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, 108 (4), 1388-94

The Steap Proteins Are Metalloreductases

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The Steap Proteins Are Metalloreductases

Robert S Ohgami et al. Blood.

Abstract

Iron and copper are essential for all organisms, assuming critical roles as cofactors in many enzymes. In eukaryotes, the transmembrane transport of these elements is a highly regulated process facilitated by the single electron reduction of each metal. Previously, we identified a mammalian ferrireductase, Steap3, critical for erythroid iron homeostasis. Now, through homology, expression, and functional studies, we characterize all 4 members of this protein family and demonstrate that 3 of them, Steap2, Steap3, and Steap4, are not only ferrireductases but also cupric reductases that stimulate cellular uptake of both iron and copper in vitro. Finally, the pattern of tissue expression and subcellular localization of these proteins suggest they are physiologically relevant cupric reductases and ferrireductases in vivo.

Figures

Figure 1.
Figure 1.
Multiple sequence alignment. Alignment of full-length Steap1, Steap2, Steap3, Steap4, Archaeoglobus fulgidus FNO, and the transmembrane region corresponding to amino acids 207 to 390 of S cerevisiae FRE1. Regions of high homology are boxed, and similar amino acids are depicted in bold. Predicted transmembrane domains of Steap family members are highlighted with solid bars. The Rossman fold motif, GXGXXA/G, is indicated by a series of asterisks. The predicted NAD(P)H binding motif is indicated by dashes. Conserved histidine residues predicted to be involved in heme binding are underlined in transmembrane domains 3 and 5.
Figure 2.
Figure 2.
Steap mRNA expression in human tissues by quantitative real-time PCR. Quantitative real-time PCR was carried out using human cDNA samples. Products are normalized to β-actin for each tissue and adjusted to a percent of the maximum value obtained for that tissue. (A) Steap1 mRNA expression; (B) Steap2 mRNA expression; (C) Steap4 mRNA expression. Each cDNA sample was amplified 3 times. Error bars represent ± 1 standard deviation (SD).
Figure 3.
Figure 3.
Steap mRNA expression by in situ hybridization. E15.5 mouse embryo sections were analyzed for Steap mRNA expression by 35S-radioactive in situ hybridization. (A) Bright field image; (B) inverted dark field negative control probe (Steap1 sense RNA); (C) Steap1 (antisense RNA), note adrenal staining (▸); (D) Steap2 (antisense RNA); (E) Steap2 higher magnification of the liver (▾) and gastroduodenal junction (▸); (F) Steap4. Checkerboard background is the result of assembling the low-power image from multiple high-power images using Compix software (see “Materials and methods”).
Figure 4.
Figure 4.
Steap subcellular localization. Colocalization of epitope-tagged Steap family members with Tf and endogenous TfR1. (A) Steap1, (B) Tf, (C) Tf-Steap1 merged; (D) Steap1, (E) TfR1, (F) TfR1-Steap1 merged; (G) Steap2, (H) Tf, (I) Tf-Steap2 merged; (J) Steap2, (K) TfR1, (L) TfR1-Steap2 merged; (M) Steap4, (N) Tf, (O) Tf-Steap4 merged; (P) Steap4, (Q) TfR1, (R) TfR1-Steap4 merged.
Figure 5.
Figure 5.
Iron and copper reductase activity in HEK-293T cells expressing Steap family members. (A) Ferrireductase activity measured in cells overexpressing Steap family members. (B) Cupric reductase activity in cells overexpressing Steap family members. (C) Ferrireductase activity measured in cells overexpressing Steap family members with only ferric iron added (▪) or ferric iron plus a 5-fold (formula image) or 10-fold (□) molar excess of cupric copper. n = 4 for each result in each panel. Error bars represent ± 1 SD.
Figure 6.
Figure 6.
Iron and copper uptake in HEK-293T cells expressing Steap family members. (A) Total iron uptake measured in cells overexpressing Steap family members. (B) Total 64Cu-copper uptake measured in cells overexpressing Steap family members. n = 4 for each result in both panels. Error bars represent ± 1 SD.

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