Asymmetric functions of a binuclear metal center within the transport pathway of a human zinc transporter ZIP4

Zhang, Tuo; Sui, Dexin; Zhang, Chi; Cole, Logan; Hu, Jian

doi:10.1096/fj.201902043r

Cited by 41 publications

(82 citation statements)

References 33 publications

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“…As shown in Figure 2A, in sharp contrast to the wild type hZIP4, none of the cells expressing any variants uptook more zinc than the blank group in which the cells were transfected with an empty vector, which means that the variants have no detectable zinc transport activity with 10 µM of Zn 2+ added in culture media. For the wild type hZIP4, the previous studies have shown that zinc transport activity reaches plateau at 10 µM of Zn 2+ under the same condition (28,29). To examine whether any activity can be detected at higher zinc concentration, the P200L variant was tested at various zinc concentrations up to 50 µM, but the results only confirmed no detectable activity ( Figure S1).…”

Section: Resultsmentioning

confidence: 83%

“…In this work, we characterized all the seven AE-causing mutations, including four homozygous mutations (C62R, P200L, Q303H and C309Y) (5,6,25,26) and three heterozygous mutations (R95C, A99T and N106K) (6,26,27), and studied their impacts on hZIP4 function. The hZIP4 variants with a Cterminal HA tag were transiently expressed in HEK293T cells and applied to the cell-based radioactive zinc transport assay (6,7,28,29). As shown in Figure 2A, in sharp contrast to the wild type hZIP4, none of the cells expressing any variants uptook more zinc than the blank group in which the cells were transfected with an empty vector, which means that the variants have no detectable zinc transport activity with 10 µM of Zn 2+ added in culture media.…”

Section: Resultsmentioning

confidence: 99%

“…It is unclear whether this discrepancy is due to different approaches used for tracing zinc transport. Radioactive 65 Zn based transport assay has been used to study transport kinetics of a variety of ZIPs, including hZIP4 (7,19,28,29,35,(40)(41)(42)(43)(44). Consistent with loss of zinc transport activity, the P200L variant of hZIP4 is shown to be barely expressed at cell surface.…”

Section: Discussionmentioning

confidence: 99%

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Structural defects caused by Acrodermatitis Enteropathica mutations in the extracellular domain account for mistrafficking and malfunction of ZIP4

Kuliyev

Zhang

Sui

et al. 2020

Preprint

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ZIP4 is a representative member of the Zrt-/Irt-like protein (ZIP) transporter family and responsible for zinc uptake from diet. Loss-of-function mutations of human ZIP4 (hZIP4) drastically reduce zinc absorption, causing a life-threatening autosomal recessive disorder, Acrodermatitis Enteropathica (AE). Although the zinc transport machinery is located in the transmembrane domain conserved in the entire ZIP family, half of the missense mutations occur in the extracellular domain (ECD) of hZIP4, which is only present in a fraction of mammalian ZIPs. How the AE-causing mutations in the ECD lead to ZIP4 malfunction has not be fully clarified. In this work, we characterized all the seven confirmed AE-causing missense mutations in hZIP4-ECD and found that the variants exhibited completely abolished zinc transport activity measured in a cell-based transport assay. Although the variants were able to be expressed in HEK293T cells, they failed to traffic to cell surface and were largely retained in the ER with immature glycosylation. When the corresponding mutations were introduced in the ECD of ZIP4 from Pteropus Alecto, a close homolog of hZIP4, the variants exhibited impaired protein folding and reduced thermal stability, which likely account for intracellular mistrafficking of the AE-associated variants and as such a total loss of zinc uptake in cells. This work provides a molecular pathogenic mechanism for AE, which lays out a basis for potential therapy using small molecular chaperones.

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Section: Resultsmentioning

confidence: 83%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Structural defects caused by Acrodermatitis Enteropathica mutations in the extracellular domain account for mistrafficking and malfunction of ZIP4

Kuliyev

Zhang

Sui

et al. 2020

Preprint

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“…In the second mode, through certain significant dynamics, the BMC is transiently exposed to both sides so that it is located in the middle of a pore with two ends opening simultaneously, a characteristic of channels. Cell-based metal transport assays using either radioactive substrate or metal-responsive fluorescence dye on the ZIPs from E.coli to multiple eukaryotes have shown Michaelis-Menten kinetics [1,3,23,31,32,[36][37][38][39][40][41], supporting the carrier mode. However, the study of BbZIP reconstituted in proteoliposomes indicated that the transport is non-saturable, which supports the channel mode [42].…”

Section: Inward-facing Conformation and Implications On Transport Mecmentioning

confidence: 97%

“…A BMC is often observed at the active site of metalloenzymes or the soluble portion of metal transporters [29,30], but rare in the middle of the transport pathway. A survey of over 17,000 ZIPs indicated that a BMC is present in many ZIPs from a variety of species, suggestive of an important role in ZIP function [31]. Mutagenesis and transport assay of HsZIP4 showed that the M1 is absolutely required for transport, whereas eliminating the M2 or occupying the M2 by a lysine residue only reduced but did not completely abolish transport activity.…”

Section: A Binuclear Metal Center (Bmc) In the Middle Of The Transpormentioning

confidence: 99%

Toward unzipping the ZIP metal transporters: structure, evolution, and implications on drug discovery against cancer

2021

The FEBS Journal

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The Zrt‐/Irt‐like protein (ZIP) family consists of divalent metal transporters, ubiquitous in all kingdoms of life. Since the discovery of the first ZIPs in the 1990s, the ZIP family has been expanding to contain tens of thousands of members playing key roles in uptake and homeostasis of life‐essential trace elements, primarily zinc, iron and manganese. Some family members are also responsible for toxic metal (particularly cadmium) absorption and distribution. Their central roles in trace element biology, and implications in many human diseases, including cancers, have elicited interest across multiple disciplines for potential applications in biomedicine, agriculture and environmental protection. In this review and perspective, selected areas under rapid progress in the last several years, including structural biology, evolution, and drug discovery against cancers, are summarised and commented. Future research to address the most prominent issues associated with transport and regulation mechanisms are also discussed.

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Effect of the zinc transporter ZupT on the virulence mechanisms of mesophilic Aeromonas salmonicida SRW‐OG1

Wang

Huang

2023

Animal Research and One Health

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As a typical psychrophilic bacterial pathogen, Aeromonas salmonicida causes furunculosis in wild and farmed freshwater and marine fish, leading to substantial economic losses in the global aquaculture industry. Previous studies have shown that A. salmonicida is unable to grow above 25°C, hence limiting its infection to cold‐water fish. However, we isolated A. salmonicida SRW‐OG1, a mesophilic pathogenic strain from the warm‐water fish Epinephelus coioides. Through RNA‐seq analysis, we observe significant upregulation of the zupT gene at 28°C. ZupT is a member of zinc‐regulated transporters and iron‐regulated transporter‐like proteins (ZIP family) and is closely associated with transcriptional regulation of virulence in certain pathogens. Consequently, our study aimed to examine the role of zupT during A. salmonicida SRW‐OG1 infection at high temperatures. Our findings demonstrate that the zupT‐RNAi strain exhibits severe growth restriction under limited Zn2+ and Fe2+ conditions. Notably, this strain shows significantly reduced mortality rates and colonization abilities. Moreover, its motility, biofilm formation, adhesion, and hemolytic activities are significantly diminished. Confocal laser scanning microscopy reveals earlier and accelerated biofilm dissociation in the zupT‐RNAi strain. Analysis of extracellular products at 36 h indicates a considerable reduction in relative extracellular protein content in the zupT‐RNAi strain. Taken together, our results highlight the vital role of the zupT gene in zinc transport and the fitness of A. salmonicida SRW‐OG1 within the host.

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Asymmetric functions of a binuclear metal center within the transport pathway of a human zinc transporter ZIP4

Cited by 41 publications

References 33 publications

Structural defects caused by Acrodermatitis Enteropathica mutations in the extracellular domain account for mistrafficking and malfunction of ZIP4

Structural defects caused by Acrodermatitis Enteropathica mutations in the extracellular domain account for mistrafficking and malfunction of ZIP4

Toward unzipping the ZIP metal transporters: structure, evolution, and implications on drug discovery against cancer

Effect of the zinc transporter ZupT on the virulence mechanisms of mesophilic Aeromonas salmonicida SRW‐OG1

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