unlike in WT mice, concurrent increases in enterocyte and liver copper levels and serum ferroxidase activity may have contributed to maintenance of iron homeostasis.

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The lumenal loop Met672-Pro707 of copper-transporting ATPase ATP7A binds metals and facilitates copper release from the intramembrane sites.

Publications

The lumenal loop Met672-Pro707 of copper-transporting ATPase ATP7A binds metals and facilitates copper release from the intramembrane sites.

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