Programmed Ribosomal Frameshifting Generates a Copper Transporter and a Copper Chaperone from the Same Gene.

Metal efflux pumps maintain ion homeostasis in the cell. The functions of the transporters are often supported by chaperone proteins, which scavenge the metal ions from the cytoplasm. Although the copper ion transporter CopA has been known in Escherichia coli, no gene for its chaperone had been identified. We show that the CopA chaperone is expressed in E. coli from the same gene that encodes the transporter. Some ribosomes translating copA undergo programmed frameshifting, terminate translation in the -1 frame, and generate the 70 aa-long polypeptide CopA(Z), which helps cells survive toxic copper concentrations. The high efficiency of frameshifting is achieved by the combined stimulatory action of a "slippery" sequence, an mRNA pseudoknot, and the CopA nascent chain. Similar mRNA elements are not only found in the copA genes of other bacteria but are also present in ATP7B, the human homolog of copA, and direct ribosomal frameshifting in vivo.

The magnesium transporter NIPAL1 is a pancreatic islet-expressed protein that conditionally impacts insulin secretion.

Type 2 diabetes is a chronic metabolic disease characterized by pancreatic ß-cell dysfunction and peripheral insulin resistance. Among individuals with type 2 diabetes, ∼30% exhibit hypomagnesemia. Hypomagnesemia has been linked to insulin resistance through reduced tyrosine kinase activity of the insulin receptor; however, its impact on pancreatic ß-cell function is unknown. In this study, through analysis of several single-cell RNA-sequencing data sets in tandem with quantitative PCR validation in both murine and human islets, we identified NIPAL1 (NIPA-like domain containing 1), encoding a magnesium influx transporter, as an islet-enriched gene. A series of immunofluorescence experiments confirmed NIPAL1's magnesium-dependent expression and that it specifically localizes to the Golgi in Min6-K8 cells, a pancreatic ß-cell-like cell line (mouse insulinoma 6 clone K8). Under varying magnesium concentrations, NIPAL1 knockdown decreased both basal insulin secretion and total insulin content; in contrast, its overexpression increased total insulin content. Although the expression, distribution, and magnesium responsiveness of NIPAL1 in α-TC6 glucagonoma cells (a pancreatic α-cell line) were similar to the observations in Min6-K8 cells, no effect was observed on glucagon secretion in α-TC6 cells under the conditions studied. Overall, these results suggest that NIPAL1 expression is regulated by extracellular magnesium and that down-regulation of this transporter decreases glucose-stimulated insulin secretion and intracellular insulin content, particularly under conditions of hypomagnesemia.

Functional overlap of two major facilitator superfamily transporter, ZIF1, and ZIFL1 in zinc and iron homeostasis.

Zinc and iron are essential micronutrients for plant growth, and their homeostasis must be tightly regulated. Previously, it has been shown that Zinc-Induced Facilitator 1 (ZIF1) is involved in basal Zn tolerance by controlling the vacuolar storage of nicotianamine (NA). However, knowledge of the functional roles of two ZIF1 paralogs, ZIF-LIKE1 (ZIFL1) and ZIFL2, in metal homeostasis remains limited. Here, we functionally characterized the roles of ZIF1, ZIFL1, and ZIFL2 in Zn and Fe homeostasis. Expression of ZIF1 and ZIFL1 was induced by both excess Zn and Fe-deficiency, and their loss-of-function led to hypersensitivity under excess Zn and Fe-deficiency, suggesting functional overlap between ZIF1 and ZIFL1. By contrast, the disruption of ZIFL2 resulted in no obvious phenotypic alteration under both conditions. Additionally, the expression of ZIFL1, but not that of ZIFL2, in the zif1 mutant partially restored the phenotype under excess Zn, suggesting that ZIF1 and ZIFL1 perform functionally redundant roles in Zn homeostasis.

Marginally reduced maternal hepatic and splenic ferroportin under severe nutritional iron deficiency in pregnancy maintains systemic iron supply.

The demand for iron is high in pregnancy to meet the increased requirements for erythropoiesis. Even pregnant females with initially iron-replete stores develop iron-deficiency anemia, due to inadequate iron absorption. In anemic females, the maternal iron supply is dedicated to maintaining iron metabolism in the fetus and placenta. Here, using a mouse model of iron deficiency in pregnancy, we show that iron recycled from senescent erythrocytes becomes a predominant source of this microelement that can be transferred to the placenta in females with depleted iron stores. Ferroportin is a key protein in the molecular machinery of cellular iron egress. We demonstrate that under iron deficiency in pregnancy, levels of ferroportin are greatly reduced in the duodenum, placenta and fetal liver, but not in maternal liver macrophages and in the spleen. Although low expression of both maternal and fetal hepcidin predicted ferroportin up-regulation in examined locations, its final expression level was very likely correlated with tissue iron status. Our results argue that iron released into the circulation of anemic females is taken up by the placenta, as evidenced by high expression of iron importers on syncytiotrophoblasts. Then, a substantial decrease in levels of ferroportin on the basolateral side of syncytiotrophoblasts, may be responsible for the reduced transfer of iron to the fetus. As attested by the lowest decrease in iron content among analyzed tissues, some part is retained in the placenta. These findings confirm the key role played by ferroportin in tuning iron turnover in iron-deficient pregnant mouse females and their fetuses.

Coaction of hepatic thioredoxin and glutathione systems in iron overload-induced oxidative stress.

In the present study, we demonstrate the coaction of thioredoxin and glutathione (GSH) systems in mouse liver against iron overload-induced oxidative stress (OS). Mice were injected intraperitoneally with an iron dextran solution twice a week for 3 weeks. Iron accumulation in mouse liver was demonstrated spectroscopically. To confirm the iron overload model in the liver, the increased gene expression levels of hepcidin (Hamp), ferroportin (Fpn1), and ferritin (Fth1), which regulate iron trafficking, were observed by a quantitative polymerase chain reaction. In the case of iron overload, the GSH level and the reduced glutathione/oxidized glutathione ratio, which represents a marker of OS, decreased significantly. An increase in the malondialdehyde level, one of the final products of the lipid peroxidation process, was observed. The gene expression of the thioredoxin system, including thioredoxin (Trx1) and thioredoxin reductase (TrxR1), was examined. Though TrxR1 expression decreased, no changes were observed in Trx1. The enzyme activity and semiquantitative protein expression of TRXR1 increased. The activity of GSH reductase and GSH peroxidase increased in the iron overload group. The gene and protein expressions of thioredoxininteracting protein, which is an indicator of the commitment of the cell to apoptosis, were elevated significantly. The increased protein expression of Bcl-2-related X protein and CASPASE-3, which is an indicator of apoptosis, increased significantly. In conclusion, excess iron accumulation in mouse liver tissue causes OS, which affects the redox state of the thioredoxin and GSH systems, inducing cell apoptosis and also ferroptosis due to increased lipid peroxidation and the depletion of GSH level.

Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.

Changes in bone matrix composition are frequently found with bone diseases and may be associated with increased fracture risk. Bone is rich in the trace element zinc. Zinc was established to play a significant role in the growth, development, and maintenance of healthy bones; however, the mechanisms underlying zinc effects on the integrity of the skeleton are poorly understood. Here, we show that the zinc receptor (ZnR)/Gpr39 is required for normal bone matrix deposition by osteoblasts. Initial analysis showed that Gpr39-deficient ( Gpr39-/-) mice had weaker bones as a result of altered bone composition. Fourier transform infrared spectroscopy analysis showed high mineral-to-matrix ratios in the bones of Gpr39-/- mice. Histologic analysis showed abnormally high numbers of active osteoblasts but normal osteoclast numbers on the surfaces of bones from Gpr39-/- mice. Furthermore, Gpr39-/- osteoblasts had disorganized matrix deposition in vitro with cultures exhibiting abnormally low collagen and high mineral contents, findings that demonstrate a cell-intrinsic role for ZnR/Gpr39 in these cells. We show that both collagen synthesis and deposition by Gpr39-/- osteoblasts are perturbed. Finally, the expression of the zinc transporter Zip13 and a disintegrin and metalloproteinase with thrombospondin motifs family of zinc-dependent metalloproteases that regulate collagen processing was downregulated in Gpr39-/- osteoblasts. Altogether, our results suggest that zinc sensing by ZnR/Gpr39 affects the expression levels of zinc-dependent enzymes in osteoblasts and regulates collagen processing and deposition.-Jovanovic, M., Schmidt, F. N., Guterman-Ram, G., Khayyeri, H., Hiram-Bab, S., Orenbuch, A., Katchkovsky, S., Aflalo, A., Isaksson, H., Busse, B., Jähn, K., Levaot, N. Perturbed bone composition and integrity with disorganized osteoblast function in zinc receptor/Gpr39-deficient mice.

Leishmania donovani inhibits ferroportin translation by modulating FBXL5-IRP2 axis for its growth within host macrophages.

Hepcidin mediated ferroportin (Fpn) degradation in macrophages is a well adopted strategy to limit iron availability towards invading pathogens. Leishmania donovani (LD), a protozoan parasite, resides within macrophage and competes with host for availing iron. Using in vitro and in vivo model of infection, we reveal that LD decreases Fpn abundance in host macrophages by hepcidin independent mechanism. Unaffected level of Fpn-FLAG in LD infected J774 macrophage confirms that Fpn down-regulation is not due its degradation. While increased Fpn mRNA but decreased protein expression in macrophages suggests blocking of Fpn translation by LD infection that is confirmed by 35 S-methionine labelling assay. We further reveal that LD blocks Fpn translation by induced binding of iron regulatory proteins (IRPs) to the iron responsive element present in its 5'UTR. Supershift analysis provides evidence of involvement of IRP2 particularly during in vivo infection. Accordingly, a significant increase in IRP2 protein expression with simultaneous decrease in its stability regulator F-box and leucine-rich repeat Protein 5 (FBXL5) is detected in splenocytes of LD-infected mice. Increased intracellular growth due to compromised expressions of Fpn and FBXL5 by specific siRNAs reveals that LD uses a novel strategy of manipulating IRP2-FBXL5 axis to inhibit host Fpn expression.

MRI reporter gene MagA suppresses transferrin receptor and maps Fe2+ dependent lung cancer.

As a magnetic resonance imaging (MRI) reporter gene, MagA has become a powerful tool to monitor dynamic gene expression and allowed concomitant high resolution anatomical and functional imaging of subcellular genetic information. Here we establish a stably expressed MagA method for lung cancer MRI. The results show that MagA can not only enhance both in vitro and in vivo MRI contrast by specifically alternating the transverse relaxation rate of water, but also inhibit the malignant growth of lung tumor. In addition, MagA can regulate magnetic nanoparticle production in grafted tissues and also suppress transferrin receptor expression by acting as an iron transporter, and meanwhile can permit iron biomineralization in the presence of mammalian iron homeostasis. This work provides experimental evidence for the safe preclinical applications of MagA as both a potential inhibitor and an MRI-based tracing tool for iron ion-dependent lung cancer.

Zinc Transporter SLC39A7/ZIP7 Promotes Intestinal Epithelial Self-Renewal by Resolving ER Stress.

Zinc transporters play a critical role in spatiotemporal regulation of zinc homeostasis. Although disruption of zinc homeostasis has been implicated in disorders such as intestinal inflammation and aberrant epithelial morphology, it is largely unknown which zinc transporters are responsible for the intestinal epithelial homeostasis. Here, we show that Zrt-Irt-like protein (ZIP) transporter ZIP7, which is highly expressed in the intestinal crypt, is essential for intestinal epithelial proliferation. Mice lacking Zip7 in intestinal epithelium triggered endoplasmic reticulum (ER) stress in proliferative progenitor cells, leading to significant cell death of progenitor cells. Zip7 deficiency led to the loss of Olfm4+ intestinal stem cells and the degeneration of post-mitotic Paneth cells, indicating a fundamental requirement for Zip7 in homeostatic intestinal regeneration. Taken together, these findings provide evidence for the importance of ZIP7 in maintenance of intestinal epithelial homeostasis through the regulation of ER function in proliferative progenitor cells and maintenance of intestinal stem cells. Therapeutic targeting of ZIP7 could lead to effective treatment of gastrointestinal disorders.

NBCe1-A Regulates Proximal Tubule Ammonia Metabolism under Basal Conditions and in Response to Metabolic Acidosis.

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes.

An immunoaffinity-based method for isolating ultrapure adult astrocytes based on ATP1B2 targeting by the ACSA-2 antibody.

Astrocytes are a major cell type in the mammalian CNS. Astrocytes are now known to play a number of essential roles in processes including synapse formation and function, as well as blood-brain barrier formation and control of cerebral blood flow. However, our understanding of the molecular mechanisms underlying astrocyte development and function is still rudimentary. This lack of knowledge is at least partly due to the lack of tools currently available for astrocyte biology. ACSA-2 is a commercially available antibody originally developed for the isolation of astrocytes from young postnatal mouse brain, using magnetic cell-sorting methods, but its utility in isolating cells from adult tissue has not yet been published. Using a modified protocol, we now show that this tool can also be used to isolate ultrapure astrocytes from the adult brain. Furthermore, using a variety of techniques (including single-cell sequencing, overexpression and knockdown assays, immunoblotting, and immunohistochemistry), we identify the ACSA-2 epitope for the first time as ATP1B2 and characterize its distribution in the CNS. Finally, we show that ATP1B2 is stably expressed in multiple models of CNS injury and disease. Hence, we show that the ACSA-2 antibody possesses the potential to be an extremely valuable tool for astrocyte research, allowing the purification and characterization of astrocytes (potentially including injury and disease models) without the need for any specialized and expensive equipment. In fact, our results suggest that ACSA-2 should be a first-choice method for astrocyte isolation and characterization.

Suppression of ferroportin expression by cadmium stimulates proliferation, EMT, and migration in triple-negative breast cancer cells.

Cadmium (Cd) has been linked to a variety of cancers, including breast cancer; however, the molecular mechanism of its carcinogenic activity is not fully understood. To this end, the present study investigated the roles of ferroportin (FPN), a prognostic marker of breast cancer, in Cd-induced stimulation of cell proliferation and cell migration. Triple-negative MDA-MB-231 cells were treated with 1-3 µM Cd. The cells exhibited significant reduction in FPN expression and concomitant increase in iron concentration. Cells treated with Cd for 8 weeks displayed elevated proliferative and migratory activities which were inversely related with FPN expression. Reduced FPN expression also resulted in EMT as indicated by an increase in the expression of E-cadherin, and a decrease in the expression of N-cadherin, Twist and Slug. Further investigation revealed that Cd suppressed FPN expression at least partially by activating TGF-ß, a known regulator of FPN expression. Taken together, these results indicate that Cd-induced stimulation of MDA-MB-231 cell proliferation, EMT, and migration is brought about by suppression of FPN expression and associated disruption of iron homeostasis.

Induction of endoplasmic reticulum stress and changes in expression levels of Zn2+-transporters in hypertrophic rat heart.

Clinical and experimental studies have shown an association between intracellular free Zn2+ ([Zn2+]i)-dyshomeostasis and cardiac dysfunction besides [Ca2+]i-dyshomeostasis. Since [Zn2+]i-homeostasis is regulated through Zn2+-transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn2+-homeostasis via alteration in Zn2+-transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3ß, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NFκB to the NFκB are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKCα and PKCα levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn2+-influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn2+]i detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn2+]i via decreased ER-[Zn2+] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn2+-transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn2+]i- besides [Ca2+]i-dyshomeostasis.

Enhancement of ε-poly-L-lysine production by overexpressing the ammonium transporter gene in Streptomyces albulus PD-1.

The antibacterial polymer ɛ-poly-L-lysine (ε-PL) has been widely used as a safe food preservative. As the synthesis of ε-PL requires a rich supply of nitrogen, the efficiency of nitrogen translocation and utilization is extremely important. The objective of this study was to improve the production of ε-PL by overexpressing the ammonium transporter gene amtB in Streptomyces albulus PD-1. Using the recombinant bacteria, the optimum carbon-to-nitrogen ratio in the synthesis stage of fermentation increased from 3 to 4.71, compared with that obtained using the wild-type strain, and the utilization efficiency of ammonium was improved too. Ultimately, the production of ε-PL increased from 22.7 to 35.7 g/L upon fed-batch cultivation in a 5 L bioreactor. Determination of the expression of the genes and enzymes associated with ammonium metabolism and ε-PL synthesis revealed that the overexpression of amtB in S. albulus PD-1 enhanced ε-PL biosynthesis by increasing the activity of the corresponding metabolic pathways. To the best of our knowledge, this is the first report on enhancing ε-PL production by overexpression of the amtB gene in an ε-PL-producing strain.

Iron Export through the Transporter Ferroportin 1 Is Modulated by the Iron Chaperone PCBP2.

Ferroportin 1 (FPN1) is an iron export protein found in mammals. FPN1 is important for the export of iron across the basolateral membrane of absorptive enterocytes and across the plasma membrane of macrophages. The expression of FPN1 is regulated by hepcidin, which binds to FPN1 and then induces its degradation. Previously, we demonstrated that divalent metal transporter 1 (DMT1) interacts with the intracellular iron chaperone protein poly(rC)-binding protein 2 (PCBP2). Subsequently, PCBP2 receives iron from DMT1 and then disengages from the transporter. In this study, we investigated the function of PCBP2 in iron export. Mammalian genomes encode four PCBPs (i.e. PCBP1-4). Here, for the first time, we demonstrated using both yeast and mammalian cells that PCBP2, but not PCBP1, PCBP3, or PCBP4, binds with FPN1. Importantly, iron-loaded, but not iron-depleted, PCBP2 interacts with FPN1. The PCBP2-binding domain of FPN1 was identified in its C-terminal cytoplasmic region. The silencing of PCBP2 expression suppressed FPN1-dependent iron export from cells. These results suggest that FPN1 exports iron received from the iron chaperone PCBP2. Therefore, it was found that PCBP2 modulates cellular iron export, which is an important physiological process.

Altered expression of Mg(2+) transport proteins during Parkinson's disease-like dopaminergic cell degeneration in PC12 cells.

Mg(2+) is an essential cation to maintain cellular functions, and intracellular Mg(2+) concentration ([Mg(2+)]i) is regulated by Mg(2+) channels and transporters. In our previous study, we demonstrated that MPP(+) elicits Mg(2+) influx across the cell membrane and Mg(2+) mobilization from mitochondria, and the resulting [Mg(2+)]i is an important determinants of the cell viability in MPP(+) model of Parkinson's disease (PD). It indicates that cellular Mg(2+) transport is one of the important factors to determine the progress of PD. However, whether the expression levels of Mg(2+) transport proteins change in the progress of PD has still been obscure. In this study, we estimated the mRNA expression levels of Mg(2+) transport proteins upon the exposure to MPP(+). In thirteen Mg(2+) transport proteins examined, mRNA expression level of SLC41A2 was increased and that of ACDP2, NIPA1 and MMgT2 were decreased. Knockdown of SLC41A2, ACDP2 or NIPA1 accelerated the MPP(+)-induced cell degeneration, and overexpression attenuated it. The decrease in the mRNA expression levels of NIPA1 and MMgT2 were also elicited by rotenone, H2O2 and FCCP, indicating that mitochondrial dysfunction related to this down-regulation. The increase in that of SLC41A2 was induced by an uncoupler, FCCP, as well as MPP(+), suggesting that it is an intrinsic protection mechanism against depolarized mitochondrial membrane potential and/or cellular ATP depletion. Our results shown here indicate that alteration of Mg(2+) transport proteins is implicated in the MPP(+) model of PD, and it affects cell degeneration.

Copper therapy reduces intravascular hemolysis and derepresses ferroportin in mice with mosaic mutation (Atp7amo-ms): An implication for copper-mediated regulation of the Slc40a1 gene expression.

Mosaic mutant mice displaying functional dysfunction of Atp7a copper transporter (the Menkes ATPase) are an established animal model of Menkes disease and constitute a convenient tool for investigating connections between copper and iron metabolisms. This model allows to explore changes in iron metabolism in suckling mutant mice suffering from systemic copper deficiency as well as in young and adult ones undergone copper therapy, which reduces lethal effect of the Atp7a gene mutation. Our recent study demonstrated that 14-day-old mosaic mutant males display blood cell abnormalities associated with intravascular hemolysis, and show disturbances in the functioning of the hepcidin-ferroportin regulatory axis, which controls systemic iron homeostasis. We thus aimed to check whether copper supplementation recovers mutants from hemolytic insult and rebalance systemic iron regulation. Copper supplementation of 14-day-old mosaic mutants resulted in the reestablishment of hematological status, attenuation of hepicidin and concomitant induction of the iron exporter ferroportin/Slc40a1 expression in the liver, down-regulated in untreated mutants. Interestingly, treatment of wild-type males with copper, induced hepcidin-independent up-regulation of ferroportin protein level in hepatic macrophages in both young and adult (6-month-old) animals. Stimulatory effect of copper on ferroportin mRNA and protein levels was confirmed in bone marrow-derived macrophages isolated from both wild-type and mosaic mutant males. Our study indicates that copper is an important player in the regulation of the Slc40a1 gene expression.

Targeting miR-21 decreases expression of multi-drug resistant genes and promotes chemosensitivity of renal carcinoma.

Renal cell carcinoma, the most common neoplasm of adult kidney, accounts for about 3% of adult malignancies and is usually highly resistant to conventional therapy. MicroRNAs are a class of small non-coding RNAs, which have been previously shown to promote malignant initiation and progression. In this study, we focused our attention on miR-21, a well described oncomiR commonly upregulated in cancer. Using a cohort of 99 primary renal cell carcinoma samples, we showed that miR-21 expression in cancer tissues was higher than in adjacent non-tumor tissues whereas no significant difference was observed with stages, grades, and metastatic outcome. In vitro, miR-21 was also overexpressed in renal carcinoma cell lines compared to HK-2 human proximal tubule epithelial cell line. Moreover, using Boyden chambers and western blot techniques, we also showed that miR-21 overexpression increased migratory, invasive, proliferative, and anti-apoptotic signaling pathways whereas opposite results were observed using an anti-miR-21-based silencing strategy. Finally, we assessed the role of miR-21 in mediating renal cell carcinoma chemoresistance and further showed that miR-21 silencing significantly (1) increased chemosensitivity of paclitaxel, 5-fluorouracil, oxaliplatin, and dovitinib; (2) decreased expression of multi-drug resistance genes; and (4) increased SLC22A1/OCT1, SLC22A2/OCT2, and SLC31A1/CTR1 platinum influx transporter expression. In conclusion, our results showed that miR-21 is a key actor of renal cancer progression and plays an important role in the resistance to chemotherapeutic drugs. In renal cell carcinoma, targeting miR-21 is a potential new therapeutic strategy to improve chemotherapy efficacy and consequently patient outcome.

Iron importers Zip8 and Zip14 are expressed in retina and regulated by retinal iron levels.

Intracellular retinal iron accumulation has been implicated in the pathogenesis of age-related macular degeneration (AMD), the leading cause of irreversible blindness among individuals over the age of 50. Ceruloplasmin/hephaestin double knockout mice (Cp/Heph DKO) and hepcidin knockout mice (Hepc KO) accumulate retinal iron and model some features of AMD. Two canonical pathways govern cellular iron import - transferrin-bound iron import and non-transferrin bound iron import. In Cp/Heph DKO and Hepc KO iron-loaded retinas, transferrin-bound iron import is downregulated. Despite this effort to reduce cellular iron burden, iron continues to accumulate in these retinas in an age-dependent manner. Quantitative RT-PCR and Western analysis were used to quantify the expression of three ferrous iron importers, Dmt1, Zip8, and Zip14, in wild-type (Wt), Cp/Heph DKO, and Hepc KO retinas. Zip8 and Zip14 protein levels were analyzed using Western analysis in mice injected intravitreally with either apo- or holo-transferrin to elucidate one possible mechanism of Zip14 regulation in the retina. Both zip8 and zip14 were expressed in the mouse retina. Paradoxically, protein levels of non-transferrin bound iron importers were upregulated in both Cp/Heph DKO and Hepc KO retinas. Intravitreal holo-transferrin injection decreased Zip 14 protein levels. These data indicate that Zip8 and Zip14 may take up increasing amounts of non-transferrin bound iron in these two mouse models of retinal iron accumulation. Their upregulation in these already iron-loaded retinas suggests a vicious cycle leading to toxicity.

Production of recombinant proteins in Escherichia coli tagged with the fusion protein CusF3H.

Recombinant protein expression in the bacterium Escherichia coli still is the number one choice for large-scale protein production. Nevertheless, many complications can arise using this microorganism, such as low yields, the formation of inclusion bodies, and the requirement for difficult purification steps. Most of these problems can be solved with the use of fusion proteins. Here, the use of the metal-binding protein CusF3H+ is described as a new fusion protein for recombinant protein expression and purification in E. coli. We have previously shown that CusF produces large amounts of soluble protein, with low levels of formation of inclusion bodies, and that proteins can be purified using IMAC resins charged with Cu(II) ions. CusF3H+ is an enhanced variant of CusF, formed by the addition of three histidine residues at the N-terminus. These residues then can bind Ni(II) ions allowing improved purity after affinity chromatography. Expression and purification of Green Fluorescent Protein tagged with CusF3H+ showed that the mutation did not alter the capacity of the fusion protein to increase protein expression, and purity improved considerably after affinity chromatography with immobilized nickel ions; high yields are obtained after tag-removal since CusF3H+ is a small protein of just 10 kDa. Furthermore, the results of experiments involving expression of tagged proteins having medium to large molecular weights indicate that the presence of the CusF3H+ tag improves protein solubility, as compared to a His-tag. We therefore endorse CusF3H+ as a useful alternative fusion protein/affinity tag for production of recombinant proteins in E. coli.