Liver, Pancreas, and Biliary TractA mutation of the Wilson disease protein, ATP7B, is degraded in the proteasomes and forms protein aggregates☆,☆☆
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.
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Cited by (53)
Role of endoplasmic reticulum stress and protein misfolding in disorders of the liver and pancreas
2019, Advances in Medical SciencesCitation 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 ResearchCitation 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.
Niemann-Pick C1 protein transports copper to the secretory compartment from late endosomes where ATP7B resides
2009, Experimental Cell ResearchKeratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components
2005, Experimental Cell ResearchAdult human liver contains intermediate-type proteasomes with different enzymatic properties
2014, Annals of HepatologyCitation 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.
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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.).
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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.