V体育安卓版 - Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official VSports app下载. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2013 Nov 12;110(46):E4279-88.
doi: 10.1073/pnas.1311749110. Epub 2013 Oct 28.

V体育安卓版 - Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain

Helena Öhrvik  1 , Yasuhiro Nose, L Kent Wood, Byung-Eun Kim, Sophie-Charlotte Gleber, Martina Ralle, Dennis J Thiele
Affiliations

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain

Helena Öhrvik et al. Proc Natl Acad Sci U S A. .

Abstract

Copper is an essential catalytic cofactor for enzymatic activities that drive a range of metabolic biochemistry including mitochondrial electron transport, iron mobilization, and peptide hormone maturation. Copper dysregulation is associated with fatal infantile disease, liver, and cardiac dysfunction, neuropathy, and anemia. Here we report that mammals regulate systemic copper acquisition and intracellular mobilization via cleavage of the copper-binding ecto-domain of the copper transporter 1 (Ctr1). Although full-length Ctr1 is critical to drive efficient copper import across the plasma membrane, cleavage of the ecto-domain is required for Ctr1 to mobilize endosomal copper stores VSports手机版. The biogenesis of the truncated form of Ctr1 requires the structurally related, previously enigmatic copper transporter 2 (Ctr2). Ctr2(-/-) mice are defective in accumulation of truncated Ctr1 and exhibit increased tissue copper levels, and X-ray fluorescence microscopy demonstrates that copper accumulates as intracellular foci. These studies identify a key regulatory mechanism for mammalian copper transport through Ctr2-dependent accumulation of a Ctr1 variant lacking the copper- and cisplatin-binding ecto-domain. .

Keywords: endosome; lysosome; platinum; protein regulation; uptake. V体育安卓版.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Loss of Ctr2 results in increased copper levels in tissue. (A) Topological models for Ctr1 and Ctr2 showing transmembrane domains and a conserved M-X3-M motif. The Ctr1 ecto-domain has two glycosylation sites and clusters of Met and His residues, with two Met and one His in the Ctr2 ecto-domain. (B) Wild-type and Ctr2−/− age-matched mice 5–7 and 20–22 mo were analyzed for tissue copper levels by ICP-MS. (C) Zn analysis as in B. Data presented as mean ± SD from two to eight mice. See Fig. S1 for generation of Ctr2 knockout mice.
Fig. 2.
Fig. 2.
Copper accumulates in intracellular deposits in Ctr2−/− mouse brain. (A) XFM images for phosphorous (P, green) and copper (Cu, red) and a magnified overlay from the cortex for a Ctr2+/+ (Upper) and a Ctr2−/− (Lower) mouse. (Scale bar, 50 μm.) The false coloring scheme for P and Cu is shown below images. The intensity of the fluorescence signal is displayed as a linear scale (P:0 − ≥11 μg/cm2; Cu:0 − ≥0.03 μg/cm2). In the P map, nuclei are visible (red arrows). The overlay for the magnified area shows an intracellular localization for the copper foci. (B) XFM image of the caudoputamen for littermate Ctr2+/+ (Upper) and Ctr2−/− (Lower) mice exhibiting two-dimensional elemental maps for phosphorous (P, green), copper (Cu, red), and a magnified overlay. Nuclei are visible in the P-map. Pencil fibers in the caudoputamen (white arrows). (See Table S1 for Cu and P concentrations in other brain regions determined by XFM.)
Fig. 3.
Fig. 3.
Ctr2−/− MEFs accumulate copper in punctae. (A) Wild-type and Ctr2−/− MEFs were analyzed for total copper and Zn levels by ICP-MS. (B) Expression of the Ctr2 cDNA (Ctr2) but not the empty vector (V) re-establishes low copper levels without changes in Zn levels. Data are presented as mean ± SD from four biological replicates. (C) Wild type (Ctr2+/+) and Ctr2−/− MEFs were stained with the membrane-permeable Cu+-specific stain CS3 and photographed by confocal microscopy. White arrowheads point to large punctate staining. (See Fig. S2 for PCR design of Ctr2 genotyping in the Ctr2 MEFs and genotyping of the Ctr2 MEFs.)
Fig. 4.
Fig. 4.
Copper accumulates in endosomes in Ctr2−/− MEFs. (A) Copper was measured (ng/mg protein) in each of three subcellular fractions from wild-type (Ctr2+/+) and Ctr2−/− MEFs (see Fig. S3A for fractions collected from the iodixanol gradient). Shown are the results (mean ± SD) from six biological replicates for each fractionation. (B) Immunoblotting of protein extracts derived from subcellular fractionation experiments outlined in Fig. S3A from wild-type and Ctr2−/− MEFs. Blots were analyzed by probing with anti-Ctr2, anti-Ctr1 (T, truncated; F, full-length), anti-Lamp1, anti-Rab4, anti-Rab5, anti-Rab7, anti-Rab9, anti-Rab11, anti-CoxIV, anti-Na/K-ATPase, and anti-GAPDH. (C) Ctr1 and Ctr2 reciprocally coimmunoprecipitate from solubilized protein extracts. HEK293T cells were transfected with the indicated expression vector or empty vector and treated with DSP crosslinker; proteins were immunoprecipitated with the indicated antibody (IP), and proteins were analyzed by immunoblotting with the indicated antibody (WB). (See Fig. S3B for co-IP without cross-linker) (D) Ctr1 and Ctr2 associate in BiFC assays. HEK293T cells were transfected with the indicated pairs of expression plasmids and photographed by fluorescence microscopy and differential interference contrast microscopy (DIC).
Fig. 5.
Fig. 5.
Ctr2−/− MEFs show Ctr1-dependent copper accumulation. (A) Silencing of Ctr1 (siCtr1) in two independent experiments, but not scrambled RNA (Sc), results in depletion of Ctr1 protein levels. Full-length Ctr1 (F) and truncated Ctr1 (T) are shown with tubulin as a loading control. (B) Silencing of Ctr1 reduces Cu accumulation in Ctr2−/− MEFs with no change in Zn levels. (C) Overexpression of Ctr2 in Ctr1−/− MEFs does not alter copper or Zn levels. (D) Silencing of Ctr2 in Ctr1−/− MEFs does not alter copper levels. Data presented as mean ± SD from four biological replicates. (E) Protein extracts from the indicated tissues from wild-type mice and Ctr2−/− littermates were immunoblotted with anti-Ctr1, anti-CoxIV, and anti-GAPDH antibody. Shown are the full-length (F) and truncated (T) forms of Ctr1. (F) Protein extracts from wild-type, Ctr2+/−, and Ctr2−/− MEFs were analyzed as in A but with anti-tubulin antibody as loading control. (G) Silencing of Ctr2 (siCtr2) in wild-type MEFs resulted in a decrease in the levels of truncated Ctr1 compared with scRNA. Extracts were analyzed as in E. (H) Overexpression of Ctr2-enhanced Ctr1 ecto-domain cleavage in wild-type cells and restored Ctr1 cleavage in Ctr2−/− cells. Cells were transfected with empty vector (V) or the vector with Ctr2 cDNA (Ctr2) and protein extracts were analyzed as in E with SOD1 detected as loading control. (I) Expression of mouse Ctr2 enhances human Ctr1 ecto-domain cleavage. HEK293T cells were transfected with an empty vector (V) or a vector with the Ctr2 cDNA (Ctr2), and protein extracts were analyzed with anti-Ctr1, anti-CCS, and anti-SOD1 antibody. (See Fig. S5 for ATP7A protein expression in wild-type and Ctr2−/− mice and MEFs, and localization of ATP7A in the Ctr2 MEFs.)
Fig. 6.
Fig. 6.
Truncated Ctr1 mobilizes copper from endosomal compartments. (A) Red arrowheads indicate the cleavage sites determined for mouse and human Ctr1 (see Fig. S6 for band identification). (B) Ectopic expression of Ctr1 or truncated Ctr1 in Ctr2−/− MEFs. Protein extract was immunoblotted with anti-Ctr1, Ctr2, β-galactosidase antibody as transfection efficiency control, and anti-actin. (C) Ctr2−/− MEFs transfected in B were stained for Cu+ with CS3, and several fields (representative fields shown) were photographed for quantitation. (D) Enumeration of CS3-positive punctae for Ctr2−/− cells transfected with an empty vector (V), Ctr2, Ctr1, or truncated Ctr1 (tCtr1). (E) Ctr2−/− MEFs were analyzed for total Cu levels by ICP-MS. Expression of the Ctr2 cDNA (Ctr2), tCtr1, and Ctr2LX3L, but not the empty expression vector (V) re-established low Cu levels. Data are presented as mean ± SD from three biological replicates. (F) A conserved Ctr1 motif is not required for Ctr2-induced cleavage of the Ctr1 ecto-domain. Protein extracts from Ctr2−/− MEFs transformed with vector (V) or a vector expressing Ctr2 or Ctr2LX3L were immunoblotted with anti-Ctr1, anti-Ctr2, or anti-SOD1 antibody. (G) Cells in F were stained and CS3-positive punctae were quantitated as in D.
Fig. 7.
Fig. 7.
Ctr2 modulates copper and cisplatin acquisition. (A) Model for Ctr1-dependent Cu+ uptake and endosomal copper mobilization by truncated Ctr1. (B) Ctr2−/− MEFs accumulate cisplatin. Wild-type and Ctr2−/− MEFs were exposed to cisplatin and copper (Left) or platinum (Pt) (Right) measured by ICP-MS. Data are presented as mean ± SD for four biological replicates. (C) Model for Ctr1-dependent cisplatin import and the modulation of Pt uptake by truncation of Ctr1.
See this image and copyright information in PMC

References

    1. Kim BE, Nevitt T, Thiele DJ. Mechanisms for copper acquisition, distribution and regulation. Nat Chem Biol. 2008;4(3):176–185. - PubMed
    1. Wang Y, Hodgkinson V, Zhu S, Weisman GA, Petris MJ. Advances in the understanding of mammalian copper transporters. Adv Nutr. 2011;2(2):129–137. - PMC - PubMed
    1. Collins JF, Prohaska JR, Knutson MD. Metabolic crossroads of iron and copper. Nutr Rev. 2010;68(3):133–147. - PMC - PubMed
    1. Barnham KJ, Bush AI. Metals in Alzheimer’s and Parkinson’s diseases. Curr Opin Chem Biol. 2008;12(2):222–228. - PubMed
    1. Kaler SG. ATP7A-related copper transport diseases-emerging concepts and future trends. Nat Rev Neurol. 2011;7(1):15–29. - PMC - PubMed

"VSports最新版本" Publication types

VSports app下载 - MeSH terms