Gastroenterology

Gastroenterology

Volume 120, Issue 4, March 2001, Pages 967-974
Gastroenterology

Liver, Pancreas, and Biliary Tract
A mutation of the Wilson disease protein, ATP7B, is degraded in the proteasomes and forms protein aggregates,☆☆

https://doi.org/10.1053/gast.2001.22543Get rights and content

Abstract

Background & Aims: Wilson disease is a genetic disorder characterized by the accumulation of copper in the body as a result of a defect of copper excretion from hepatocytes. The intracellular localization of the Wilson disease gene product, ATP7B, was recently identified as the late endosomes. Various mutations have been documented in patients with Wilson disease. The clinical manifestations vary greatly among the patients; however, there is little information on the genotype-phenotype correlation. Methods: We investigated the distribution of a common ATP7B mutant His1069Gln and a mutant Asp1270Ser by expressing the mutants tagged with green fluorescent protein in Huh7 and HEK293 cells. Intracellular organelles were visualized by fluorescence microscopy. Results: Although the wild-type ATP7B and Asp1270Ser mutant localized in the late endosomes, His1069Gln mutant did not locate in the late endosomes and was degraded by the proteasomes in the cytoplasm. Furthermore, His1069Gln formed aggresomes composed of the degradates and intermediate filaments at the microtubule-organizing center. These aggresomes were similar to Mallory bodies on electron microscopy. Conclusions: The different protein properties of ATP7B mutants may explain the variety of clinical spectrums in patients with Wilson disease.

GASTROENTEROLOGY 2001;120:967-974

Section snippets

Cells

Huh7 and HEK293 cells were cultured in Dulbecco's modified Eagle medium (Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (Wako Pure Chemical Industries, Osaka, Japan), penicillin (100 U/mL, crystalline penicillin G Meiji; Meiji Seika Kaisya, Tokyo, Japan), and streptomycin (0.1 mg/mL, Meiji Seika Kaisya) at 37°C in 5% CO2. Copper concentration in the medium measured by direct colorimetric assay was below the detectable level (<5 μg/dL). At 24 hours after plating, complementary DNAs

Distribution of ATP7B in Huh7 cells

Immunofluorescent signals of ATP7B in untransfected or ATP7B cDNA–transfected cells showed a juxtanuclear punctate pattern as did GFP-ATP7B (data not shown).8 When GFP-ATP7B–transfected cells were labeled with the anti-ATP7B antibody, the GFP signals were identical to the signals produced by the anti-ATP7B antibody as described previously (data not shown).8 GFP alone was observed throughout the cells, when pEGFP-C2 was transfected (data not shown).8 We investigated the relationship between

Discussion

We have examined the intracellular distributions of 2 ATP7B proteins with the disease-specific mutation, H1069Q and N1270S, tagged with GFP. GFP-N1270S localized in the late endosomes similar to wild-type ATP7B, because it colocalized with lamp 1 but not with calnexin, GalT, or cathepsin D. GFP signals in GFP-H1069Q–transfected cells were observed in the cytoplasm and nucleus, and were not recognized by anti-ATP7B antibody. Furthermore, ALLN, MG 132, and lactacystine, proteasome inhibitors,

Acknowledgements

The authors thank Dr. J. T. August for providing the anti-lamp1 antibody, and M. Inayoshi for expert technical assistance.

References (35)

  • RW Carrell et al.

    Conformational disease

    Lancet

    (1997)
  • I. Sternlieb

    Copper and zinc

  • PC Bull et al.

    The Wilson's disease gene is a putative copper transporting P-type ATPase similar to the Menkes gene

    Nat Genet

    (1993)
  • K Petrukhin et al.

    Mapping, cloning and genetic characterization of the region containing the Wilson's disease gene

    Nat Genet

    (1993)
  • RE Tanzi et al.

    The Wilson's disease gene is a copper transporting ATPase with homology to the Menkes disease gene

    Nat Genet

    (1993)
  • XL Yang et al.

    Two forms of Wilson disease protein produced by alternative splicing are localized in distinct cellular compartments

    Biochem J

    (1997)
  • AS Payne et al.

    Functional expression of the Wilson disease protein reveals mislocalization and impaired copper-dependent trafficking of the common H1069Q mutation

    Proc Natl Acad Sci U S A

    (1998)
  • Cited by (53)

    • Role of endoplasmic reticulum stress and protein misfolding in disorders of the liver and pancreas

      2019, Advances in Medical Sciences
      Citation Excerpt :

      The degradation via the ubiquitin proteasome system is a common path without taking into account the glycosylation status of misfolded or aggregated proteins in the ER. A number of reports provide evidence that protein folding, processing and endocytic trafficking are compromised as part of the Wilson´s disease pathology [94,95]. This finding appears to be largely dependent on the type of mutation.

    • Copper induces hepatocyte injury due to the endoplasmic reticulum stress in cultured cells and patients with Wilson disease

      2016, Experimental Cell Research
      Citation Excerpt :

      It has been described that the gene product of ATP7B, which is copper-transporting ATPase, is localized at the trans-Golgi network in hepatocytes and functions as the copper transporter [9,10]. However, we have reported that ATP7B locates in the late endosomes of hepatocytes [11–15]. Although this ATP7B localization remains a matter of controversy, dysfunction of this transporter induces excess copper accumulation in the body, particularly in the liver and brain.

    • Adult human liver contains intermediate-type proteasomes with different enzymatic properties

      2014, Annals of Hepatology
      Citation Excerpt :

      Hepatocytes constitute about 60% of the total hepatic cell population. To study the involvement of the ubiquitin/proteasome-system in hepatocytes during various patho-physiological processes HuH7 hepatocarcinoma cells were used as a model cell line.41 –45 However, nothing is known about the feature of proteasomes in this cell line.

    View all citing articles on Scopus

    Supported in part by a grant-in-aid (12670535) from the Ministry of Education of Japan and a grant from the Alumni Association of Kurume University School of Medicine (to M.H.).

    ☆☆

    Address requests for reprints to: Masaru Harada, M.D., Ph.D., Second Department of Medicine, Kurume University School of Medicine, 67 Asahi-Machi, Kurume 830-0011, Japan. Fax: (81) 942-34-2623.

    View full text