Increased Copper Metallothionein in Menkes Cultured Skin Fibroblasts

Labadie, Gundula U; Hirschhorn, Kurt; Katz, Safiana; Beratis, Nicholas G.

doi:10.1203/00006450-198103000-00012

Cited by 51 publications

(19 citation statements)

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“…Metallothioneins have been identified as the major intracellular metal-binding protein in a variety of normal (23) and mutant (2,5,6,24,25,32,34) tissues. An excessive accumulation of cadmium in kinky hair syndrome fibroblasts was invoked to support the postulate of a mutant metallothionein in this disorder (6), and additional work in those cells (6) suggested the existence of structurally abnormal metallothioneins with decreased affinity for copper.…”

Section: Discussionmentioning

confidence: 99%

Trace Metal Metabolism in Cultured Skin Fibroblasts of the Mottled Mouse: Response to Metallothionein Inducers

Packman

O'Toole

1984

Pediatr Res

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ABSTRACT. Menkes' kinky hair syndrome is a lethal Xlinked disorder marked by tissue-specific increases in copper content. An animal model of kinky hair syndrome is provided by mice mutant at the X-linked mottled locus. The basic defect is unknown. In order to discriminate among potential etiologies, we asked whether the expression of the mottled mutation causes abnormalities in the metabolism of trace metals other than copper in hemizygous mottled (blotchy) cultured skin fibroblasts, and whether we can differentiate mutant and normal cells according to their response to metal inducers of metallothionein. Blotchy fibroblasts accumulated up to 12 times more "Cu than control (littermate) cells, over time and over a range of 6 4 C~ concentrations. A saturable high affinity component to accumulation over a fixed time interval was revealed in these studies. While uptake kinetics were indistinguishable in mutant and control cells, the patterns of 64Cu exit differed. In both cell types, the rate of release of a rapidly exchangeable fraction of newly acquired 'j4Cu was similar. However, in mutant cells, a larger fraction of recently accumulated 6 4 C~ is retained. In contrast to the results for "CU, accumulation and exit of 65Zn and '09Cd were not distinguishable in mutants and controls. With exposure to either a strong (cadmium) or weaker (zinc) inducer of metallothionein, "CU accumulation was increased in normal cells, while there was no change from the already elevated level of 6 4 C~ accumulation in blotchy cells. In contrast, the effects of metal inducers of metallothionein on 65Zn or '09Cd accumulation in mutant cells were indistinguishable from the effects on controls. The present observations reveal that blotchy fibroblasts exhibit normal accumulation and exit of metals other than copper, and evince a differential response to metallothionein inducers, which response is also limited to the accumulation of copper. When considered together with the larger body of data on the kinky hair syndrome and mottled mutation, we infer that the defect in both species resides in the function of a thionein or other protein specifically binding copper; or in an altered transport system specifically affecting copper. (Pediatr Res 18:1282-1286, 1984

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Section: Discussionmentioning

confidence: 99%

Trace Metal Metabolism in Cultured Skin Fibroblasts of the Mottled Mouse: Response to Metallothionein Inducers

Packman

O'Toole

1984

Pediatr Res

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“…The folding of the domains was examined by solution NMR at equal protein concentrations. The 1 H, 15 N-HSQC (heteronuclear single quantum coherence) spectrum of the Gly 875 Adomain showed excellent chemical shift dispersion, characteristic of a well-folded protein (Fig. 5B).…”

Section: Atp7b-arg 875 Is Mislocalized Under Basal Conditions But Tramentioning

confidence: 99%

“…One may expect that high cellular copper would stabilize ATP7B-Arg 875 , precluding abnormal behavior, except that extra copper may not be easily available to ATP7B because of up-regulation of the endogenous high-affinity copper chelator metallothionein (14). To clarify this issue, we used Menkes fibroblasts, which, similar to WD liver, accumulate copper on prolonged exposure and up-regulate metallothionein (15). We first compared copper levels (by atomic absorption spectroscopy) in Menkes fibroblasts grown in basal medium (when ATP7B-Arg 875 is targeted to the ER) after treatment for 4 h with 5 μM copper (when ATP7B traffics to the TGN) and after treating cells for 24 h with 50-100 μM copper [to overload cells with copper and induce metallothionein (15)].…”

Section: Extra Copper Is Not Required For Atp7b-arg 875 Biosynthesis Butmentioning

confidence: 99%

“…To clarify this issue, we used Menkes fibroblasts, which, similar to WD liver, accumulate copper on prolonged exposure and up-regulate metallothionein (15). We first compared copper levels (by atomic absorption spectroscopy) in Menkes fibroblasts grown in basal medium (when ATP7B-Arg 875 is targeted to the ER) after treatment for 4 h with 5 μM copper (when ATP7B traffics to the TGN) and after treating cells for 24 h with 50-100 μM copper [to overload cells with copper and induce metallothionein (15)]. We then transfected copperloaded cells with ATP7B-Arg 875 using our standard protocol and characterized its intracellular localization.…”

Section: Extra Copper Is Not Required For Atp7b-arg 875 Biosynthesis Butmentioning

confidence: 99%

“…The ATP7B fragment (residues 789-907) encoding the A-domain with Arg or Gly at position 875 was cloned into the pTXB1 vector and expressed in E. coli. Proteins were metabolically labeled with 15 NH 4 Cl; purified on chitin beads; cleaved using 2-mercaptoethane sulfonate sodium (Sigma); and dialyzed into 50 mM Na-phosphate (pH 7.0), 50 mM arginine, and 50 mM glutamate. The 1 H, 15 N-HSQC spectra were recorded with 0.2 mM protein.…”

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Cellular copper levels determine the phenotype of the Arg ⁸⁷⁵ variant of ATP7B/Wilson disease protein

Gupta¹,

Bhattacharjee²,

Dmitriev

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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In human disorders, the genotype-phenotype relationships are often complex and influenced by genetic and/or environmental factors. Wilson disease (WD) is a monogenic disorder caused by mutations in the copper-transporting P-type ATPase ATP7B. WD shows significant phenotypic diversity even in patients carrying identical mutations; the basis for such diverse manifestations is unknown. We demonstrate that the 2623A/G polymorphism (producing the Gly 875 →Arg substitution in the A-domain of ATP7B) drastically alters the intracellular properties of ATP7B, whereas copper reverses the effects. Under basal conditions, the common Gly 875 variant of ATP7B is targeted to the trans-Golgi network (TGN) and transports copper into the TGN lumen. In contrast, the Arg 875 variant is located in the endoplasmic reticulum (ER) and does not deliver copper to the TGN. Elevated copper corrects the ATP7B-Arg 875 phenotype. Addition of only 0.5-5 μM copper triggers the exit of ATP7B-Arg 875 from the ER and restores copper delivery to the TGN. Analysis of the recombinant A-domains by NMR suggests that the ER retention of ATP7B-Arg 875 is attributable to increased unfolding of the Arg 875 -containing A-domain. Copper is not required for the folding of ATP7B-Arg 875 during biosynthesis, but it stabilizes protein and stimulates its activity. A chemotherapeutical drug, cisplatin, that mimics a copper-bound state of ATP7B also corrects the "disease-like" phenotype of ATP7B-Arg 875 and promotes its TGN targeting and transport function. We conclude that in populations harboring the Arg 875 polymorphism, the levels of bioavailable copper may play a vital role in the manifestations of WD.trafficking | cisplatin | phenotypic variability

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Regulation of Copper Metabolism in the Mottled Mouse

Packman¹

1987

Arch Dermatol

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Menkes' kinky-hair syndrome is an X-linked recessive neurodegenerative and connective-tissue disorder, with decreased serum copper and ceruloplasmin-copper oxidase concentrations and tissue-specific increases in copper content. Clinical manifestations can be related to relative copper deficiency and reduced activity of cuproenzymes in multiple organs. An animal model is provided by mice hemizygous for mutant alleles, such as the blotchy allele, at the X-linked mottled locus. This locus may be homologous in mouse and man. The basic defect is unknown but has been thought to reside in the regulation of the function or synthesis of metallothioneins. In the blotchy mouse and in cultured skin fibroblasts derived therefrom, we showed that the mutation specifically affects the metabolism of copper and not other trace metals. Excessive accumulation and abnormal (reduced) exit kinetics were demonstrated for copper but not for the related trace metals cadmium and zinc. While metallothionein-I messenger RNA (mRNA) concentrations were elevated in blotchy fibroblasts, the elevations in metallothionein-I mRNA in response to metallothionein inducers (cadmium, copper) were similar in blotchy and control cells. Further, metallothionein-I mRNA levels were indistinguishable in mutant and control fibroblasts containing equivalent intracellular copper concentrations. Finally, metallothionein-I mRNA content was not elevated in blotchy kidneys at early developmental stages, before storage of excessive copper. The aggregate data suggest that the basic defect in the blotchy mouse--and, by analogy, in Menkes' syndrome--does not reside in defective modulation of metallothionein function and does not cause abnormal regulation of metallothionein synthesis.

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Increased Copper Metallothionein in Menkes Cultured Skin Fibroblasts

Cited by 51 publications

References 2 publications

Trace Metal Metabolism in Cultured Skin Fibroblasts of the Mottled Mouse: Response to Metallothionein Inducers

Trace Metal Metabolism in Cultured Skin Fibroblasts of the Mottled Mouse: Response to Metallothionein Inducers

Cellular copper levels determine the phenotype of the Arg ⁸⁷⁵ variant of ATP7B/Wilson disease protein

Regulation of Copper Metabolism in the Mottled Mouse

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Increased Copper Metallothionein in Menkes Cultured Skin Fibroblasts

Cited by 51 publications

References 2 publications

Trace Metal Metabolism in Cultured Skin Fibroblasts of the Mottled Mouse: Response to Metallothionein Inducers

Trace Metal Metabolism in Cultured Skin Fibroblasts of the Mottled Mouse: Response to Metallothionein Inducers

Cellular copper levels determine the phenotype of the Arg 875 variant of ATP7B/Wilson disease protein

Regulation of Copper Metabolism in the Mottled Mouse

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Cellular copper levels determine the phenotype of the Arg ⁸⁷⁵ variant of ATP7B/Wilson disease protein