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

Abstract

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...

The cotton fiber zinc-binding domain of cellulose synthase A1 from Gossypium hirsutum displays rapid turnover in vitro and in vivo

“…In fact, MET has been shown to transfer zinc atoms to zinc-depleted sorbitol dehydrogenase in a redox-dependent manner (33). It has also been proposed that the MT͞T system serves as both a cellular reservoir for zinc and a controlled system that can supply different amounts of zinc to apoproteins according to demand (34,35). The biological specificity of this system seems to be embedded not in the recognition between MET and apoproteins, but rather in signals effecting a change of the cellular redox state (33,34,36,37).…”

“…Preliminary experiments using [ 35 S]methionine indicated that 400 M cycloheximide was sufficient to inhibit completely the incorporation of methionine into proteins (data not shown). To measure the effects of cycloheximide on longer-term levels of total protein, 20-DPA cotton cultured fibers (39) were incubated in the absence and presence of 30 Ci͞ml [ 35 S]methionine by using EasyTag EXPRESS 35 S protein-labeling mix (PerkinElmer) in the presence or absence of 400 M cycloheximide. All samples, including controls, contained 0.01% DMSO.…”

Microarray analysis of metallothioneins in human diseases—A review

“…It was shown that regulating the availability of zinc the thionein/Zn-MT conjugate pair modulates the DNA-binding activity of zinc finger transcription factors [28]. The feasibility of exchanging of Cd 2+ bound to transcription factors to Zn 2+ by MT thereby restoring the DNA-binding activity of transcription factors is an important event of metal detoxification [28]. Under physiological conditions, the role of metallothionein is zinc reservoir for zinc-dependent proteins.…”

Interprotein metal exchange between transcription factor IIIa and apo-metallothionein

“…Studies of the reaction of apoMT and one of its component peptide sequences with Cd-carbonic anhydrase showed that both were remarkably effective in competing for protein-bound Cd 2+ [31,32]. There is evidence that, in vitro, Cd 2+ can substitute for Zn 2+ in zinc-finger structures and that such substitution can change the activities of the finger domains [22,28,33]. Therefore, this study has also examined how Cd-TFIIIA reacts with apo-or Zn 7 -MT.…”

“…More recently, two reports have described chemical properties of the reaction of selected Znfinger peptides with apoMT or the α-domain of metallothionein [27,28]. The present experiments were undertaken to gain insight into the chemical properties of the reaction of Zn-TFIIIA with apoMT.…”