Zinc binding ligands and cellular zinc trafficking: apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1.
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 U87mg 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 24h 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 Zn-proteome. They also show that TPEN is a robust intracellular chelator of proteomic Zn(2+).
Rana
Ujala
U
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, United States.
Kothinti
Rajendra
R
Meeusen
Jeffrey
J
Tabatabai
Niloofar M
NM
Krezoski
Susan
S
Petering
David H
DH
D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002850:Chromatography, Gel; D024202:Electrophoretic Mobility Shift Assay; D005029:Ethylenediamines / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008668:Metallothionein / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
D002352:Carrier Proteins; D005029:Ethylenediamines; D016329:Sp1 Transcription Factor; C021267:zinc thionein; C017800:zinc-binding protein; D008668:Metallothionein; D005978:Glutathione; D015032:Zinc; C044387:N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine