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

Abstract

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+ .

Toxic metal proteomics: Reaction of the mammalian zinc proteome with Cd2+

“…A previous study had shown that under approximately stoichiometric conditions EDTA reacts with LLC-PK 1 Zn-proteome to extract about 30% of its Zn 2+ [22]. That result was repeated in the present experiment.…”

“…For kinetic or thermodynamic reasons, it is largely unreactive with proteomic Zn 2+ bound to native proteins. It does compete for much of the adventitiously bound Zn 2+ to form Zn(ZQ) 2 and also appears to bind the rest in the form of ternary complexes, which are revealed by a blue-shift in the fluorescence emission spectrum of Zn(ZQ) 2 from 490 nm to 470 nm [19]: $$\mathrm{normalZ}\mathrm{normaln}-\text{proteome}•\mathrm{normalZ}\mathrm{normaln}+2\mathrm{normalZ}\mathrm{normalQ}\rightleftarrows \mathrm{normalZ}\mathrm{normalQ}-\mathrm{normalZ}\mathrm{normaln}-\text{proteome}•\mathrm{normalZ}\mathrm{normaln}-\mathrm{normalZ}\mathrm{normalQ}$$ Also, as documented in previous studies and below in Figure 5a, 4 μM apometallothionein, which provided 28 μM in Zn 2+ binding sites, removed little if any Zn 2+ from 28 μM Zn-proteome to form Zn-MT [21,22]. Thus, as with ZQ, Zn-proteome is mostly unreactive with an endogenous ligand, apo-MT, that also has a large binding constant for Zn 2+ , 10 11.2 [16].…”

Zinc is an essential trace element for spermatogenesis

“…CA is also known to be a Zn-binding protein, and its activity is dependent on the Zn concentration (38). Additionally, the removal of Zn from Zn-protein complexes extracted from human U87 human glioblastomaastrocytoma cells by TPEN inhibited the function of the transcription factor, Sp1 (39). Although there is currently no information on effects of TPEN on CA activity, we speculate that TPEN may inhibit sperm motility by sequestering Zn away from this enzyme in sperm.…”

A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana

“…16 A C-terminal TAP-tag under the control of the 35S promoter was cloned without the stop codon into the vector pFGC5941. 26 The plant expression vector was transformed into Agrobacterium tumefaciens strain GV3101. Arabidopsis thaliana was transformed using the floral dip method for A. tumefaciens-mediated gene transfer.…”