Human Zinc Research Articles

Effect of Pooling Practices and Time Postpartum of Milk Donations on the Energy, Macronutrient, and Zinc Concentrations of Resultant Donor Human Milk Pools

To characterize the macronutrient, energy, and zinc composition of pasteurized donor human milk pools and evaluate how composition varies based on pooling practices and "time postpartum" (ie, elapsed time from parturition to expression date) of individual milk donations. The Mothers' Milk Bank (Arvada, Colorado) donated 128 donor human milk pools. Caloric density was assessed via mid-infrared spectroscopy, and zinc concentration was measured by atomic absorption spectroscopy. Pool time postpartum was calculated as the unweighted average of the time postpartum of all milk donations included in any given pool. Time postpartum of donor human milk pools ranged from 3days to 9.8months. The majority (91%) of donor human milk pools included milk from either 1 donor or 2 donors. Pool energy density ranged from 14.7 to 23.1kcal/oz, and protein ranged from 0.52 to 1.43g/dL. Milk zinc concentrations were higher in preterm pools and were negatively correlated with pool time postpartum. We present an equation that estimates donor human milk pool zinc content based on time postpartum and explains 49% of the variability in zinc concentrations (P<.0001). Including more donors in donor human milk pools decreased the variability in protein, but not zinc, concentrations. Donor human milk pools were lower in calories than is normally assumed in standard human milk fortification practices. Zinc concentrations were related to donor human milk time postpartum and were on average insufficient to meet preterm and term infants' needs without fortification or supplementation.

Polymorphisms in the human PRDM9 gene may lead to meiotic arrest and azoospermia

PRDM9 is responsible for directing the location of programmed double strand breaks and subsequent crossover events between homologous chromosomes during meiosis in human gametes. Prdm9 is also essential for meiosis and fertility in mice, with PRDM9 knockout males displaying complete azoospermia. PRDM9 contains a rapidly evolving DNA binding zinc finger array that is coded by a ∼84 nucleotide repeating unit mini-satellite sequence. The human A-type allele, which accounts for ∼90% of the alleles in the human population, contains 13 repeating units. We sought to develop a s strategy to effectively genotype this mini-satellite with PacBio sequencing and to determine whether PRDM9 variation, including mini-satellite length polymorphisms, is associated with infertility in the human male. Observational study. Using a normospermic human control, a two-step polymerase chain reaction (PCR) protocol was established to successfully amplify the PRDM9 zinc finger array mini-satellite and sequence it using both Sanger and PacBio next generation sequencing, which has the advantage of circular consensus sequencing and long reads. We next amplified PRDM9 from the genomic DNA of 48 azoospermic men and 5 controls. The samples were visually analyzed with gel electrophoresis, and potential mutant alleles that had an atypical band appearance were compared to the wild type. PacBio sequencing results from the normospermic control were found to be an identical match to the known human A allele, confirming our ability to effectively genotype the mini-satellite array in a single PacBio run. Gel electrophoresis of PCR amplified PRDM9 alleles from azoospermic men identified 6 potential mutant variants of distinct mini-satellite lengths differing from the A-allele. We have developed a protocol to effectively genotype the human PRDM9 zinc finger array mini-satellite to evaluate a potential etiology of azoospermia in the infertile human male. We found 6 potential PRDM9 alleles differing from the known A-allele in a small (n=48) azoospermia cohort. Current/ongoing research includes applying our PacBio sequencing protocol to genotype all 48 azoospermic men in comparison to a control group and evaluate if there is an association between PRDM9 mini-satellite repeat length, polymorphisms, or de novo mutations and azoospermia.

A functional Cis-eQTL locus in lncRNA ZNRD1-AS1 contributes to the susceptibility of endometrial cancer.

Endometrial cancer (EC) remains one of the most common gynecologic malignancies worldwide. However, the exact etiology is still unknown. Human Zinc ribbon domain containing 1 (ZNRD1) was involved in carcinogenesis and progression of multiple cancers, including EC. ZNRD1-AS1, a long noncoding RNA (lncRNA) located in the upstream of ZNRD1, has been reported as an essential component in carcinogenesis. However, the underlying relations of ZNRD1-AS1 with development of EC remain obscure. This study aims to evaluate the potential role of ZNRD1-AS1 and Cis-eQTL loci of ZNRD1 in the occurrence of EC. We first evaluated the expression of ZNRD1-AS1 and ZNRD1 among EC tissues and corresponding normal tissues using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Then, to reveal the underlying mechanisms, we investigated the associations between Cis-eQTL loci of ZNRD1 in ZNRD1-AS1 and the susceptibility of EC. Further, in vitro experiments were conducted to evaluate the regulation role of rs9261204 on the expression of ZNRD1gene. Higher expression of ZNRD1-AS1 and lower expression of ZNRD1 were detected in the EC tissues, compared to the normal tissues. Minor allele of rs9261204 was significantly associated with increased risk of EC (OR: 1.31; 95% CI: 1.08-1.6; p=0.007) [corrected]. Furthermore, in vitro experiments confirmed that Ishikawa cells with rs9261204 G allele had lower mRNA level of ZNRD1, compared to the A allele. Our findings first showed the contribution of LncRNA ZNRD1-AS1 and Cis-eQTL loci of ZNRD1 to the development of EC. Further studies incorporating larger populations and functional assays are warranted.

Dietary zinc and the control of Streptococcus pneumoniae infection

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.

Characterization of the human zinc finger nfx‑1‑type containing 1 encoding ZNFX1 gene and its response to 12‑O‑tetradecanoyl‑13‑acetate in HL‑60 cells.

Human promyelocytic HL‑60 cells can be differentiated into macrophage‑like cells by treatment with 12‑O‑tetra decanoylphorbol‑13‑acetate (TPA). Certain 5' upstream regions of the zinc finger protein (ZNF)‑encoding genes contain duplicated GGAA motifs, which are frequently found in the TPA‑responding gene promoter regions. To examine transcriptional responses to TPA, 5'flanking regions of human zinc finger CCCH‑type containing, antiviral, ZNF252, ZNF343, ZNF555, ZNF782 and zinc finger nfx‑1‑type containing 1 (ZNFX1) genes were isolated by polymerase chain reaction (PCR) and ligated into a multiple‑cloning site of the pGL4.10[luc2] vector. Transient transfection and a luciferase assay revealed that the ZNFX1 promoter most prominently responded to the TPA treatment. Deletion and point mutation experiments indicated that the duplicated GGAA motif in the 100‑bp region positively responded to TPA. In addition, reverse transcription‑quantitative PCR and western blotting showed that the mRNA and protein of ZNFX1 accumulate during the differentiation of HL‑60 cells. These results indicated that expression of the TPA‑inducible ZNFX1 gene, which belongs to the group of interferon‑responsive genes, is regulated by the cis‑action of the duplicated GGAA motif.

Low Expression of GLIS2 Gene Might Associate with Radiosensitivity of Gastric Cancer.

Human gene GLIS family zinc finger 2 (GLIS2) is a member of GLI-similar zinc finger protein family. Previous studies indicated GLIS2 gene involved in tumorigenesis mechanisms. However, the association between GLIS2 expression and radiosensitivity of gastric cancer has not been well understood. In this study, we used the gastric cancer database in TCGA, and significant association was observed between the low expression of GLIS2 and radiosensitivity of patients with gastric cancer. The adjusted HR values for radiotherapy were 0.162(0.035-0.756) and 0.089(0.014-0.564), with p values 0.021 and 0.010, respectively, in training and testing data, for these patients with low expression of GLIS2, while for patients with high expression of GLIS2, there was no significant survival difference between radiotherapy and nonradiotherapy groups. The adjusted HR were 0.676(0.288-1.586) and 0.508(0.178-1.450), with p values 0.368 and 0.206 in training and testing data, respectively. Further study showed that, for low expression patients, radiotherapy did not significantly increase new tumor event rate and disease progression rate, which partially supported our assumption. These results suggested that low expression of GLIS2 might significantly associate with the radiosensitivity of patients with gastric cancer. The GLIS2 gene might be a potential effective molecular marker of gastric cancer for precise radiotherapy.

Shaping of Drosophila Neural Cell Lineages Through Coordination of Cell Proliferation and Cell Fate by the BTB-ZF Transcription Factor Tramtrack-69.

Cell diversity in multicellular organisms relies on coordination between cell proliferation and the acquisition of cell identity. The equilibrium between these two processes is essential to assure the correct number of determined cells at a given time at a given place. Using genetic approaches and correlative microscopy, we show that Tramtrack-69 (Ttk69, a Broad-complex, Tramtrack and Bric-à-brac - Zinc Finger (BTB-ZF) transcription factor ortholog of the human promyelocytic leukemia zinc finger factor) plays an essential role in controlling this balance. In the Drosophila bristle cell lineage, which produces the external sensory organs composed by a neuron and accessory cells, we show that ttk69 loss-of-function leads to supplementary neural-type cells at the expense of accessory cells. Our data indicate that Ttk69 (1) promotes cell cycle exit of newborn terminal cells by downregulating CycE, the principal cyclin involved in S-phase entry, and (2) regulates cell-fate acquisition and terminal differentiation, by downregulating the expression of hamlet and upregulating that of Suppressor of Hairless, two transcription factors involved in neural-fate acquisition and accessory cell differentiation, respectively. Thus, Ttk69 plays a central role in shaping neural cell lineages by integrating molecular mechanisms that regulate progenitor cell cycle exit and cell-fate commitment.

Zinc Absorption From Agronomically Biofortified Wheat Is Similar to Post-Harvest Fortified Wheat and Is a Substantial Source of Bioavailable Zinc in Humans

Limited data exist on human zinc absorption from wheat biofortified via foliar (FBW) or root (hydroponically fortified wheat, HBW) zinc application. Stable isotope labels added at point of consumption (extrinsic labeling) might not reflect absorption from native zinc obtained by intrinsic labeling. We measured fractional and total zinc absorption (FAZ, TAZ) in FBW and HBW wheat, compared with control wheat (CW) and fortified wheat (FW). The effect of labeling method was assessed in HBW (study 1), and the effect of milling extraction rate (EXR, 80% and 100%) in FBW (studies 2 and 3). Generally healthy adults (n=71, age: 18-45 y, body mass index: 18.5-25 kg/m2) were allocated to 1 of the studies, in which they served as their own controls. In study 1, men and women consumed wheat porridges colabeled intrinsically and extrinsically with 67Zn and 70Zn. In studies 2 and 3, women consumed wheat flatbreads (chapatis) labeled extrinsically. Zinc absorption was measured with the oral to intravenous tracer ratio method with a 4-wk wash-out period between meals. Data were analyzed with linear mixed models. In study 1 there were no differences in zinc absorption from extrinsic versus intrinsic labels in either FW or HBW. Similarly, FAZ and TAZ from FW and HBW did not differ. TAZ was 70-76% higher in FW and HBW compared with CW (P<0.01). In studies 2 and 3, TAZ from FW and FBW did not differ but was 20-48% higher compared with CW (P<0.001). Extraction rate had no effect on TAZ. Colabeling demonstrates that extrinsic zinc isotopic labels can be used to accurately quantify zinc absorption from wheat in humans. Biofortification through foliar zinc application, root zinc application, or fortification provides higher TAZ compared with unfortified wheat. In biofortified wheat, extraction rate (100-80%) has a limited impact on total zinc absorption. These studies were registered on clinicaltrials.gov (NCT01775319).

Unraveling the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2)

The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–translocation pore. Interestingly, the H63A substitution completely abrogated pH sensitivity, and substitutions of Glu-67 and Phe-269 altered the pH and voltage modulation of transport. In contrast, substitution of Glu-106, which might be part of a dimerization interface, altered pH but not voltage modulation. Substitution of Phe-269, located close to the substrate-binding site, also affected substrate selectivity. These findings were supported by an additional model of ZIP2 that was based on the structure of a prokaryotic homolog, Bordetella bronchiseptica ZrT/Irt-like protein (bbZIP), and in silico pKa calculations. We also found that residues Glu-179, His-175, His-202, and Glu-276 are directly involved in the coordination of the substrate metal ion. We noted that, unlike bbZIP, human ZIP2 is predicted to harbor a single divalent metal-binding site, with the charged side chain of Lys-203 replacing the second bound ion. Our results provide the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport, identify the substrate-binding site, and suggest a structure-based transport mechanism for the ZIP2 transporter.

Quantifying the Oligomeric State of hZIP4 on the Surface of Cells.

The human (h) zinc transporter ZIP4 is expressed on the plasma membrane and functions to increase cytosolic zinc levels. Mutations in hZIP4 cause the disease acrodermatitis enteropathica. Dysfunction in the regulation of hZIP4 has also been indicated in solid tissue cancers, including pancreatic and prostate cancer. Although structural studies of the extracellular domain and computational modeling of the membrane domain suggest hZIP4 exists as a dimer, the oligomerization status of hZIP4 in the plasma membrane of mammalian cells has not been directly quantified in vivo. Here, the oligomeric state of hZIP4 expressed in HEK293 cells was quantified using fluorescence correlation spectroscopy. hZIP4 was tagged with eGFP, and by comparing brightness values (ε) of monomer and tandem eGFP constructs to that of an hZIP4/eGFP, we show that hZIP4 is a dimer. Determining that hZIP4 is a dimer is an important step toward understanding the function and processing of the protein, which can provide more insight into how diseases affected by hZIP4 occur and can be managed.

Concomitant disorder and high-affinity zinc binding in the human zinc- and iron-regulated transport protein 4 intracellular loop.

The human zinc- and iron-regulated transport protein 4 (hZIP4) protein is the major plasma membrane protein responsible for the uptake of zinc in the body, and as such it plays a key role in cellular zinc homeostasis. hZIP4 plasma membrane levels are regulated through post-translational modification of its large, disordered, histidine-rich cytosolic loop (ICL2) in response to intracellular zinc concentrations. Here, structural characteristics of the isolated disordered loop region, both in the absence and presence of zinc, were investigated using nuclear magnetic resonance (NMR) spectroscopy. NMR chemical shifts, coupling constants and temperature coefficients of the apoprotein, are consistent with a random coil with minor propensities for transient polyproline Type II helices and β-strand in regions implicated in post-translational modifications. The ICL2 protein remains disordered upon zinc binding, which induces exchange broadening. Paramagnetic relaxation enhancement experiments reveal that the histidine-rich region in the apoprotein makes transient tertiary contacts with predicted post-translational modification sites. The residue-specific data presented here strengthen the relationship between hZIP4 post-translational modifications, which impact its role in cellular zinc homeostasis, and zinc sensing by the intracellular loop. Furthermore, the zinc sensing mechanism employed by the ICL2 protein demonstrates that high-affinity interactions can occur in the presence of conformational disorder.

Genomic Characterization of the Zinc Transcriptional Regulatory Element Reveals Potential Functional Roles of ZNF658.

The zinc transcriptional regulatory element (ZTRE) is a newly reported binding motif for human zinc finger protein ZNF658, which alters gene expression in response to cellular zinc. The ZTRE has two nucleotide components-the palindromic flanking pairs and the bridging "N" bases between these flanks that range in number from 0 to 100. There are 12 pairs of ZTRE flanks (designated A-L). Three thousand five hundred twenty-five genes contain one or more ZTREs - 1000 to + 200bp from their transcriptional start site (TSS). ZTRE-E is observed at a greater frequency, and ZTRE containing 25 bridging bases are less frequent, within - 200bp from the TSS. The genes with ZTREs in this range are enriched in processes that may compensate zinc deficiency, while other genes with ZTREs outside this range are enriched in transcriptional activation processes. The division of ZTREs into two groups may imply a dual role of ZNF658, similar to the homologous yeast protein Zap1, via binding to low or high affinity sequences dependent upon cellular zinc. The KLF/Sp1-family binding motif is prevalent within the ZTRE "N" bridging bases, suggesting ZNF658 may compete with Sp1-like transactivators to suppress transcription.

An extracellular histidine-containing motif in the zinc transporter ZIP4 plays a role in zinc sensing and zinc-induced endocytosis in mammalian cells

Zinc is an essential trace element that serves as a cofactor for enzymes in critical biochemical processes and also plays a structural role in numerous proteins. Zinc transporter ZIP4 (ZIP4) is a zinc importer required for dietary zinc uptake in the intestine and other cell types. Studies in cultured cells have reported that zinc stimulates the endocytosis of plasma membrane-localized ZIP4 protein, resulting in reduced cellular zinc uptake. Thus, zinc-regulated trafficking of ZIP4 is a key means for regulating cellular zinc homeostasis, but the underlying mechanisms are not well understood. In this study, we used mutational analysis, immunoblotting, HEK293 cells, and immunofluorescence microscopy to identify a histidine-containing motif (398HTH) in the first extracellular loop that is required for high sensitivity to low zinc concentrations in a zinc-induced endocytic response of mouse ZIP4 (mZIP4). Moreover, using synthetic peptides with selective substitutions and truncated mZIP4 variants, we provide evidence that histidine residues in this motif coordinate a zinc ion in mZIP4 homodimers at the plasma membrane. These findings suggest that 398HTH is an important zinc-sensing motif for eliciting high-affinity zinc-stimulated endocytosis of mZIP4 and provide insight into cellular mechanisms for regulating cellular zinc homeostasis in mammalian cells.

Soil Zinc Is Associated with Serum Zinc But Not with Linear Growth of Children in Ethiopia.

To our knowledge, the relationships among soil zinc, serum zinc and children’s linear growth have not been studied geographically or at a national level in any country. We use data from the cross-sectional, nationally representative Ethiopian National Micronutrient Survey (ENMS) (n = 1776), which provided anthropometric and serum zinc (n = 1171) data on children aged 6–59 months. Soil zinc levels were extracted for each child from the digital soil map of Ethiopia, developed by the Africa Soil Information Service. Children’s linear growth was computed using length/height and age converted into Z-scores for height-for-age. Multi-level mixed linear regression models were used for the analysis. Nationally, 28% of children aged 6–59 months were zinc deficient (24% when adjusted for inflammation) and 38% were stunted. Twenty percent of households in the ENMS were located on zinc-deficient soils. Soil zinc (in mg/kg) was positively associated with serum zinc (in µg/dL) (b = 0.9, p = 0.020) and weight-for-height-Z-score (b = 0.05, p = 0.045) but linear growth was not associated with soil zinc (p = 0.604) or serum zinc (p = 0.506) among Ethiopian preschool children. Intervention studies are needed to determine whether there are causal links between soil and human zinc status.

Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct.

The large-scale identification of putative alternative promoters study shows more than 52% of human genes are regulated by alternative promoters. The human myc-associated zinc finger protein (SAF/MAZ) gene have SAF-1 and SAF-3 variants transcripted from two transcription start sites (TSSs). By using SAF/MAZ promoter as a model, we set up an approach to probe how the alternative promoters are regulated in real time. We have constructed the bichromatic fluorescent reporter driven by SAF/MAZ 5′-proximal promoter plasmids from which transactivation status of SAF-1 and SAF-3 alternative promoter could be monitored by EGFP and DsRed expression respectively. The results showed that the SAF-3 expression is regulated by alternative promoters. When the bichromatic fluorescent reporter was driven by −1692/+277 or −1401/+277 SAF/MAZ promoter the dominant expression of SAF-3 would be observed in comparison with SAF-1 expression. We also identified that Elk-1 is an inhibitory transcription factor for SAF-3 expression. The temporal diversity of SAF-1 and SAF-3 expressions can be observed via bichromatic fluorescent reporters. These imply that the bichromatic fluorescent reporter driven by alternative promoter construct might be a useful tool for decoding the temporal regulatory repertoire of alternative promoter in human genes.

Selective Inhibition of HIF1α Expression by ZnSO4 Has Antitumoral Effects in Human Melanoma.

Zinc as an essential trace metal is a ubiquitous component of various molecules of the cell. Studies indicated that it may modulate functions of various cancer cell types, and can even inhibit metastasis formation in experimental models. In melanoma, zinc was shown to affect melanin production and to induce apoptosis. Using human melanoma cell lines, we have tested the effects of ZnSO4 on cell proliferation, survival, migration as well as in vivo on experimental liver colony formation. We have found that ZnSO4 has antiproliferative and proapoptotic effects in vitro. In SCID mice intraperitoneal administration of ZnSO4 specifically inhibited liver colony formation without affecting primary tumor growth. To reveal the molecular mechanisms of action of zinc in human melanoma, we have tested mRNA expression of zinc finger transcription factors and found a strong inhibitory effect on HIF1α, as compared to WT1 whereas HIF2α and MTF1 expression was unaffected. Immunohistochemical detection of HIF1α protein in liver metastases confirmed its decreased nuclear expression after in vivo ZnSO4 treatment. These data indicate that in human melanoma zinc administration may have an antimetastatic effect due to a selective downregulation of HIF1α.

Zinc application enhances grain zinc density in genetically-zinc-biofortified wheat grown on a low-zinc calcareous soil

Human zinc (Zn) deficiency is a worldwide problem, especially in developing countries due to the prevalence of cereals in the diet. Among different alleviation strategies, genetic Zn biofortification is considered a sustainable approach. However, it may depend on Zn availability from soils. We grew Zincol-16 (genetically-Zn-biofortified wheat) and Faisalabad-08 (widely grown standard wheat) in pots with (8 mg kg−1) or without Zn application. The cultivars were grown in a low-Zn calcareous soil. The grain yield of both cultivars was significantly (P≤0.05) increased with that without Zn application. As compared to Faisalabad-08, Zincol-16 had 23 and 41% more grain Zn concentration respectively at control and applied rate of Zn. Faisalabad-08 accumulated about 18% more grain Zn concentration with Zn than Zincol-16 without Zn application. A near target level of grain Zn concentration (36 mg kg−1) was achieved in Zincol-16 only with Zn fertilisation. Over all, the findings clearly signify the importance of agronomic Zn biofortification of genetically Zn-biofortified wheat grown on a low-Zn calcareous soil.

Demonstrating aspects of multiscale modeling by studying the permeation pathway of the human ZnT2 zinc transporter.

Multiscale modeling provides a very powerful means of studying complex biological systems. An important component of this strategy involves coarse-grained (CG) simplifications of regions of the system, which allow effective exploration of complex systems. Here we studied aspects of CG modeling of the human zinc transporter ZnT2. Zinc is an essential trace element with 10% of the proteins in the human proteome capable of zinc binding. Thus, zinc deficiency or impairment of zinc homeostasis disrupt key cellular functions. Mammalian zinc transport proceeds via two transporter families: ZnT and ZIP; however, little is known about the zinc permeation pathway through these transporters. As a step towards this end, we herein undertook comprehensive computational analyses employing multiscale techniques, focusing on the human zinc transporter ZnT2 and its bacterial homologue, YiiP. Energy calculations revealed a favorable pathway for zinc translocation via alternating access. We then identified key residues presumably involved in the passage of zinc ions through ZnT2 and YiiP, and functionally validated their role in zinc transport using site-directed mutagenesis of ZnT2 residues. Finally, we use a CG Monte Carlo simulation approach to sample the transition between the inward-facing and the outward-facing states. We present our structural models of the inward- and outward-facing conformations of ZnT2 as a blueprint prototype of the transporter conformations, including the putative permeation pathway and participating residues. The insights gained from this study may facilitate the delineation of the pathways of other zinc transporters, laying the foundations for the molecular basis underlying ion permeation. This may possibly facilitate the development of therapeutic interventions in pathological states associated with zinc deficiency and other disorders based on loss-of-function mutations in solute carriers.

Defining the human C2H2 zinc finger degrome targeted by thalidomide analogs through CRBN.

The small molecules thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of the transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) by recruiting a Cys2-His2 (C2H2) zinc finger domain to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase. We screened the human C2H2 zinc finger proteome for degradation in the presence of thalidomide analogs, identifying 11 zinc finger degrons. Structural and functional characterization of the C2H2 zinc finger degrons demonstrates how diverse zinc finger domains bind the permissive drug-CRBN interface. Computational zinc finger docking and biochemical analysis predict that more than 150 zinc fingers bind the drug-CRBN complex in vitro, and we show that selective zinc finger degradation can be achieved through compound modifications. Our results provide a rationale for therapeutically targeting transcription factors that were previously considered undruggable.

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

Solute carrier family 30 member 8 (SLC30A8), 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 SLC30A8 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.