Nucleophilic Distribution of Metallothionein in Human Tumor Cells

Woo, Elizabeth S.; Kondo, Yukihiro; Watkins, Simon C.; Hoyt, Dale G.; Lazo, John S.

doi:10.1006/excr.1996.0146

Cited by 44 publications

(31 citation statements)

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“…Alternatively, it is possible that the two proteins are isolated from one another by compartmentalization, Zn 3 -Sp1 in the nucleus and apo-MT in the cytoplasm. In support of this possibility, numerous publications report cytoplasmic or nuclear localization of immunoreactive MT in a variety of tissues and cell types as well as conditions that foster a shift in its location between these compartments [39][40][41][42][43][44]. Because antibodies to MT generally detect both metal-bound and apo-MT, such studies do not provide needed information on the intracellular location of metal ion unsaturated MT.…”

Section: Discussionmentioning

confidence: 99%

Zinc binding ligands and cellular zinc trafficking: Apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

Rana¹,

Kothinti²,

Meeusen³

et al. 2008

Journal of Inorganic Biochemistry

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Many cell types contain metal-ion unsaturated metallothionein (MT). Considering the Zn 2+ binding affinity of metallothionein, the existence of this species in the intracellular environment constitutes a substantial "thermodynamic sink." Indeed, the mM concentration of glutathione may be thought of in the same way. In order to understand how apo-MT and the rest of the Zn-proteome manage to co-exist, experiments examined the in vitro reactivity of Zn-proteome with apo-MT, glutathione (GSH), and a series of common Zn 2+ chelating agents including N,N,N',N'-(2-pyridylethyl) ethylenediammine (TPEN), EDTA, and [(2,2'-oxyproplylene-dinitrilo]tetraacetic acid (EGTA). Less than 10% of Zn-proteome from U87 mg cells reacted with apo-MT or GSH. In contrast, each of the synthetic chelators was 2−3 times more reactive. TPEN, a cell permeant reagent, also reacted rapidly with both Zn-proteome and Zn-MT in LLC-PK 1 cells. Taking a specific zinc finger protein for further study, apo-MT, GSH, and TPEN inhibited the binding of Zn 3 -Sp1 with its cognate DNA site (GC-1) in the sodium-glucose co-transporter promoter of mouse kidney. In contrast, preformation of Zn 3 -Sp1-(GC-1) prevented reaction with apo-MT and GSH; TPEN remained active but at a higher concentration. Whereas, Zn 3 -Sp1 is active in cells containing apo-MT and GSH, exposure of LLC-PK 1 cells to TPEN for 24 h largely inactivated its DNA binding activity. The results help to rationalize the steady state presence of cellular apo-MT in the midst of the many, diverse members of the Znproteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn 2+ .

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Section: Discussionmentioning

confidence: 99%

Zinc binding ligands and cellular zinc trafficking: Apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

Rana¹,

Kothinti²,

Meeusen³

et al. 2008

Journal of Inorganic Biochemistry

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“…Concentration of MT in the nucleus is reported to be energy-dependent and a means for supplying zinc to this organelle (53). These studies place MT at a site with a high requirement for zinc and also where pathological conditions associated with cadmium-induced genotoxicity or carcinogenesis (54) and interactions with electrophiles such as antitumor drugs (55) can occur.…”

Section: Discussionmentioning

confidence: 99%

Modulation of DNA Binding of a Tramtrack Zinc Finger Peptide by the Metallothionein-Thionein Conjugate Pair

Roesijadi¹,

Bogumil²,

Vašák³

et al. 1998

Journal of Biological Chemistry

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The ability of metallothionein (MT) to modulate DNA binding by a two-finger peptide of Tramtrack (TTK), a CCHH zinc transcription factor, was investigated using metal-bound and metal-deficient forms of rabbit MT-2 and the TTK peptide. Thionein inhibited DNA binding by zinc-bound TTK, and Zn-MT restored DNA-binding by zinc-deficient apo-TTK. "Free" zinc at low concentrations was as effective as Zn-MT in restoring DNA binding by apopeptide but was inhibitory at concentrations equal to zinc bound to 2 mol eq and higher of Zn-MT. Substitution of cadmium for zinc reduced the affinity of the peptide for its DNA binding site. This effect was reversed by incubation with Zn-MT. The circular dichroic spectra of the TTK peptide indicated that zinc removal resulted in loss of ␣-helical structures, which are sites of DNA contact points. Reconstitution with cadmium resulted in stoichiometric substitution of 2 mol of Cd/mol of peptide but not recovery of ␣-helical structures. Incubation of Cd-TTK with Zn-MT restored the secondary structure expected for zinc-bound TTK. The ability of Zn-MT and thionein to restore or inhibit DNAbinding by TTK was associated with effects on the metallation status of the peptide and related alterations in its secondary structure.The low molecular weight metal-binding protein metallothionein (MT) 1 is proposed to have functions in metal ion regulation and detoxification (1) and as a scavenger of free radicals (2). Roles for MT in dynamic intermolecular metal exchange reactions are consistent with the high thermodynamic stability (3) and high kinetic lability of its metal binding sites (4, 5). Metal exchange can also be facilitated by cellular redox couples such as ). An early focus on enzyme activation by Zn-MT implicated MT as a zinc donor to apometallic forms of metalloenzymes such as aldolase, thermolysin, and carbonic anhydrase (10) and pyridoxal kinase (11). Recent studies on the effect of MTs on interactions between zinc transcription factors and their cognate DNAs implicate MTs in gene regulation. Incubation of metal-free thionein with CCHH zinc finger transcription factors Sp1 (12) and TFIIIA (13) inhibits DNA binding, suggesting zinc abstraction by thionein as a mechanism for regulating gene expression. Reciprocal zinc exchange is suggested as a mechanism for regulating gene expression in a study in which Zn-MT and thionein could activate or inhibit DNA binding by the estrogen receptor (14), a CCCC zinc finger protein.Zinc transcription factors appear uniquely qualified as potential partner molecules in metal exchange reactions with MT. Reported stability constants for zinc binding by zinc finger peptides and proteins are in a similar range, i.e. within 1 or 2 orders of magnitude, as that of . Additionally, it appears that the kinetics for zinc exchange in these structures are also relatively rapid (18). Direct examination of the metal transfer process has shown that zinc can be transferred from an ␣-domain peptide of human MT-2 (␣-hMT-2) to a peptide derived from the third finger of Sp1...

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“…MT functions as a donor of zinc ions for transcription factors and enzymes involved in the processes of DNA and protein synthesis. This is confirmed not only by elevated MT levels in hyperplasia but also by MT translocation from cytoplasm to cell nucleus during DNA synthesis (S phase) (Woo et al, 1996;Jasani & Schmid, 1997). In neoplastic tissues, the level of MT was shown to be proportionally elevated with the concentration of zinc ions Florianczyk et al, 2006).…”

Section: Role Of Mt In a Neoplastic Processmentioning

confidence: 88%