Publications by authors named "Dax Fu"

27 Publications

  • Page 1 of 1

Novel autoantibodies to the β-cell surface epitopes of ZnT8 in patients progressing to type-1 diabetes.

J Autoimmun 2021 Aug 12;122:102677. Epub 2021 Jun 12.

Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD, USA. Electronic address:

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by autoimmune destruction of insulin-producing β-cells in pancreatic islets. Seroconversions to islet autoantibodies (IAbs) precede the disease onset by many years, but the role of humoral autoimmunity in the disease initiation and progression are unclear. In the present study, we identified a new IAb directed to the extracellular epitopes of ZnT8 (ZnT8ec) in newly diagnosed patients with T1D, and demonstrated immunofluorescence staining of the surface of human β-cells by autoantibodies to ZnT8ec (ZnT8ecA). With the assay specificity set on 99th percentile of 336 healthy controls, the ZnT8ecA positivity rate was 23.6% (74/313) in patients with T1D. Moreover, 30 children in a longitudinal follow up of clinical T1D development were selected for sequential expression of four major IAbs (IAA, GADA, IA-2A and ZnT8icA). Among them, 10 children were ZnT8ecA positive. Remarkably, ZnT8ecA was the earliest IAb to appear in all 10 children. The identification of ZnT8ec as a cell surface target of humoral autoimmunity in the earliest phase of IAb responses opens a new avenue of investigation into the role of IAbs in the development of β-cell autoimmunity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jaut.2021.102677DOI Listing
August 2021

Zinc transporters and their functional integration in mammalian cells.

J Biol Chem 2021 Jan-Jun;296:100320. Epub 2021 Jan 22.

Department of Physiology, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, Maryland, USA. Electronic address:

Zinc is a ubiquitous biological metal in all living organisms. The spatiotemporal zinc dynamics in cells provide crucial cellular signaling opportunities, but also challenges for intracellular zinc homeostasis with broad disease implications. Zinc transporters play a central role in regulating cellular zinc balance and subcellular zinc distributions. The discoveries of two complementary families of mammalian zinc transporters (ZnTs and ZIPs) in the mid-1990s spurred much speculation on their metal selectivity and cellular functions. After two decades of research, we have arrived at a biochemical description of zinc transport. However, in vitro functions are fundamentally different from those in living cells, where mammalian zinc transporters are directed to specific subcellular locations, engaged in dedicated macromolecular machineries, and connected with diverse cellular processes. Hence, the molecular functions of individual zinc transporters are reshaped and deeply integrated in cells to promote the utilization of zinc chemistry to perform enzymatic reactions, tune cellular responsiveness to pathophysiologic signals, and safeguard cellular homeostasis. At present, the underlying mechanisms driving the functional integration of mammalian zinc transporters are largely unknown. This knowledge gap has motivated a shift of the research focus from in vitro studies of purified zinc transporters to in cell studies of mammalian zinc transporters in the context of their subcellular locations and protein interactions. In this review, we will outline how knowledge of zinc transporters has been accumulated from in-test-tube to in-cell studies, highlighting new insights and paradigm shifts in our understanding of the molecular and cellular basis of mammalian zinc transporter functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbc.2021.100320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7949119PMC
January 2021

Down-regulation of the islet-specific zinc transporter-8 (ZnT8) protects human insulinoma cells against inflammatory stress.

J Biol Chem 2019 11 7;294(45):16992-17006. Epub 2019 Oct 7.

Department of Physiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205

Zinc transporter-8 (ZnT8) primarily functions as a zinc-sequestrating transporter in the insulin-secretory granules (ISGs) of pancreatic β-cells. Loss-of-function mutations in ZnT8 are associated with protection against type-2 diabetes (T2D), but the protective mechanism is unclear. Here, we developed an in-cell ZnT8 assay to track endogenous ZnT8 responses to metabolic and inflammatory stresses applied to human insulinoma EndoC-βH1 cells. Unexpectedly, high glucose and free fatty acids did not alter cellular ZnT8 levels, but proinflammatory cytokines acutely, reversibly, and gradually down-regulated ZnT8. Approximately 50% of the cellular ZnT8 was localized to the endoplasmic reticulum (ER), which was the primary target of the cytokine-mediated ZnT8 down-regulation. Transcriptome profiling of cytokine-exposed β-cells revealed an adaptive unfolded protein response (UPR) including a marked immunoproteasome activation that coordinately degraded ZnT8 and insulin over a 1,000-fold cytokine concentration range. RNAi-mediated knockdown protected cells against cytokine cytotoxicity, whereas inhibiting immunoproteasomes blocked cytokine-induced ZnT8 degradation and triggered a transition of the adaptive UPR to cell apoptosis. Hence, cytokine-induced down-regulation of the ER ZnT8 level promotes adaptive UPR, acting as a protective mechanism that decongests the ER burden of ZnT8 to protect β-cells from proapoptotic UPR during chronic low-grade inflammation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.010937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851310PMC
November 2019

Water molecules mediate zinc mobility in the bacterial zinc diffusion channel ZIPB.

J Biol Chem 2019 09 18;294(36):13327-13335. Epub 2019 Jul 18.

Department of Physiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205. Electronic address:

Regulated ion diffusion across biological membranes is vital for cell function. In a nanoscale ion channel, the active role of discrete water molecules in modulating hydrodynamic behaviors of individual ions is poorly understood because of the technical challenge of tracking water molecules through the channel. Here we report the results of a hydroxyl radical footprinting analysis of the zinc-selective channel ZIPB from the Gram-negative bacterium, Irradiating ZIPB by microsecond X-ray pulses activated water molecules to form covalent hydroxyl radical adducts at nearby residues, which were identified by bottom-up proteomics to detect residues that interact either with zinc or water in response to zinc binding. We found a series of residues exhibiting reciprocal changes in water accessibility attributed to alternating zinc and water binding. Mapping these residues to the previously reported crystal structure of ZIPB, we identified a water-reactive pathway that superimposed on a zinc translocation pathway consisting of two binuclear metal centers and an interim zinc-binding site. The cotranslocation of zinc and water suggested that pore-lining residues undergo a mode switch between zinc coordination and water binding to confer zinc mobility. The unprecedented details of water-mediated zinc transport identified here highlight an essential role of solvated waters in driving zinc coordination dynamics and transmembrane crossing.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA119.009239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737223PMC
September 2019

Induction of the metal transporter ZIP8 by interferon gamma in intestinal epithelial cells: Potential role of metal dyshomeostasis in Crohn's disease.

Biochem Biophys Res Commun 2019 07 29;515(2):325-331. Epub 2019 May 29.

Department of Medicine, Division of Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

Transition metals are required for intestinal homeostasis and provide essential nutrients for the resident microbiota. Abnormalities in metal homeostasis are common in Crohn's disease (CD), but remain poorly defined and causes appear multifactorial. There has been renewed interest in understanding these mechanisms with the discovery of an association between a coding variant in SLC39A8 (rs13107325; ZIP8 A391T) and increased CD risk. SLC39A8 encodes the protein ZIP8, a metal transporter that is induced under inflammatory stimuli; however, studies of its gut-specific functions are lacking. Here, we show that SLC39A8 mRNA is differentially expressed in active CD with a high positive correlation with markers of disease severity, including CXCL8, TNFα, IFNγ, and calprotectin. SLC39A8 expression exhibits a negative correlation with SLC39A4 and SLC39A5, two key zinc importers in absorptive enterocytes, and a lack of correlation with two manganese transporters, SLC39A14 and SLC11A2. Immunohistochemistry demonstrates ZIP8 expression in intestinal epithelial cells and immune cells of the lamina propria. Patients with CD exhibit variable patterns of ZIP8 subcellular localization within IECs. In ileal enteroids, SLC39A8 was induced by IFNγ and IFNγ + TNFα, but not by TNFα alone, independent of NF-κB activation. IFNγ also down-regulated SLC39A5. To explore the functional implications of disease-associated genetic variation, in over-expression experiments in HEK293A cells, ZIP8 A391T was associated with increased TNFα-induced NF-κB activation, consistent with a loss of negative regulation. Taken together, these results suggest a potential role for ZIP8 in intestinal inflammation, induced by IFNγ in the intestinal epithelial compartment, and that perturbations in negative regulation of NF-κB by ZIP8 A391T may contribute to CD pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2019.05.137DOI Listing
July 2019

Highly specific monoclonal antibodies for allosteric inhibition and immunodetection of the human pancreatic zinc transporter ZnT8.

J Biol Chem 2018 10 4;293(42):16206-16216. Epub 2018 Sep 4.

From the Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and

Solute carrier family 30 member 8 (), encoding the pancreatic zinc transporter ZnT8, is a susceptibility gene for type 2 diabetes (T2D). Reducing ZnT8 transport activity or down-regulating its cellular expression is hypothesized to be an antidiabetogenic strategy mimicking the protective effect of haploinsufficiency in humans. However, research tools to inhibit ZnT8 activity and measure cellular ZnT8 levels are not available. Here, we report the identification of two anti-ZnT8 mAbs applicable to addressing these unmet needs. Both mAbs exhibited subnanomolar affinities for human ZnT8 and were selective against homologous zinc transporters with distinct cross-species reactivities and epitope recognition. We showed that antigen-binding fragments (Fabs) protected ZnT8 from unfolding and inhibited ZnT8-mediated zinc transport in proteoliposomes. Negative-stain EM revealed a ternary binding complex of a ZnT8 monomer and two different Fabs at a 1:1:1 stoichiometry. Moreover, dual bindings of two different mAbs to a single ZnT8 protein multiplied the individual anti-ZnT8 specificities, enabling quantification of cellular ZnT8 levels by homogeneous time-resolved fluorescence (HTRF). Our results demonstrate the utilities of the two generated mAbs as allosteric inhibitors and highly specific biosensors of human ZnT8.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.005136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6200924PMC
October 2018

A subclass of serum anti-ZnT8 antibodies directed to the surface of live pancreatic β-cells.

J Biol Chem 2018 01 28;293(2):579-587. Epub 2017 Nov 28.

From the Department of Physiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205,

The islet-specific zinc transporter ZnT8 is a major self-antigen found in insulin granules of pancreatic β-cells. Frequent insulin secretion exposes ZnT8 to the cell surface, but the humoral antigenicity of the surface-displayed ZnT8 remains unknown. Here we show that a membrane-embedded human ZnT8 antigen triggered a vigorous immune response in knock-out mice. Approximately 50% of serum immunoreactivities toward ZnT8 were mapped to its transmembrane domain that is accessible to extracellular ZnT8 antibody (ZnT8A). ZnT8A binding was detected on live rat insulinoma INS-1E cells, and the binding specificity was validated by a CRISPR/Cas9 mediated knock-out. Applying established ZnT8A assays to purified serum antibodies from patients with type 1 diabetes, we detected human ZnT8A bound to live INS-1E cells, whereas a knock-out specifically reduced the surface binding. Our results demonstrate that ZnT8 is a cell surface self-antigen, raising the possibility of a direct involvement in antibody-mediated β-cell dysfunction and cytotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA117.000195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5767863PMC
January 2018

Proteoliposome-based full-length ZnT8 self-antigen for type 1 diabetes diagnosis on a plasmonic platform.

Proc Natl Acad Sci U S A 2017 09 5;114(38):10196-10201. Epub 2017 Sep 5.

Department of Chemistry, Bio-X, and the Biophysics Program, Stanford University, Stanford, CA 94305;

Identified as a major biomarker for type 1 diabetes (T1D) diagnosis, zinc transporter 8 autoantibody (ZnT8A) has shown promise for staging disease risk and disease diagnosis. However, existing assays for ZnT8 autoantibody (ZnT8A) are limited to detection by soluble domains of ZnT8, owing to difficulties in maintaining proper folding of a full-length ZnT8 protein outside its native membrane environment. Through a combined bioengineering and nanotechnology approach, we have developed a proteoliposome-based full-length ZnT8 self-antigen (full-length ZnT8 proteoliposomes; PLR-ZnT8) for efficient detection of ZnT8A on a plasmonic gold chip (pGOLD). The protective lipid matrix of proteoliposomes improved the proper folding and structural stability of full-length ZnT8, helping PLR-ZnT8 immobilized on pGOLD (PLR-ZnT8/pGOLD) achieve high-affinity capture of ZnT8A from T1D sera. Our PLR-ZnT8/pGOLD exhibited efficient ZnT8A detection for T1D diagnosis with ∼76% sensitivity and ∼97% specificity ( = 307), superior to assays based on detergent-solubilized full-length ZnT8 and the C-terminal domain of ZnT8. Multiplexed assays using pGOLD were also developed for simultaneous detection of ZnT8A, islet antigen 2 autoantibody, and glutamic acid decarboxylase autoantibody for diagnosing T1D.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1711169114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5617307PMC
September 2017

Coupling of Insulin Secretion and Display of a Granule-resident Zinc Transporter ZnT8 on the Surface of Pancreatic Beta Cells.

J Biol Chem 2017 03 27;292(10):4034-4043. Epub 2017 Jan 27.

From the Department of Physiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205

The islet-specific zinc transporter ZnT8 mediates zinc enrichment in the insulin secretory granules of the pancreatic beta cell. This granular zinc transporter is also a major self-antigen found in type 1 diabetes patients. It is not clear whether ZnT8 can be displayed on the cell surface and how insulin secretion may regulate the level of ZnT8 exposure to extracellular immune surveillance. Here we report specific antibody binding to the extracellular surface of rat insulinoma INS-1E cells that stably expressed a tagged human zinc transporter ZnT8. Flow cytometry analysis after fluorescent antibody labeling revealed strong correlations among the levels of ZnT8 expression, its display on the cell surface, and glucose-stimulated insulin secretion (GSIS). Glucose stimulation increased the surface display of endogenous ZnT8 from a basal level to 32.5% of the housekeeping Na/K ATPase on the cell surface, thereby providing direct evidence for a GSIS-dependent surface exposure of the ZnT8 self-antigen. Moreover, the variation in tagged-ZnT8 expression and surface labeling enabled sorting of heterogeneous beta cells to subpopulations that exhibited marked differences in GSIS with parallel changes in endogenous ZnT8 expression. The abundant surface display of endogenous ZnT8 and its coupling to GSIS demonstrated the potential of ZnT8 as a surface biomarker for tracking and isolating functional beta cells in mixed cell populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.772152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354490PMC
March 2017

Lipid-tuned Zinc Transport Activity of Human ZnT8 Protein Correlates with Risk for Type-2 Diabetes.

J Biol Chem 2016 12 8;291(53):26950-26957. Epub 2016 Nov 8.

From the Department of Physiology, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205 and

Zinc is a critical element for insulin storage in the secretory granules of pancreatic beta cells. The islet-specific zinc transporter ZnT8 mediates granular sequestration of zinc ions. A genetic variant of human ZnT8 arising from a single nonsynonymous nucleotide change contributes to increased susceptibility to type-2 diabetes (T2D), but it remains unclear how the high risk variant (Arg-325), which is also a higher frequency (>50%) allele, is correlated with zinc transport activity. Here, we compared the activity of Arg-325 with that of a low risk ZnT8 variant (Trp-325). The Arg-325 variant was found to be more active than the Trp-325 form following induced expression in HEK293 cells. We further examined the functional consequences of changing lipid conditions to mimic the impact of lipid remodeling on ZnT8 activity during insulin granule biogenesis. Purified ZnT8 variants in proteoliposomes exhibited more than 4-fold functional tunability by the anionic phospholipids, lysophosphatidylcholine and cholesterol. Over a broad range of permissive lipid compositions, the Arg-325 variant consistently exhibited accelerated zinc transport kinetics versus the Trp-form. In agreement with the human genetic finding that rare loss-of-function mutations in ZnT8 are associated with reduced T2D risk, our results suggested that the common high risk Arg-325 variant is hyperactive, and thus may be targeted for inhibition to reduce T2D risk in the general populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.764605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207130PMC
December 2016

The PP-motif in luminal loop 2 of ZnT transporters plays a pivotal role in TNAP activation.

Biochem J 2016 09 14;473(17):2611-21. Epub 2016 Jun 14.

Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan

Secretory and membrane-bound zinc-requiring enzymes are thought to be activated by binding zinc in the early secretory pathway. One such enzyme, tissue-non-specific alkaline phosphatase (TNAP), is activated through a two-step mechanism, via protein stabilization and subsequent enzyme activation through metalation, by ZnT5-ZnT6 heterodimers or ZnT7 homodimers. However, little is known about the molecular basis underlying the activation process. In the present study, we found that the di-proline motif (PP-motif) in luminal loop 2 of ZnT5 and ZnT7 is important for TNAP activation. TNAP activity was significantly reduced in cells lacking ZnT5-ZnT6 heterodimers and ZnT7 homodimers [triple knockout (TKO) cells]. The decreased TNAP activity was restored by expressing hZnT5 with hZnT6 or hZnT7, but significantly less so (almost 90% less) by expressing mutants thereof in which the PP-motif was mutated to alanine (PP-AA). In TKO cells, overexpressed hTNAP was not completely activated, and it was converted less efficiently into the holo form by expressing a PP-AA mutant of hZnT5 with hZnT6, whose defects were not restored by zinc supplementation. The zinc transport activity of hZnT7 was not significantly impaired by the PP-AA mutation, indicating that the PP-motif is involved in the TNAP maturation process, although it does not control zinc transport activity. The PP-motif is highly conserved in ZnT5 and ZnT7 orthologues, and its importance for TNAP activation is conserved in the Caenorhabditis elegans hZnT5 orthologue CDF5. These results provide novel molecular insights into the TNAP activation process in the early secretory pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1042/BCJ20160324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557410PMC
September 2016

Visualizing the kinetic power stroke that drives proton-coupled zinc(II) transport.

Nature 2014 Aug 22;512(7512):101-4. Epub 2014 Jun 22.

1] Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA [2] Department of Physiology, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.

The proton gradient is a principal energy source for respiration-dependent active transport, but the structural mechanisms of proton-coupled transport processes are poorly understood. YiiP is a proton-coupled zinc transporter found in the cytoplasmic membrane of Escherichia coli. Its transport site receives protons from water molecules that gain access to its hydrophobic environment and transduces the energy of an inward proton gradient to drive Zn(II) efflux. This membrane protein is a well-characterized member of the family of cation diffusion facilitators that occurs at all phylogenetic levels. Here we show, using X-ray-mediated hydroxyl radical labelling of YiiP and mass spectrometry, that Zn(II) binding triggers a highly localized, all-or-nothing change of water accessibility to the transport site and an adjacent hydrophobic gate. Millisecond time-resolved dynamics reveal a concerted and reciprocal pattern of accessibility changes along a transmembrane helix, suggesting a rigid-body helical re-orientation linked to Zn(II) binding that triggers the closing of the hydrophobic gate. The gated water access to the transport site enables a stationary proton gradient to facilitate the conversion of zinc-binding energy to the kinetic power stroke of a vectorial zinc transport. The kinetic details provide energetic insights into a proton-coupled active-transport reaction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nature13382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4144069PMC
August 2014

Intact functional fourteen-subunit respiratory membrane-bound [NiFe]-hydrogenase complex of the hyperthermophilic archaeon Pyrococcus furiosus.

J Biol Chem 2014 Jul 23;289(28):19364-72. Epub 2014 May 23.

From the Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229,

The archaeon Pyrococcus furiosus grows optimally at 100 °C by converting carbohydrates to acetate, CO2, and H2, obtaining energy from a respiratory membrane-bound hydrogenase (MBH). This conserves energy by coupling H2 production to oxidation of reduced ferredoxin with generation of a sodium ion gradient. MBH is encoded by a 14-gene operon with both hydrogenase and Na(+)/H(+) antiporter modules. Herein a His-tagged MBH was expressed in P. furiosus and the detergent-solubilized complex purified under anaerobic conditions by affinity chromatography. Purified MBH contains all 14 subunits by electrophoretic analysis (13 subunits were also identified by mass spectrometry) and had a measured iron:nickel ratio of 15:1, resembling the predicted value of 13:1. The as-purified enzyme exhibited a rhombic EPR signal characteristic of the ready nickel-boron state. The purified and membrane-bound forms of MBH both preferentially evolved H2 with the physiological donor (reduced ferredoxin) as well as with standard dyes. The O2 sensitivities of the two forms were similar (half-lives of ∼ 15 h in air), but the purified enzyme was more thermolabile (half-lives at 90 °C of 1 and 25 h, respectively). Structural analysis of purified MBH by small angle x-ray scattering indicated a Z-shaped structure with a mass of 310 kDa, resembling the predicted value (298 kDa). The angle x-ray scattering analyses reinforce and extend the conserved sequence relationships of group 4 enzymes and complex I (NADH quinone oxidoreductase). This is the first report on the properties of a solubilized form of an intact respiratory MBH complex that is proposed to evolve H2 and pump Na(+) ions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.567255DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4094048PMC
July 2014

Metalloproteomics: challenges and prospective for clinical research applications.

Authors:
Dax Fu Lydia Finney

Expert Rev Proteomics 2014 Feb 16;11(1):13-9. Epub 2014 Jan 16.

Department of Physiology, Johns Hopkins School of Medicine, 202 Physiology Building, 725 North Wolfe Street, Baltimore, MD 21205, USA.

Metals are essential cofactors, utilized in many critical cellular processes. For example, zinc is important in insulin biosynthesis and may play a role in Alzheimer's disease, but much of how the zinc-mediated process remains unknown. Knowing which metal is in which protein at a given point in time would lead to new insights into how metals work in biological systems. New tools are being developed to investigate the biochemistry and cell biology of metals, with potential for biomedical applications. In this report, we consider the promise and limitations of metalloproteins detection techniques. We provide a brief overview of the techniques available and a discussion of the technical challenges to biomedical applications, with particular focus on what must be overcome for the potential of these approaches to be achieved.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1586/14789450.2014.876365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4598182PMC
February 2014

Histidine pairing at the metal transport site of mammalian ZnT transporters controls Zn2+ over Cd2+ selectivity.

Proc Natl Acad Sci U S A 2012 May 23;109(19):7202-7. Epub 2012 Apr 23.

Department of Physiology, Morphology, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva 84105, Israel.

Zinc and cadmium are similar metal ions, but though Zn(2+) is an essential nutrient, Cd(2+) is a toxic and common pollutant linked to multiple disorders. Faster body turnover and ubiquitous distribution of Zn(2+) vs. Cd(2+) suggest that a mammalian metal transporter distinguishes between these metal ions. We show that the mammalian metal transporters, ZnTs, mediate cytosolic and vesicular Zn(2+) transport, but reject Cd(2+), thus constituting the first mammalian metal transporter with a refined selectivity against Cd(2+). Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminate between Zn(2+) and Cd(2+). A phylogenetic comparison between the tetrahedral metal transport motif of YiiP and ZnTs identifies a histidine at the mammalian site that is critical for metal selectivity. Residue swapping at this position abolished metal selectivity of ZnTs, and fully reconstituted selective Zn(2+) transport of YiiP. Finally, we show that metal selectivity evolves through a reduction in binding but not the translocation of Cd(2+) by the transporter. Thus, our results identify a unique class of mammalian transporters and the structural motif required to discriminate between Zn(2+) and Cd(2+), and show that metal selectivity is tuned by a coordination-based mechanism that raises the thermodynamic barrier to Cd(2+) binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1200362109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3358884PMC
May 2012

Selective electrodiffusion of zinc ions in a Zrt-, Irt-like protein, ZIPB.

J Biol Chem 2010 Dec 28;285(50):39013-20. Epub 2010 Sep 28.

Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn(2+) and Cd(2+), whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M110.180620DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2998139PMC
December 2010

Structural basis for autoregulation of the zinc transporter YiiP.

Nat Struct Mol Biol 2009 Oct 13;16(10):1063-7. Epub 2009 Sep 13.

Biology Department, Brookhaven National Laboratory, Upton, New York, USA.

Zinc transporters have crucial roles in cellular zinc homeostatic control. The 2.9-A resolution structure of the zinc transporter YiiP from Escherichia coli reveals a richly charged dimer interface stabilized by zinc binding. Site-directed fluorescence resonance energy transfer (FRET) measurements and mutation-activity analysis suggest that zinc binding triggers hinge movements of two electrically repulsive cytoplasmic domains pivoting around four salt bridges situated at the juncture of the cytoplasmic and transmembrane domains. These highly conserved salt bridges interlock transmembrane helices at the dimer interface, where they are well positioned to transmit zinc-induced interdomain movements to reorient transmembrane helices, thereby modulating coordination geometry of the active site for zinc transport. The cytoplasmic domain of YiiP is a structural mimic of metal-trafficking proteins and the metal-binding domains of metal-transporting P-type ATPases. The use of this common structural module to regulate metal coordination chemistry may enable a tunable transport activity in response to cytoplasmic metal fluctuations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/nsmb.1662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2758918PMC
October 2009

Structure of the zinc transporter YiiP.

Authors:
Min Lu Dax Fu

Science 2007 Sep 23;317(5845):1746-8. Epub 2007 Aug 23.

Department of Biology, Brookhaven National Laboratory, Upton, NY 11973, USA.

YiiP is a membrane transporter that catalyzes Zn2+/H+ exchange across the inner membrane of Escherichia coli. Mammalian homologs of YiiP play critical roles in zinc homeostasis and cell signaling. Here, we report the x-ray structure of YiiP in complex with zinc at 3.8 angstrom resolution. YiiP is a homodimer held together in a parallel orientation through four Zn2+ ions at the interface of the cytoplasmic domains, whereas the two transmembrane domains swing out to yield a Y-shaped structure. In each protomer, the cytoplasmic domain adopts a metallochaperone-like protein fold; the transmembrane domain features a bundle of six transmembrane helices and a tetrahedral Zn2+ binding site located in a cavity that is open to both the membrane outer leaflet and the periplasm.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.1143748DOI Listing
September 2007

The structural basis of water permeation and proton exclusion in aquaporins.

Authors:
Dax Fu Min Lu

Mol Membr Biol 2007 Sep-Dec;24(5-6):366-74

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.

Aquaporins (AQPs) represent a ubiquitous class of integral membrane proteins that play critical roles in cellular osmoregulations in microbes, plants and mammals. AQPs primarily function as water-conducting channels, whereas members of a sub-class of AQPs, termed aquaglyceroporins, are permeable to small neutral solutes such as glycerol. While AQPs facilitate transmembrane permeation of water and/or small neutral solutes, they preclude the conduction of protons. Consequently, openings of AQP channels allow rapid water diffusion down an osmotic gradient without dissipating electrochemical potentials. Molecular structures of AQPs portray unique features that define the two central functions of AQP channels: effective water permeation and strict proton exclusion. This review describes AQP structures known to date and discusses the mechanisms underlying water permeation, proton exclusion and water permeability regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/09687680701446965DOI Listing
November 2007

Binding and transport of metal ions at the dimer interface of the Escherichia coli metal transporter YiiP.

Authors:
Yinan Wei Dax Fu

J Biol Chem 2006 Aug 21;281(33):23492-502. Epub 2006 Jun 21.

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.

YiiP is a representative member of the cation diffusion facilitator (CDF) family, a class of ubiquitous metal transporters that play an essential role in metal homeostasis. Recently, a pair of Zn2+/Cd2+-selective binding sites has been localized to two highly conserved aspartyl residues (Asp157), each in a 2-fold-symmetry-related transmembrane segment 5 (TM5) of a YiiP homodimer. Here we report the functional and structural interactions between Asp157 and yet another highly conserved Asp49 in the TM2. Calorimetric binding analysis indicated that Asp49 and Asp157 contribute to a common Cd2+ binding site in each subunit. Copper phenanthroline oxidation of YiiP(D49C), YiiP(D157C), and YiiP(D49C/D157C) yielded inter- and intra-subunit cross-links among Cys49 and Cys157, consistent with the spatial proximity of two (Asp49-Asp157) sites at the dimer interface. Hg2+ binding to YiiP(D49C) or YiiP(D49C/D157C) also yielded a Cys49-Hg2+-Cys49 biscysteinate complex across the dimer interface, further establishing the interfacial location of a (Asp49-Asp157)2 bimetal binding center. Two bound Cd2+ ions were found transported cooperatively with a sigmoidal dependence on the Cd2+ concentration (n = 1.4). The binding affinity, transport cooperativity, and rate were modestly reduced by either a D49C or D157C mutation, but greatly diminished when all the bidentate aspartate O-ligands in (Asp49-Asp157)2 were replaced by the monodentate cysteine S-ligands. The functional significance of these findings is discussed based on the unique coordination chemistry of aspartyl residues and a model for the translocation pathway of metal ions at the YiiP dimer interface.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M602254200DOI Listing
August 2006

Crystal structure of AqpZ tetramer reveals two distinct Arg-189 conformations associated with water permeation through the narrowest constriction of the water-conducting channel.

J Biol Chem 2006 Jan 20;281(1):454-60. Epub 2005 Oct 20.

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.

AqpZ is a homotetramer of four water-conducting channels that facilitate rapid water movements across the plasma membrane of Escherichia coli. Here we report a 3.2 angstroms crystal structure of the tetrameric AqpZ (tAqpZ). All channel-lining residues in the four monomeric channels are found orientated in nearly identical positions with one marked exception at the narrowest channel constriction, where the side chain of a highly conserved Arg-189 adopts two distinct conformational orientations. In one of the four monomers, the guanidino group of Arg-189 points toward the periplasmic vestibule, opening up the constriction to accommodate the binding of a water molecule through a tridentate H-bond. In the other three monomers, the Arg-189 guanidino group bends over to form an H-bond with carbonyl oxygen of the Thr-183, thus occluding the channel. Therefore, the tAqpZ structure reveals two distinct Arg-189 confirmations associated with water permeation through the channel constrictions. Alternation between the two Arg-189 conformations disrupts continuous flow of water, thus regulating the open probability of the water pore. Further, the difference in Arg-189 displacements is correlated with a strong electron density found between the first transmembrane helices of two open channels, suggesting that the observed Arg-189 conformations are stabilized by asymmetrical subunit interactions in tAqpZ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M508926200DOI Listing
January 2006

Selective metal binding to a membrane-embedded aspartate in the Escherichia coli metal transporter YiiP (FieF).

Authors:
Yinan Wei Dax Fu

J Biol Chem 2005 Oct 27;280(40):33716-24. Epub 2005 Jul 27.

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.

The cation diffusion facilitators (CDF) are a ubiquitous family of metal transporters that play important roles in homeostasis of a wide range of divalent metal cations. Molecular identities of substrate-binding sites and their metal selectivity in the CDF family are thus far unknown. By using isothermal titration calorimetry and stopped-flow spectrofluorometry, we directly examined metal binding to a highly conserved aspartate in the Escherichia coli CDF transporter YiiP (FieF). A D157A mutation abolished a Cd2+-binding site and impaired the corresponding Cd2+ transport. In contrast, substitution of Asp-157 with a cysteinyl coordination residue resulted in intact Cd2+ binding as well as full transport activity. A similar correlation was found for Zn2+ binding and transport, suggesting that Asp-157 is a metal coordination residue required for binding and transport of Cd2+ and Zn2+. The location of Asp-157 was mapped topologically to the hydrophobic core of transmembrane segment 5 (TM-5) where D157C was found partially accessible to thiol-specific labeling of maleimide polyethylene-oxide biotin. Binding of Zn2+ and Cd2+, but not Fe2+, Hg2+, Co2+, Ni2+, Mn2+, Ca2+, and Mg2+, protected D157C from maleimide polyethylene-oxide biotin labeling in a concentration-dependent manner. Furthermore, isothermal titration calorimetry analysis of YiiP(D157A) showed no detectable change in Fe2+ and Hg2+ calorimetric titrations, indicating that Asp-157 is not a coordination residue for Fe2+ and Hg2+ binding. Our results provided direct evidence for selective binding of Zn2+ and Cd2+ for to the highly conserved Asp-157 and defined its functional role in metal transport.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M506107200DOI Listing
October 2005

Oligomeric state of the Escherichia coli metal transporter YiiP.

J Biol Chem 2004 Sep 16;279(38):39251-9. Epub 2004 Jul 16.

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.

YiiP is a 32.9-kDa metal transporter found in the plasma membrane of Escherichia coli (Chao, Y., and Fu, D. (2004) J. Biol. Chem. 279, 17173-17180). Here we report the determination of the YiiP oligomeric state in detergent-lipid micelles and in membranes. Molecular masses of YiiP solubilized with dodecyl-, undecyl-, decyl-, or nonyl-beta-d-maltoside were measured directly using size-exclusion chromatography coupled with laser light-scattering photometry, yielding a mass distribution of YiiP homo-oligomers within a narrow range (68.0-68.8 kDa) that equals the predicted mass of a YiiP dimer within experimental error. The detergent-lipid masses associated with YiiP in the mixed micelles were found to increase from 135.5 to 232.6 kDa, with an apparent correlation with the alkyl chain length of the maltoside detergents. Cross-linking the detergent-solubilized YiiP with 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) resulted in a dimeric cross-linked product in an EDC concentration-dependent manner. The oligomeric state of the purified YiiP in reconstituted membranes was determined by electron microscopic analysis of two-dimensional YiiP crystals in negative stain. A projection structure calculated from measurable optical diffractions to 25 A revealed a pseudo-2-fold symmetry within a molecular boundary of approximately 75 x 40 A, indicative of the presence of YiiP dimers in membranes. These data provide direct structural evidence for a dimeric association of YiiP both in detergent-lipid micelles and in the reconstituted lipid bilayer. The functional relevance of the dimeric association in YiiP is discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M407044200DOI Listing
September 2004

Crystallization and preliminary crystallographic analysis of the Escherichia coli water channel AqpZ.

Acta Crystallogr D Biol Crystallogr 2004 Mar 25;60(Pt 3):561-3. Epub 2004 Feb 25.

Biology Department, Brookhaven National Laboratory, Upton, NY 11973-5000, USA.

AqpZ is a 24 kDa integral membrane protein that facilitates water movement across the plasma membrane of Escherichia coli. In this study, the first crystallization and preliminary X-ray analysis of AqpZ are described. AqpZ was overexpressed and purified with a yield of 13 mg of purified AqpZ per litre of cell culture. The purified AqpZ was shown to be a monodisperse species consisting of tetrameric protein-detergent complexes. A crystallization condition for producing diffraction-quality crystals was identified. Initial X-ray analysis indicated that the diffraction limit of AqpZ extended to 3.6 A. Crystals were found to belong to space groups P4(1)22 or P4(3)22, with unit-cell parameters a = b = 119.04, c = 380.23 A.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1107/S090744490302972XDOI Listing
March 2004

Thermodynamic studies of the mechanism of metal binding to the Escherichia coli zinc transporter YiiP.

Authors:
Yang Chao Dax Fu

J Biol Chem 2004 Apr 11;279(17):17173-80. Epub 2004 Feb 11.

Department of Biology, Brookhaven National Laboratory, Upton, New York 11973, USA.

Sequence homology of the Escherichia coli YiiP places it within the family of cation diffusion facilitators, a family of membrane transporters that play a central role in regulating cellular zinc homeostasis. Here we describe the first thermodynamic and mechanistic studies of metal binding to a cation diffusion facilitator. Isothermal titration calorimetric analyses of the purified YiiP and binding competitions among Zn(2+), Cd(2+), and Hg(2+) revealed a mutually competitive binding site common to three metal ions and a set of noncompetitive binding sites, including one Cd(2+) site, one Hg(2+) site, and at least one Zn(2+) site, to which the binding of Zn(2+) exhibited partial inhibitions of both Cd(2+) and Hg(2+) bindings. Lowering the pH from 7.0 to 5.5 inhibited binding of Zn(2+) and Cd(2+) to the common site. Further, the enthalpy change of the Cd(2+) binding to the common site was found to be related linearly to the ionization enthalpy of the pH buffer with a slope corresponding to the release of 1.23 H(+) for each Cd(2+) binding. These H(+) effects are consistent with a coupled deprotonation process upon binding of Zn(2+) and Cd(2+). Modification of histidine residues by diethyl pyrocarbonate specifically inhibited Zn(2+) binding to the common binding site, indicating that the mechanism of binding-deprotonation coupling involves a histidine residue(s).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M400208200DOI Listing
April 2004

Kinetic study of the antiport mechanism of an Escherichia coli zinc transporter, ZitB.

Authors:
Yang Chao Dax Fu

J Biol Chem 2004 Mar 10;279(13):12043-50. Epub 2004 Jan 10.

Department of Biology, Building 463, Brookhaven National Laboratory, Upton, NY 11973, USA.

ZitB is a member of the cation diffusion facilitator (CDF) family that mediates efflux of zinc across the plasma membrane of Escherichia coli. We describe the first kinetic study of the purified and reconstituted ZitB by stopped-flow measurements of transmembrane fluxes of metal ions using a metal-sensitive fluorescent indicator encapsulated in proteoliposomes. Metal ion filling experiments showed that the initial rate of Zn2+ influx was a linear function of the molar ratio of ZitB to lipid and was related to the concentration of Zn2+ or Cd2+ by a hyperbola with a Michaelis-Menten constant (K(m)) of 104.9 +/- 5.4 microm and 90.1 +/- 3.7 microm, respectively. Depletion of proton stalled Cd2+ transport down its diffusion gradient, whereas tetraethylammonium ion substitution for K+ did not affect Cd2+ transport, indicating that Cd2+ transport is coupled to H+ rather than to K+. H+ transport was inferred by the H+ dependence of Cd2+ transport, showing a hyperbolic relationship with a Km of 19.9 nm for H+. Applying H+ diffusion gradients across the membrane caused Cd2+ fluxes both into and out of proteoliposomes against the imposed H(+) gradients. Likewise, applying outwardly oriented membrane electrical potential resulted in Cd2+ efflux, demonstrating the electrogenic effect of ZitB transport. Taken together, these results indicate that ZitB is an antiporter catalyzing the obligatory exchange of Zn2+ or Cd2+ for H+. The exchange stoichiometry of metal ion for proton is likely to be 1:1.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M313510200DOI Listing
March 2004

The structure of GlpF, a glycerol conducting channel.

Novartis Found Symp 2002 ;245:51-61; discussion 61-5, 165-8

Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA.

The passage of water or small neutral solutes across the cell membrane in animals, plants and bacteria is facilitated by a family of homologous membrane channels, variously known as aquaporins though perhaps more correctly as aquaglyceroporins. The glycerol facilitator (GlpF) is a 28 kDa aquaglyceroporin that catalyses transmembrane diffusion of glycerol and certain linear polyhydric alcohols in Escherichia coli. X-ray crystallographic analysis of GlpF to 2.2 A resolution revealed an alpha-barrel structure, surrounded by six full-length transmembrane helices and two half-spanning helices that are joined head-to-head in the middle of the membrane. These helices are arranged to a quasi twofold manner relative to the central membrane plane, where highly conserved residues make helix-to-helix contacts that stabilize the relative position and orientation of the helices in the structure. This sequence-structure correlation suggests that the evolutionary divergence of aquaporins and aquaglyceroporins is constrained by a conserved structural framework within which specialized function may be developed. Three glycerol molecules were resolved in the central channel through the GlpF monomer, thereby defining a transmembrane channel for glycerol permeation. The structure of glycerol GlpF complex provides insight into the chemical basis for transmembrane selective permeability.
View Article and Find Full Text PDF

Download full-text PDF

Source
November 2002
-->