Cellular Metal Ion Homeostasis Research Articles

Elemental Analysis for the Characterization of Antimicrobial Effects.

To address the mounting resistance challenge, novel antibiotics and unprecedented mechanisms of action are urgently needed. In this context, metals have attracted attention in two distinct ways: First, the bacterial metal ion homeostasis is essential for many cellular processes, making it a putatively lucrative antibiotic target. Metal ions are, for example, cofactors for enzymes, and they contribute to signaling and transport processes or to energy metabolism. Possible antibacterial strategies include, for example, depletion of accessible essential metals by sequestration or disruption of metal ion homeostasis by ionophores that transport ions across membranes. Second, organometallic antibiotics that contain metals as integral structural elements can provide unique chemistry with unique modes of action. Since many metal-containing structures used in synthetic chemistry are unprecedented in nature, such antibiotics could circumvent existing mechanisms of resistance. Here, we present a method for quantification of cellular metal/metalloid levels and outline the procedures necessary for antibiotic treatment of Bacillus subtilis, subsequent sample preparation, elemental analysis, and data evaluation. This approach allows to investigate disturbances of the cellular metal ion homeostasis, as well as the localization and quantitation of antibiotics that contain metals rarely found in biological systems, overall aiding in the elucidation of antibiotic mechanisms of action.

FERONIA-like receptor 1-mediated calcium ion homeostasis is involved in the immune response

Calcium (Ca2+) is the most abundant divalent cation in plants, and cellular levels of Ca2+, which functions as a nutrient and secondary messenger, play a critical role in plant immunity. In the present study, we found that FERONIA-like receptor 1 (FLR1) positively regulates Magnaporthe oryzae resistance and that expression of FLR1 is strongly induced in response to Ca2+ deficiency. In addition, the Ca content in the shoots of flr1 was lower than that in wild-type, and the M. oryzae-sensitive phenotype of the flr1 mutant was not rescued by exogenous application of Ca2+. Moreover, RNA sequencing revealed 2,697 differentially expressed genes (DEGs) in the flr1 mutant compared with wild-type, and some of these DEGs are involved in cellular metal ion homeostasis and transition metal ion homeostasis. Changes in expression of overlapping genes between the flr1 mutant and in plants under low-Ca2+ treatment were consistent in terms of direction, indicating that FLR1 is involved in Ca2+ homeostasis. In summary, we detected FLR1-mediated resistance to M. oryzae, a phenomenon associated with Ca2+ homeostasis.

Integrated ionomic and transcriptomic dissection reveals the core transporter genes responsive to varying cadmium abundances in allotetraploid rapeseed

BackgroundOilseed rape (B. napus L.) has great potential for phytoremediation of cadmium (Cd)-polluted soils due to its large plant biomass production and strong metal accumulation. Soil properties and the presence of other soluble compounds or ions, cause a heterogeneous distribution of Cd.ResultsThe aim of our study was to reveal the differential responses of B. napus to different Cd abundances. Herein, we found that high Cd (50 μM) severely inhibited the growth of B. napus, which was not repressed by low Cd (0.50 μM) under hydroponic culture system. ICP-MS assays showed that the Cd2+ concentrations in both shoots and roots under 50 μM Cd were over 10 times higher than those under 0.50 μM Cd. Under low Cd, the concentrations of only shoot Ca2+/Mn2+ and root Mn2+ were obviously changed (both reduced); under high Cd, the concentrations of most cations assayed were significantly altered in both shoots and roots except root Ca2+ and Mg2+. High-throughput transcriptomic profiling revealed a total of 18,021 and 1408 differentially expressed genes under high Cd and low Cd conditions, respectively. The biological categories related to the biosynthesis of plant cell wall components and response to external stimulus were over-accumulated under low Cd, whereas the terms involving photosynthesis, nitrogen transport and response, and cellular metal ion homeostasis were highly enriched under high Cd. Differential expression of the transporters responsible for Cd uptake (NRAMPs), transport (IRTs and ZIPs), sequestration (HMAs, ABCs, and CAXs), and detoxification (MTPs, PCR, MTs, and PCSs), and some other essential nutrient transporters were investigated, and gene co-expression network analysis revealed the core members of these Cd transporters. Some Cd transporter genes, especially NRAMPs and IRTs, showed opposite responsive patterns between high Cd and low Cd conditions.ConclusionsOur findings would enrich our understanding of the interaction between essential nutrients and Cd, and might also provide suitable gene resources and important implications for the genetic improvement of plant Cd accumulation and resistance through molecular engineering of these core genes under varying Cd abundances in soils.

Dysregulation of Magnesium Transport Protects Bacillus subtilis against Manganese and Cobalt Intoxication

Transition metals are essential for life but are toxic when in excess. Metal ion intoxication may result from the mismetallation of essential metal-dependent enzymes with a noncognate metal. To begin to identify enzymes and processes that are susceptible to mismetallation, we have selected for strains with increased resistance to Mn(II) and Co(II). In Bacillus subtilis, cells lacking the MntR metalloregulator are exquisitely sensitive to Mn(II) but can easily become resistant by acquiring mutations affecting the MntH Mn(II) importer. Using transposon mutagenesis, and starting with an mntR mntH strain, we recovered mariner insertions that inactivated the mpfA gene encoding a putative Mg(II) efflux system. Loss of MpfA leads to elevated intracellular Mg(II), increased sensitivity to high Mg(II), and reduced Mn(II) sensitivity. Consistently, we also recovered an insertion disrupting the mgtE riboswitch, which normally restricts expression of the major Mg(II) importer. These results suggest that Mn(II) intoxication results from disruption of a Mg(II)-dependent enzyme or process. Mutations that inactivate MpfA were also recovered in a selection for Co(II) resistance beginning with sensitized strains lacking the major Co(II) efflux pump, CzcD. Since both Mn(II) and Co(II) may mismetallate iron-dependent enzymes, we repeated the selections under conditions of iron depletion imposed by expression of the Listeria monocytogenes FrvA iron exporter. Under conditions of iron depletion, a wider variety of suppressor mutations were recovered, but they still point to a central role for Mg(II) in maintaining metal ion homeostasis.IMPORTANCE Cellular metal ion homeostasis is tightly regulated. When metal ion levels are imbalanced, or when one metal is at toxic levels, enzymes may bind to the wrong metal cofactor. Enzyme mismetallation can impair metabolism, lead to new and deleterious reactions, and cause cell death. Beginning with Bacillus subtilis strains genetically sensitized to metal intoxication through loss of efflux or by lowering intracellular iron, we identified mutations that suppress the deleterious effects of excess Mn(II) or Co(II). For both metals, mutations in mpfA, encoding a Mg(II) efflux pump, suppressed toxicity. These mutant strains have elevated intracellular Mg(II), suggesting that Mg(II)-dependent processes are very sensitive to disruption by transition metals.

Live-Cell Imaging of Physiologically Relevant Metal Ions Using Genetically Encoded FRET-Based Probes.

Essential biochemical reactions and processes within living organisms are coupled to subcellular fluctuations of metal ions. Disturbances in cellular metal ion homeostasis are frequently associated with pathological alterations, including neurotoxicity causing neurodegeneration, as well as metabolic disorders or cancer. Considering these important aspects of the cellular metal ion homeostasis in health and disease, measurements of subcellular ion signals are of broad scientific interest. The investigation of the cellular ion homeostasis using classical biochemical methods is quite difficult, often even not feasible or requires large cell numbers. Here, we report of genetically encoded fluorescent probes that enable the visualization of metal ion dynamics within individual living cells and their organelles with high temporal and spatial resolution. Generally, these probes consist of specific ion binding domains fused to fluorescent protein(s), altering their fluorescent properties upon ion binding. This review focuses on the functionality and potential of these genetically encoded fluorescent tools which enable monitoring (sub)cellular concentrations of alkali metals such as K+, alkaline earth metals including Mg2+ and Ca2+, and transition metals including Cu+/Cu2+ and Zn2+. Moreover, we discuss possible approaches for the development and application of novel metal ion biosensors for Fe2+/Fe3+, Mn2+ and Na+.

The role of metal ions in the virulence and viability of bacterial pathogens

Metal ions fulfil a plethora of essential roles within bacterial pathogens. In addition to acting as necessary cofactors for cellular proteins, making them indispensable for both protein structure and function, they also fulfil roles in signalling and regulation of virulence. Consequently, the maintenance of cellular metal ion homeostasis is crucial for bacterial viability and pathogenicity. It is therefore unsurprising that components of the immune response target and exploit both the essentiality of metal ions and their potential toxicity toward invading bacteria. This review provides a brief overview of the transition metal ions iron, manganese, copper and zinc during infection. These essential metal ions are discussed in the context of host modulation of bioavailability, bacterial acquisition and efflux, metal-regulated virulence factor expression and the molecular mechanisms that contribute to loss of viability and/or virulence during host-imposed metal stress.

Transcriptomic Analysis of THP‐1 Macrophages Exposed to Lipoprotein Hydrolysis Products Generated by Lipoprotein Lipase

Macrophage lipoprotein lipase (LPL) induces lipid accumulation and promotes atherosclerosis. However, the effects of lipoprotein hydrolysis products generated by LPL on macrophage-derived foam cell formation are not clearly understood. Thus, we analyzed the transcriptomic response to hydrolysis products via microarray analyses on RNA isolated from human THP-1 macrophages incubated with total lipoprotein hydrolysis products generated by LPL. The expression of 183 transcripts was significantly upregulated and 133 transcripts were significantly downregulated. Bioinformatics analyses revealed that there was a significant over-representation of genes involved in cell cycling, stress response, type I interferon signaling, cellular metal ion homeostasis, sterol metabolism, and nuclease activity. Interestingly, transcripts for 63 small nucleolar RNA were significantly upregulated. We verified the microarray data by quantitative real-time PCR and found that the expression of SNORA56, as well as the expression of genes associated with the cell cycle (PCNA and DKC1 variant 3), stress response (ATF3), type I interferon signaling (IFITM1), and lipid metabolism (CD36 and PLIN2) were significantly affected by LPL hydrolysis products. To determine if the free fatty acid (FFA) component of total lipoprotein hydrolysis products is sufficient to alter the expression of these genes, THP-1 macrophages were also incubated with the total FFA or individual classes of the FFA component. The gene regulation by the FFA component did not mimic that of the hydrolysis products, suggesting that the regulation of gene expression in THP-1 macrophages depends on the specific combination and concentration of lipid species present in the hydrolysis products, and not solely on FFA.

Domain Selection in Metallothionein 1A: Affinity-Controlled Mechanisms of Zinc Binding and Cadmium Exchange.

Mammalian metallothioneins (MTs) are small, metal binding proteins implicated in cellular metal ion homeostasis and heavy metal detoxification. Divalent, metal-saturated MTs form two distinct domains; the N-terminal β domain binds three metals using nine Cys residues, and the C-terminal α domain binds four metals with 11 Cys residues. Domain selection during zinc binding and cadmium exchange to human MT1A was examined using a series of competition reactions with mixtures of the isolated domain fragments. These experiments were conducted at two biologically significant pH conditions where MTs exist in vivo. Neither zinc binding nor cadmium exchange showed any significant degree of specificity or selectivity based on detailed analysis of electrospray ionization mass spectrometric and circular dichroic data. Under acidic conditions, zinc binding and cadmium exchange showed slight α domain selectivity because of the increased preference for cooperative clustering of the α domain. Modeling of the reactions showed that at both physiological (7.4) and acidic (5.8) pHs, zinc binding and cadmium exchanges occur essentially randomly between the two fragments. The metal binding affinity distributions between the domain fragments are comingled and not significantly separated as required for a domain specific mechanism. The models show rather that the order of the binding events follows the order of the binding affinities that are distributed across both domains and that this can be considered quantitatively by the KF(Cd)/KF(Zn) binding constant ratio for each metal bound.

Hippocampal transcriptional and neurogenic changes evoked by combination yohimbine and imipramine treatment.

Adjunct α2-adrenoceptor antagonism is a potential strategy to accelerate the behavioral effects of antidepressants. Co-administration of the α2-adrenoceptor antagonist yohimbine hastens the behavioral and neurogenic effects of the antidepressant imipramine. We examined the transcriptional targets of short duration (7days), combination treatment of yohimbine and imipramine (Y+I) within the adult rat hippocampus. Using microarray and qPCR analysis we observed functional enrichment of genes involved in intracellular signaling cascades, plasma membrane, cellular metal ion homeostasis, multicellular stress responses and neuropeptide signaling pathways in the Y+I transcriptome. We noted reduced expression of the α2A-adrenoceptor (Adra2a), serotonin 5HT2C receptor (Htr2c) and the somatostatin receptor 1 (Sstr1), which modulate antidepressant action. Further, we noted a regulation of signaling pathway genes like inositol monophosphatase 2 (Impa2), iodothyronine deiodinase 3 (Dio3), regulator of G-protein signaling 4 (Rgs4), alkaline ceramidase 2 (Acer2), doublecortin-like kinase 2 (Dclk2), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (Nfkbia) and serum/glucocorticoid-regulated kinase 1 (Sgk1), several of which are implicated in the pathophysiology of mood disorders. Comparative analysis revealed an overlap in the hippocampal regulation of Acer2, Nfkbia, Sgk1 and Impa2 between Y+I treatment, the fast-acting electroconvulsive seizure (ECS) paradigm, and the slow-onset chronic (21days) imipramine treatment. Further, Y+I treatment enhanced the quiescent neural progenitor pool in the hippocampal neurogenic niche similar to ECS, and distinct from chronic imipramine treatment. Taken together, our results provide insight into the molecular and cellular targets of short duration Y+I treatment, and identify potential leads for the development of rapid-action antidepressants.

Paraquat reduces natural killer cell activity via metallothionein induction

Paraquat (PQ), one of the most widely used herbicides, has been used for several decades in agriculture. Some studies suggest that PQ has effects on the immune system. Moreover, previous studies have shown that PQ imparted some immunosuppressive effects. In the present study, cytotoxicity assays using splenic NK cells from mice treated for 28 days with PQ (at 0.2, 1, and 5 mg/kg) were performed to determine whether PQ altered the function of NK cells. Given that PQ was expected to induce an immunosuppressive effect, it was hypothesized that a gene involved in cellular metal ion homeostasis, metallothionein-1 (MT-1), could play an important role in this outcome. This belief was based on the fact that MT1 encodes a protein responsible for zinc ion homeostasis, and that a reduction in free zinc ion levels impairs NK cell function. The results showed that PQ treatments led to increased MT expression in several organs (liver, kidneys, testes) and in splenocytes, caused a reduction of both free zinc ions in sera and in free intracellular zinc, and reduced the expression of GATA-3, a zinc-finger transcription factor important for maturation and activity of T-cells and NK cells. These results provide a basis for a new molecular mechanism to describe potential immunosuppressive effects of PQ in vivo.

Assessment of crosstalks between the Snf1 kinase complex and sphingolipid metabolism in S. cerevisiae via systems biology approaches

Sphingolipids are essential building blocks of the plasma membranes and are highly bioactive in the regulation of diverse cellular functions and pathological processes, a fact which renders the sphingolipid metabolism an important research area. In this study, a computational framework was recruited for the reconstruction of a functional interaction network for sphingolipid metabolism in Baker's yeast, SSN. Gene Ontology (GO) annotations were integrated with functional interaction data of the BIOGRID database and the reconstructed protein interaction network was subjected to topological and descriptive analyses. SSN was of a scale-free nature, following a power law model with γ=1.41. Prominent processes of SSN revealed that the reconstructed network encapsulated the involvement of sphingolipid metabolism in vital cellular processes such as energy homeostasis, cell growth and/or death and synthesis of building blocks. To investigate the potential of SSN for predicting signal transduction pathways regulating and/or being regulated by sphingolipid biosynthesis in yeast, a case study involving the S. cerevisiae counterpart of AMP-activated protein kinase, the Snf1 kinase complex, was conducted. The mutant strain lacking the catalytic α subunit, snf1Δ/snf1Δ, had elevated inositol phosphorylceramide and mannosyl-inositol phosphorylceramide levels, and decreased mannosyl-diinositol phosphorylceramide levels compared to the wild type strain, revealing that Snf1p has a regulatory role in the sphingolipid metabolism. Transcriptome data belonging to that strain available in the literature were mapped onto SSN and the correlated SSN was further investigated to evaluate the possible crosstalk machineries where sphingolipids and Snf1p function in coordination, in other words the crosstalk points between sphingolipid-mediated and Snf1 kinase signalling. The subsequent investigation of the discovered candidate crosstalk processes by performing sensitivity experiments imply a tight interconnection between sphingolipids and Snf1p in the regulation of calcineurin activity, cellular metal ion homeostasis and response to cell wall and endoplasmic reticulum stresses in yeast.

Transcriptomic Characterization of Temperature Stress Responses in Larval Zebrafish

Temperature influences nearly all biochemical, physiological and life history activities of fish, but the molecular mechanisms underlying the temperature acclimation remains largely unknown. Previous studies have identified many temperature-regulated genes in adult tissues; however, the transcriptional responses of fish larvae to temperature stress are not well understood. In this study, we characterized the transcriptional responses in larval zebrafish exposed to cold or heat stress using microarray analysis. In comparison with genes expressed in the control at 28°C, a total of 2680 genes were found to be affected in 96 hpf larvae exposed to cold (16°C) or heat (34°C) for 2 and 48h and most of these genes were expressed in a temperature-specific and temporally regulated manner. Bioinformatic analysis identified multiple temperature-regulated biological processes and pathways. Biological processes overrepresented among the earliest genes induced by temperature stress include regulation of transcription, nucleosome assembly, chromatin organization and protein folding. However, processes such as RNA processing, cellular metal ion homeostasis and protein transport and were enriched in genes up-regulated under cold exposure for 48 h. Pathways such as mTOR signalling, p53 signalling and circadian rhythm were enriched among cold-induced genes, while adipocytokine signalling, protein export and arginine and praline metabolism were enriched among heat-induced genes. Although most of these biological processes and pathways were specifically regulated by cold or heat, common responses to both cold and heat stresses were also found. Thus, these findings provide new interesting clues for elucidation of mechanisms underlying the temperature acclimation in fish.

Protein carbonylation and metal-catalyzed protein oxidation in a cellular perspective

Proteins can become oxidatively modified in many different ways, either by direct oxidation of amino acid side chains and protein backbone or indirectly by conjugation with oxidation products of polyunsaturated fatty acids and carbohydrates. While reversible oxidative modifications are thought to be relevant in physiological processes, irreversible oxidative modifications are known to contribute to cellular damage and disease. The most well-studied irreversible protein oxidation is carbonylation. In this work we first examine how protein carbonylation occurs via metal-catalyzed oxidation (MCO) in vivo and in vitro with an emphasis on cellular metal ion homeostasis and metal binding. We then review proteomic methods currently used for identifying carbonylated proteins and their sites of modification. Finally, we discuss the identified carbonylated proteins and the pattern of carbonylation sites in relation to cellular metabolism using the mitochondrion as a case story.