ZIP Transporters Research Articles

ZIP7 contributes to the pathogenesis of diabetic cardiomyopathy by suppressing mitophagy in mouse hearts

BackgroundAlthough the exact role of mitophagy in the pathogenesis of diabetic cardiomyopathy (DCM) caused by type 2 diabetes mellitus (T2DM) remains controversial, recent studies revealed inhibition of mitophagy exacerbates cardiac injury in DCM. The zinc transporter ZIP7 has been reported to be upregulated by high glucose in cardiomyocytes and ZIP7 upregulation leads to inhibition of mitophagy in mouse hearts in the setting of ischemia/reperfusion. Nevertheless, little is known about the role of ZIP7 and its relationship with mitophagy in DCM caused by T2DM.MethodsT2DM was induced with high-fat diet (HFD) and streptozotocin. The cardiac-specific ZIP7 conditional knockout (ZIP7 cKO) mice were generated by adopting CRISPR/Cas9 system. Cardiac function was evaluated with echocardiography. Mitophagy was assessed by detecting mito-LC3II, mitoKeima, and mitoQC. Reactive oxygen species (ROS) were detected with DHE and mitoB.ResultsZIP7 was upregulated by T2DM in mouse hearts and ZIP7 cKO reduced mitochondrial ROS generation in mouse hearts with T2DM. Mitophagy was suppressed by T2DM in mouse hearts, which was prevented by ZIP7 cKO. T2DM inhibited PINK1 and Parkin accumulation in cardiac mitochondria, an effect that was prevented by ZIP7 cKO, pointing to that ZIP7 upregulation mediates T2DM-induced suppression of mitophagy by inhibiting the PINK1/Parkin pathway. T2DM induced mitochondrial hyperpolarization and decrease of mitochondrial Zn2+ and this was blocked by ZIP7 cKO, indicating that upregulation of ZIP7 leads to mitochondrial hyperpolarization by reducing Zn2+ within mitochondria. Finally, ZIP7 cKO prevented cardiac dysfunction and fibrosis caused by T2DM.ConclusionsZIP7 upregulation mediates the inhibition of mitophagy by T2DM in mouse hearts by suppressing the PINK1/Parkin pathway. Reduction of mitochondrial Zn2+ due to upregulation of ZIP7 accounts for the inhibition of the PINK1/Parkin pathway. Prevention of ZIP7 upregulation is essential for the treatment of T2DM-induced cardiomyopathy.

Expression of Manganese Transporters ZIP8, ZIP14, and ZnT10 in Brain Barrier Tissues.

Manganese (Mn) is an essential trace mineral for brain function, but excessive accumulation can cause irreversible nervous system damage, highlighting the need for proper Mn balance. ZIP14, ZnT10, and ZIP8 are key transporters involved in maintaining Mn homeostasis, particularly in the absorption and excretion of Mn in the intestine and liver. However, their roles in the brain are less understood. The blood-cerebrospinal fluid barrier and the blood-brain barrier, formed by the choroid plexus and brain blood vessels, respectively, are critical for brain protection and brain metal homeostasis. This study identified ZIP14 on the choroid plexus epithelium, and ZIP8 and ZnT10 in brain microvascular tissue. We show that despite significant Mn accumulation in the CSF of Znt10 knockout mice, ZIP14 expression levels in the blood-cerebrospinal fluid barrier remain unchanged, indicating that ZIP14 does not have a compensatory mechanism for regulating Mn uptake in the brain in vivo. Additionally, Mn still enters the CSF without ZIP14 when systemic levels rise. This indicates that alternative transport mechanisms or compensatory pathways ensure Mn balance in the CSF, shedding light on potential strategies for managing Mn-related disorders.

356 Exploring the manganese requirement of feedlot steers

Abstract Angus-cross steers [n = 144; body weight (BW) = 359 kg ± 13.4] were used to assess the effect of dietary Mn and steroidal implants on trace mineral (TM) status, and liver and serum metabolites related to N metabolism. Steers (n = 6/pen) were stratified by BW in a 3 × 2 factorial. GrowSafe bunks recorded individual feed intake (experimental unit = steer; n = 24/treatment). Dietary treatments included (MANG; 8 pens per treatment; Mn as MnSO4): 1) no supplemental Mn (analyzed 14 mg Mn/kg DM; Mn0); 2) 20 mg supplemental Mn/kg DM (Mn20); 3) 50 mg supplemental Mn/kg DM (Mn50). Within MANG, steers received a steroidal implant treatment (IMP) on d 0: 1) no implant; NO; or 2) combination implant (Revalor-200; REV). Liver biopsies for TM analysis and qPCR, and blood for serum urea-N (SUN) analysis were collected on d 0, 20, 40, and 77. Liver TM, serum metabolite, enzyme activity, and gene expression data were analyzed as repeated measures using PROC MIXED of SAS 9.4. The Correlation Procedure of SAS 9.4 was utilized to assess correlations between liver arginase activity, SUN concentrations, and liver Mn concentrations. No MANG × IMP effects were noted (P ≥ 0.12) for growth or carcass measures. Steroidal implants increased final BW (FBW), overall average daily gain (ADG), and gain to feed (G:F; P ≤ 0.01). Dietary Mn did not influence FBW, overall ADG, or overall G:F (P ≥ 0.14). Liver Mn concentration increased with supplemental Mn (P = 0.01). An IMP × DAY effect was noted for liver Mn (P = 0.01) where NO and REV were similar on d 0 but NO cattle increased liver Mn from d 0 to 20 while REV liver Mn decreased. Relative expression of liver Mn transporter ZnT10 tended to be least (MANG; P = 0.10) in Mn20 while Mn0 and Mn100 were similar. Relative expression of liver Mn transporter ZIP8 was not influenced by MANG, IMP, or MANG × IMP (P ≥ 0.23). Liver arginase activity was not influenced by MANG, IMP, MANG × DAY, IMP × DAY, or MANG × IMP (P ≥ 0.15) but tended to decrease over time (DAY; P = 0.10). On d 0, 20, and 40, arginase activity was correlated (r ≤ 0.33; P ≤ 0.01) with SUN and liver Mn. Relative expression of MnSOD in the liver was greater in REV (P = 0.03) compared with NO and within a MANG × IMP effect (P = 0.01) REV increased liver MnSOD activity. This study indicates current NASEM Mn recommendations are adequate to meet demands of finishing beef cattle given a steroidal implant. This study also reveals how implants and supplemental Mn influence liver enzyme activity and genes related to arginine metabolism, urea synthesis, antioxidant capacity, and TM homeostasis.

Crohn's Disease-Associated Pathogenic Mutation in the Manganese Transporter ZIP8 Shifts the Ileal and Rectal Mucosal Microbiota Implicating Aberrant Bile Acid Metabolism.

A pathogenic mutation in the manganese transporter ZIP8 (A391T; rs13107325) increases the risk of Crohn's disease. ZIP8 regulates manganese homeostasis and given the shared need for metals between the host and resident microbes, there has been significant interest in alterations of the microbiome in carriers of ZIP8 A391T. Prior studies have not examined the ileal microbiome despite associations between ileal disease and ZIP8 A391T. Here, we used the Pediatric Risk Stratification Study (RISK) cohort to perform a secondary analysis of 16S ribosomal RNA gene sequencing data obtained from ileal and rectal mucosa to study associations between ZIP8 A391T carrier status and microbiota composition. We found sequence variants mapping to Veillonella were decreased in the ileal mucosa of ZIP8 A391T carriers. Prior human studies have demonstrated the sensitivity of Veillonella to bile acid abundance. We therefore hypothesized that bile acid homeostasis is differentially regulated in carriers of ZIP8 A391T. Using a mouse model of ZIP8 A391T, we demonstrate an increase in total bile acids in the liver and stool and decreased fibroblast growth factor 15 (Fgf15) signaling, consistent with our hypothesis. We confirmed dysregulation of FGF19 in the 1000IBD cohort, finding that plasma FGF19 levels are lower in ZIP8 A391T carriers with ileocolonic Crohn's disease. In the search for genotype-specific therapeutic paradigms for patients with Crohn's disease, these data suggest targeting the FGF19 pathway in ZIP8 A391T carriers. Aberrant bile acid metabolism may precede development of Crohn's disease and prioritize study of the interactions between manganese homeostasis, bile acid metabolism and signaling, and complicated ileal Crohn's disease.

Aberrant N-glycosylation is a therapeutic target in carriers of a common and highly pleiotropic mutation in the manganese transporter ZIP8.

The treatment of defective glycosylation in clinical practice has been limited to patients with rare and severe phenotypes associated with congenital disorders of glycosylation (CDG). Carried by approximately 5% of the human population, the discovery of the highly pleiotropic, missense mutation in a manganese transporter ZIP8 has exposed under-appreciated roles for Mn homeostasis and aberrant Mn-dependent glycosyltransferases activity leading to defective N-glycosylation in complex human diseases. Here, we test the hypothesis that aberrant N-glycosylation contributes to disease pathogenesis of ZIP8 A391T-associated Crohn's disease. Analysis of N-glycan branching in intestinal biopsies demonstrates perturbation in active Crohn's disease and a genotype-dependent effect characterized by increased truncated N-glycans. A mouse model of ZIP8 391-Thr recapitulates the intestinal glycophenotype of patients carrying mutations in ZIP8. Borrowing from therapeutic strategies employed in the treatment of patients with CDGs, oral monosaccharide therapy with N-acetylglucosamine ameliorates the epithelial N-glycan defect, bile acid dyshomeostasis, intestinal permeability, and susceptibility to chemical-induced colitis in a mouse model of ZIP8 391-Thr. Together, these data support ZIP8 391-Thr alters N-glycosylation to contribute to disease pathogenesis, challenging the clinical paradigm that CDGs are limited to patients with rare diseases. Critically, the defect in glycosylation can be targeted with monosaccharide supplementation, providing an opportunity for genotype-driven, personalized medicine.

Unlocking the brain’s zinc code: implications for cognitive function and disease

Zn2+ transport across neuronal membranes relies on two classes of transition metal transporters: the ZnT (SLC30) and ZIP (SLC39) families. These proteins function to decrease and increase cytosolic Zn2+ levels, respectively. Dysfunction of ZnT and ZIP transporters can alter intracellular Zn2+ levels resulting in deleterious effects. In neurons, imbalances in Zn2+ levels have been implicated as risk factors in conditions such as Alzheimer’s disease and neurodegeneration, highlighting the pivotal role of Zn2+ homeostasis in neuropathologies. In addition, Zn2+ modulates the function of plasma membrane proteins, including ion channels and receptors. Changes in Zn2+ levels, on both sides of the plasma membrane, profoundly impact signaling pathways governing cell development, differentiation, and survival. This review is focused on recent developments of neuronal Zn2+ homeostasis, including the impact of Zn2+ dyshomeostasis in neurological disorders, therapeutic approaches, and the increasingly recognized role of Zn2+ as a neurotransmitter in the brain.

Manganese-dependent transcription regulation by MntR and PerR in Thermus thermophilus HB8.

Bacteria contain conserved mechanisms to control the intracellular levels of metal ions. Metalloregulatory transcription factors bind metal cations and play a central role in regulating gene expression of metal transporters. Often, these transcription factors regulate transcription by binding to a specific DNA sequence in the promoter region of target genes. Understanding the preferred DNA-binding sequence for transcriptional regulators can help uncover novel gene targets and provide insight into the biological role of the transcription factor in the host organism. Here, we identify consensus DNA-binding sequences and subsequent transcription regulatory networks for two metalloregulators from the ferric uptake regulator (FUR) and diphtheria toxin repressor (DtxR) superfamilies in Thermus thermophilus HB8. By homology search, we classify the DtxR homolog as a manganese-specific, MntR (TtMntR), and the FUR homolog as a peroxide-sensing, PerR (TtPerR). Both transcription factors repress separate ZIP transporter genes invivo, and TtPerR acts as a bifunctional transcription regulator by activating the expression of ferric and hemin transport systems. We show TtPerR and TtMntR bind DNA in the presence of manganese invitro and invivo; however, TtPerR is unable to bind DNA in the presence of iron, likely due to iron-mediated histidine oxidation. Unlike canonical PerR homologs, TtPerR does not appear to contribute to peroxide detoxification. Instead, the TtPerR regulon and DNA binding sequence are more reminiscent of Fur or Mur homologs. Collectively, these results highlight the similarities and differences between two metalloregulatory superfamilies and underscore the interplay of manganese and iron in transcription factor regulation.

The Zn2+ transporter ZIP7 enhances endoplasmic-reticulum-associated protein degradation and prevents neurodegeneration in Drosophila

Proteotoxic stress drives numerous degenerative diseases. Cells initially adapt to misfolded proteins by activating the unfolded protein response (UPR), including endoplasmic-reticulum-associated protein degradation (ERAD). However, persistent stress triggers apoptosis. Enhancing ERAD is a promising therapeutic approach for protein misfolding diseases. The ER-localized Zn2+ transporter ZIP7 is conserved from plants to humans and required for intestinal self-renewal, Notch signaling, cell motility, and survival. However, a unifying mechanism underlying these diverse phenotypes was unknown. In studying Drosophila border cell migration, we discovered that ZIP7-mediated Zn2+ transport enhances the obligatory deubiquitination of proteins by the Rpn11 Zn2+ metalloproteinase in the proteasome lid. In human cells, ZIP7 and Zn2+ are limiting for deubiquitination. In a Drosophila model of neurodegeneration caused by misfolded rhodopsin (Rh1), ZIP7 overexpression degrades misfolded Rh1 and rescues photoreceptor viability and fly vision. Thus, ZIP7-mediated Zn2+ transport is a previously unknown, rate-limiting step for ERAD invivo with therapeutic potential in protein misfolding diseases.

Possible involvement of zinc transporter ZIP13 in myogenic differentiation

Ehlers–Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13G64D) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function.

Genetic control of MRI contrast using the manganese transporter Zip14.

Gene-expression reporter systems, such as green fluorescent protein, have been instrumental to understanding biological processes in living organisms at organ system, tissue, cell, and molecular scales. More than 30 years of work on developing MRI-visible gene-expression reporter systems has resulted in a variety of clever application-specific methods. However, these techniques have not yet been widely adopted, so a general-purpose expression reporter is still required. Here, we demonstrate that the manganese ion transporter Zip14 is an in vivo MRI-visible, flexible, and robust gene-expression reporter to meet this need. Plasmid constructs consisting of a cell type-specific promoter, gene coding for human Zip14, and a histology-visible tag were packaged into adeno-associated viruses. These viruses were intracranially injected into the mouse brain. Serial in vivo MRI was performed using a vendor-supplied 3D-MPRAGE sequence. No additional contrast agents were administered. Animals were sacrificed after the last imaging timepoint for immunohistological validation. Neuron-specific overexpression of Zip14 produced substantial and long-lasting changes in MRI contrast. Using appropriate viruses enabled both anterograde and retrograde neural tracing. Expression of Zip14 in astrocytes also enabled MRI of glia populations in the living mammalian brain. The flexibility of this system as an MRI-visible gene-expression reporter will enable many applications of serial, high-resolution imaging of gene expression for basic science and therapy development.

ZIP transporters-regulated Zn2+ homeostasis: A novel determinant of human diseases.

As an essential trace element for organisms, zinc participates in various physiological processes, such as RNA transcription, DNA replication, cell proliferation, and cell differentiation. The destruction of zinc homeostasis is associated with various diseases. Zinc homeostasis is controlled by the cooperative action of zinc transporter proteins that are responsible for the influx and efflux of zinc. Zinc transporter proteins are mainly categorized into two families: Zrt/Irt-like protein (SLC39A/ZIP)family and zinc transporter (SLC30A/ZNT)family. ZIP transporters contain 14 members, namely ZIP1-14, which can be further divided into four subfamilies. Currently, ZIP transporters-regulated zinc homeostasis is one of the research hotspots. Cumulative evidence suggests that ZIP transporters-regulated zinc homeostasis may cause physiological dysfunction and contribute to the onset and progression of diverse diseases, such as cancers, neurological diseases, and cardiovascular diseases. In this review, we initially discuss the structure and distribution of ZIP transporters. Furthermore, we comprehensively review the latest research progress of ZIP transporters-regulated zinc homeostasis in diseases, providing a new perspective into new therapeutic targets for treating related diseases.

Palliative Effect of Combined Application of Zinc and Selenium on Reproductive Injury Induced by Tripterygium Glycosides in Male Rats.

The long-term use of tripterygium glycosides (TG) can lead to male reproductive damage. Research indicates that zinc and selenium exhibit a synergistic effect in the male reproductive system, with the combined preparation demonstrating superior therapeutic effects compared to individual preparations. The purpose of this study was to explore the specific mechanism by which zinc and selenium mitigate reproductive toxicity induced by TG in male rats. Rats were randomly assigned to three groups: control group (C group), model group (M group, receiving TG at 30 mg/kg/day), and model + zinc + selenium group (ZS group). The ZS group was also given TG gavage for the first 4 weeks. Starting from the fifth week until the conclusion of the eighth week, the ZS group received an additional protective treatment of 10 mg/kg/day Zn and 0.1 mg/kg/day Se 4 h after TG administration. Following euthanasia, blood samples, rat testis, and epididymis tissues were collected for further experiments. Combined zinc-selenium treatment corrects the imbalance of zinc-selenium homeostasis in testicular tissue induced by TG. This is achieved by upregulating the expression of metal transcription factor (MTF1) and zinc transporters ZIP8 and ZIP14 and downregulating the expression of ZnT10. Improvement of zinc and selenium homeostasis enhanced the expression of zinc-containing enzymes (ADH, LDH, and ALP) and selenoproteins (GPx1 and SELENOP) in the testis. At the same time, zinc and selenium mitigate TG-induced reproductive damage by promoting the activity of antioxidant enzymes and upregulating the expression of proteins associated with the oxidative stress pathway, including Nrf2, Keap1, HO-1, PI3K, and p-AKT.

Protein S-palmitoylation enhances profibrotic signaling in response to cadmium

Cadmium (Cd) is a naturally occurring, toxic environmental metal found in foods. Humans do not have an efficient mechanism for Cd elimination; thus, Cd burden in humans increases with age. Cell and mouse studies show that Cd burden from low environmental levels of exposure impacts lung cell metabolism, proliferation signaling and cell growth as part of disease-promoting profibrotic responses in the lungs. Prior integrative analysis of metabolomics and transcriptomics identified the zDHHC11 transcript as a central functional hub in response to Cd dose. zDHHC11 encodes a protein S-palmitoyltransferase, but no evidence is available for effects of Cd on protein S-palmitoylation. In the present research, we studied palmitoylation changes in response to Cd and found increased protein S-palmitoylation in human lung fibroblasts that was inhibited by 2-bromopalmitate (2-BP), an irreversible palmitoyltransferase inhibitor. Mass spectrometry-based proteomics showed palmitoylation of proteins involved in divalent metal transport and in fibrotic signaling. Mechanistic studies showed that 2-BP inhibited palmitoylation of divalent metal ion transporter ZIP14 and also inhibited cellular Cd uptake. Transcription analyses showed that Cd stimulated transforming growth factor (TGF)-β1 and β3 expression within 8 h and lung fibrotic markers α-smooth muscle actin, matrix metalloproteinase-2, and collagen 1α1 gene expression and that these effects were blocked by 2-BP. Because 2-BP also blocked palmitoylation of proteins controlled by TGFβ1, these results show that palmitoylation impacts Cd-dependent fibrotic signaling both by enhancing cellular Cd accumulation and by supporting post-translational processing of TGFβ1-dependent proteins.

Supplementing two wheat genotypes with ZnSO4 and ZnO nanoparticles showed differential mitigation of Cd phytotoxicity by reducing Cd absorption, preserving root cellular ultrastructure, and regulating metal-transporter gene expression

Cadmium (Cd) contamination is a serious challenge in agricultural soils worldwide, resulting in Cd entering the food chain mainly through plant-based food and threatening human health. Minimizing Cd bioaccumulation in wheat is an important way to prevent Cd hazards to humans. Hydroponic and pot experiments were conducted to comprehensively evaluate the effects of zinc sulfate (ZnSO4) and zinc oxide nanoparticles (nZnO) on Cd uptake, translocation, subcellular distribution, cellular ultrastructure, and gene expression in two wheat genotypes that differ in grain Zn accumulation. Results showed that high-dose nZnO significantly reduced root Cd concentration (52.44%∼56.85%) in two wheats, in contrast to ZnSO4. The S216 exhibited higher tolerance to Cd compared to Z797. Importantly, Zn supplementation enhanced Cd sequestration into vacuoles and binding to cell walls, which conferred stability to ultracellular structures and photosynthetic apparatus. Down-regulation of influx transporter (TaHMA2 and TaLCT1) and up-regulation of efflux transporters (TaTM20 and TaHMA3) in Z797 might contribute to Zn-dependent alleviation of Cd toxicity and enhance its Cd tolerance. Down-regulation of ZIP transporters (TaZIP3, -5, and -7) might contribute to an increase in root Zn concentration and inhibit Cd absorption. Additionally, soil Zn provided an effective strategy for the reduction of grain Cd concentrations in both wheats, with a reduction of 26%∼32% (high ZnSO4) and 11%∼67% (high nZnO), respectively. Collectively, these findings provide new insights and perspectives on the mechanisms of Cd mitigation in wheats with different Zn fertilizers and demonstrate that the effect of nZnO in mitigating Cd stress is greater than that of ZnSO4 fertilizers.

Abstract 14145: The Zinc Transporter ZIP7 Plays a Critical Role in Ferrotposis Induced by Ischemia/Reperfusion in Mouse Hearts

Background: Although ferroptosis has been proposed to play a role in myocardial ischemia/reperfusion (I/R) injury, the exact molecular mechanism by which ferroptosis is induced by I/R remains unclear. Hypothesis: We tested the hypothesis that upregulation of the zinc transporter ZIP7 at reperfusion triggers ferroptosis by downregulating the mitochondrial GPX4 (mtGPX4) via the guanine-rich sequence-binding factor 1 (GRSF1) and increasing mitochondrial iron accumulation through inhibition of the mitochondrial iron importer degradation. Aims: To determine if ZIP7 plays a role in I/R-induced ferroptosis and to investigate the underlying molecular mechanism. Methods: Mouse hearts were subjected to I/R in vivo . The cardiac-specific Slc39a7 conditional knockout mice (ZIP7 cKO) were generated by adopting the CRISPR/Cas9 system. Adult mouse cardiomyocytes were isolated enzymatically. Ferroptosis was evaluated by detecting PTGS2, MDA, and BODIPY C11. Mitochondrial Fe 2+ was determined with Mito-FerroGreen fluorescent probe. Results: I/R upregulated ZIP7 expression and induced ferroptosis as indicated by the increases in PTGS2 mRNA expression (290.90 ± 52.78 % of control), MDA (152.10 ± 15.07 % of control) and BODIPY C11 (130.68 ± 5.52 % of control) levels, which was prevented by ZIP7 cKO. ZIP7 cKO also mitigated doxorubicin-induced ferroptosis. ZIP7 cKO upregulated mtGPX4 expression (123.40±3.26 % of I/R) and increased mitochondrial GRSF1 expression (150.90±8.51 % of I/R) at reperfusion. In addition, knockout of GRSF1 inhibited GPX4 expression (54.73±5.44 % of control). Moreover, ZIP7 cKO prevented the accumulation of Fe 2+ within mitochondria (44.15±2.46 % of I/R) and promoted the degradation of SLC25A37 (62.57±8.03% of I/R) and SLC25A28 (69.82±3.10 % of I/R) at reperfusion. Conclusions: Upregulation of ZIP7 contributes to the induction of ferroptosis at reperfusion presumably by reducing mtGPX4 expression via GRSF1and increasing Fe 2+ accumulation within mitochondria through inhibition of SLC25A37 and SLC25A28 degradation.

Rational engineering of an elevator-type metal transporter ZIP8 reveals a conditional selectivity filter critically involved in determining substrate specificity

Engineering of transporters to alter substrate specificity as desired holds great potential for applications, including metabolic engineering. However, the lack of knowledge on molecular mechanisms of substrate specificity hinders designing effective strategies for transporter engineering. Here, we applied an integrated approach to rationally alter the substrate preference of ZIP8, a Zrt-/Irt-like protein (ZIP) metal transporter with multiple natural substrates, and uncovered the determinants of substrate specificity. By systematically replacing the differentially conserved residues with the counterparts in the zinc transporter ZIP4, we created a zinc-preferring quadruple variant (Q180H/E343H/C310A/N357H), which exhibited largely reduced transport activities towards Cd2+, Fe2+, and Mn2+ whereas increased activity toward Zn2+. Combined mutagenesis, modeling, covariance analysis, and computational studies revealed a conditional selectivity filter which functions only when the transporter adopts the outward-facing conformation. The demonstrated approach for transporter engineering and the gained knowledge about substrate specificity will facilitate engineering and mechanistic studies of other transporters.

Structural mechanism of intracellular autoregulation of zinc uptake in ZIP transporters

Zinc is an essential micronutrient that supports all living organisms through regulating numerous biological processes. However, the mechanism of uptake regulation by intracellular Zn2+ status remains unclear. Here we report a cryo-electron microscopy structure of a ZIP-family transporter from Bordetella bronchiseptica at 3.05 Å resolution in an inward-facing, inhibited conformation. The transporter forms a homodimer, each protomer containing nine transmembrane helices and three metal ions. Two metal ions form a binuclear pore structure, and the third ion is located at an egress site facing the cytoplasm. The egress site is covered by a loop, and two histidine residues on the loop interact with the egress-site ion and regulate its release. Cell-based Zn2+ uptake and cell growth viability assays reveal a negative regulation of Zn2+ uptake through sensing intracellular Zn2+ status using a built-in sensor. These structural and biochemical analyses provide mechanistic insight into the autoregulation of zinc uptake across membranes.

Study on the involvement of Zinc in homeostasis under intestinal Inflammation

Background & Aim: Zinc is one of the essential trace elements, and adequate amounts of zinc are crucial for good health. It is thought that zinc is absorbed primarily in the small intestine and also plays an important role in maintaining intestinal homeostasis. Small intestine is a key organ involved in digestion/absorption and is also sensitive to intestinal bacteria and easily inflamed. Alkaline phosphatase (ALP), which has zinc as its active center, detoxifies lipopolysaccharide and is thought to be involved in the suppression of small intestinal inflammation. In this study, we investigated the role of zinc in small intestinal inflammation using a mouse model, as well as the transporters ZIP4 and ZIP14, which are involved in ALP and zinc absorption. Materials & Methods: Male ICR mice were used. Indomethacin (IM, 12 mg/kg s.c.) was administered to mice to induce small intestinal inflammation. Four days prior to IM administration, zinc sulfate heptahydrate (Zn) was administered twice daily for 6 days. Forty-eight hours after IM administration, the animals were sacrificed and the lesion area was measured. Hematoxylin and eosin staining was performed. Myeloperoxidase (MPO) activity in the ileum was measured as an indicator of the degree of inflammation. ZIP4 and ZIP14 mRNA expression levels were examined by RT-PCR. ALP activity of the ileal was examined. IEC-6 cells, a rat small intestinal epithelial cell line, were seeded in 96-well plates, and Zn was applied 24 hours after seeding, and IM was added 24 hours after seeding. Cell viability was measured 24 hours after the addition of IM. Results: After administration of IM, bloody stools were observed from 24 hours after administration. At 48 hours after IM administration, disruption of villi structure and shortening of small intestine length were observed in the jejunum and ileum, and MPO activity was significantly increased. ZIP4 and ZIP14 mRNA expression levels were decreased in the small intestine 48 hours after IM treatment, and ALP activity was increased compared to the Vehicle group. On the other hand, pretreatment with Zn suppressed the occurrence of small intestinal inflammation, and the disruption of small intestinal villi structure was weak. The increase in MPO activity induced by IM tended to be suppressed by Zn pre-administration. ZIP4 and ZIP14 mRNA expression in Zn pre-administration group was same as in the IM alone group, and ALP activity was higher than in the IM alone group. IM treatment of IEC-6 also decreased cell viability in a concentration-dependent manner, which was inhibited by pretreatment with Zn in a concentration-dependent manner. Conclusion: These results suggest that zinc is effective in preventing the occurrence of NSAID-induced small intestinal inflammation. ZIP4 and ZIP14 are normally highly expressed in the intestinal epithelium and contribute greatly to zinc absorption, but the amount of zinc absorption may be decreased during inflammation induc University This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

ZIP8 impacts protease activity in macrophages in response to Streptococcus pneumoniae

Abstract Zinc (Zn) is essential for immune function and Zn deficiency increases the incidence of community acquired pneumonia. The Zn transporter ZIP8 is required by myeloid cells to defend against pathogens. Previously, we reported that S. pneumoniae infected ZIP8 knockout mice (Zip8KO) have increased bacterial burden and mortality and that Zip8KO bone marrow derived macrophages (BMDMs) have reduced phagocytic activity. Others have shown that Zn induces cathepsin expression via the TFEB pathway. However, it is unknown whether ZIP8 impacts TFEB-dependent phagolysosomal processing in macrophages. Based on this, we hypothesized that ZIP8 facilitates phagolysosomal processing and eradication of bacteria via TFEB. BMDMs were derived from wild type and Zip8KO mice and then exposed to S. pneumoniae(MOI=10) for 1 hour. At 6–18 hours post challenge we assessed cathepsin B and L mRNA and protein levels as well as total protease, cathepsin B and L activity. In conjunction, we measured p-TFEB and total TFEB proteins levels. Active TFEB and cathepsin B protein levels were significantly lower in Zip8KO BMDMs with no difference in cathepsin L. Strikingly, Zip8KO BMDMs had significantly reduced total protease, cathepsin B and cathepsin L activity which also corresponded with decreased mRNA levels. Based on these findings we predict that ZIP8 loss in macrophages impairs phagolysosomal processing and bacterial eradication in a TFEB-dependent manner. Overall, this warrants future studies given the high incidence of Zn deficiency and the rs13107325 defective ZIP8 allele in humans. National Heart, Lung and Blood Institute #1R01HL156952-01-A1 (DLK)

Loss of hepatic manganese transporter ZIP8 disrupts serum transferrin glycosylation and the glutamate-glutamine cycle

BackgroundZIP8, encoded by SLC39A8, is a membrane transporter that facilitates the cellular uptake of divalent biometals including zinc (Zn), manganese (Mn), and iron (Fe). The hepatic system has long been accepted as the central modulator for whole-body biometal distribution. Earlier investigations suggest the propensity of ZIP8 to prioritize Mn influx, as opposed to Fe or Zn, in hepatocytes. Hepatic ZIP8 Mn transport is crucial for maintaining homeostasis of various Mn-dependent metalloenzymes and their associated pathways. Herein, we hypothesize that a drastic decrease in systemic Mn, via the loss of hepatic ZIP8, disrupts two unique cellular pathways, post-translational glycosylation and the glutamate-glutamine cycle. MethodsZIP8 liver-specific knockout (LSKO) mice were chosen in an attempt to substantially decrease whole-body Mn levels. To further elucidate the role of Mn in serum glycosylation, a Mn-deficient diet was adopted in conjunction with the LSKO mice to model a near-complete loss of systemic Mn. After the treatment course, transferrin sialylation profiles were determined using imaged capillary isoelectric focusing (icIEF). We also investigated the role of Mn in the glutamate-glutamine cycle; the conversion of glutamate to glutamine in F/F and LSKO mice was assessed by the glutamine/glutamate ratio in cerebrospinal fluid (CSF) via HPLC-MS. An open-field study was ultimately conducted to check if these mice displayed atypical behavior. ResultsTwo major biological pathways were found to be significantly altered due to the loss of hepatic ZIP8. We identified a disparity between F/F and LSKO transferrin sialylation profiles that were exacerbated under a Mn-deficient diet. Additionally, we discovered a neurotransmitter imbalance between the levels of glutamine and glutamate, exclusive to LSKO mice. This was characterized by the decreased glutamine/glutamate ratio in CSF. Secondary to the neurotransmitter alteration, LSKO mice exhibited an increase in locomotor activity in an open-field. ConclusionOur model successfully established a connection between the loss of hepatic ZIP8 and two Mn-dependent cellular pathways, namely, protein glycosylation and the glutamate-glutamine cycle.