Homeostasis Of Essential Metal Ions Research Articles

Spectroscopic, Electrochemical, and Biological Assays of Copper-Binding Molecules for Screening of Different Drugs and Plant Extracts against Neurodegenerative Disorders.

Neurodegenerative disorders, caused by prone-to-aggregation proteins, such as Alzheimer disease or Huntington disease, share other traits such as disrupted homeostasis of essential metal ions, like copper. In this context, in an attempt to identify Cu2+ chelating agents, we study several organic compounds (ethylenediaminetetraacetic acid, phenylenediamine, metformin, salicylate, and trehalose) and organic extracts obtained from Bacopa monnieri L., which has been used in Ayurvedic therapies and presented a broad spectrum of biological properties. For this purpose, UV–visible spectroscopy analysis and electrochemical measurements were performed. Further, biological assays were performed in Caenorhabditis elegans models of polyQ toxicity, in an attempt to obtain better insights on neurodegenerative disorders.

Gestational Cd Exposure in the CD-1 Mouse Sex-Specifically Disrupts Essential Metal Ion Homeostasis.

In CD-1 mice, gestational-only exposure to cadmium (Cd) causes female-specific hepatic insulin resistance, metabolic disruption, and obesity. To evaluate whether sex differences in uptake and changes in essential metal concentrations contribute to metabolic outcomes, placental and liver Cd and essential metal concentrations were quantified in male and female offspring perinatally exposed to 500 ppb CdCl2. Exposure resulted in increased maternal liver Cd+2 concentrations (364 µg/kg) similar to concentrations found in non-occupationally exposed human liver. At gestational day (GD) 18, placental Cd and manganese concentrations were significantly increased in exposed males and females, and zinc was significantly decreased in females. Placental efficiency was significantly decreased in GD18-exposed males. Increases in hepatic Cd concentrations and a transient prenatal increase in zinc were observed in exposed female liver. Fetal and adult liver iron concentrations were decreased in both sexes, and decreases in hepatic zinc, iron, and manganese were observed in exposed females. Analysis of GD18 placental and liver metallothionein mRNA expression revealed significant Cd-induced upregulation of placental metallothionein in both sexes, and a significant decrease in fetal hepatic metallothionein in exposed females. In placenta, expression of metal ion transporters responsible for metal ion uptake was increased in exposed females. In liver of exposed adult female offspring, expression of the divalent cation importer (Slc39a14/Zip14) decreased, whereas expression of the primary exporter (Slc30a10/ZnT10) increased. These findings demonstrate that Cd can preferentially cross the female placenta, accumulate in the liver, and cause lifelong dysregulation of metal ion concentrations associated with metabolic disruption.

The S. mutans mntE gene encodes a manganese efflux transporter.

Streptococcus mutans is a colonizer of the human dentition, and under conditions of dysbiosis is the primary causative agent of dental caries. The pathogenic potential of S. mutans depends, in part, on its ability to regulate the transport of metal ions across the plasma membrane to maintain intracellular metal ion homeostasis. Research in our laboratory has focused on the Mn2+ -specific SloC lipoprotein importer and its regulator encoded by the S. mutans sloR gene. Herein, we used a bioinformatics approach to identify a gene on the S. mutans UA159 chromosome, SMU_1176, as a metal ion efflux transporter that contributes to S. mutans manganese ion homeostasis. Metal ion sensitivity assays performed with the wild-type S. mutans UA159 strain and an isogenic SMU_1176 insertion-deletion mutant, called GMS3000, revealed significantly heightened sensitivity of GMS3000 to MnSO4 challenge. 54 Mn uptake experiments support the accumulation of 54 Mn in GMS3000 cell pellets when compared to 54 Mn concentrations in UA159 or in a complemented strain of GMS3000, called GMS3001. Inductively coupled plasma mass spectrometry (ICP-MS) studies were performed in parallel to quantify intracellular manganese concentrations in these strains, the results of which corroborate the 54 Mn uptake studies, and support the SMU_1176 gene product as a Mn2+ efflux protein. Expression profiling experiments revealed de-repression of SMU_1176 gene transcription in the SloR-deficient GMS584 strain of S. mutans, especially under high manganese conditions. In conclusion, the S. mutans SMU_1176 gene, which we renamed mntE, is a manganese efflux transporter that contributes to essential metal ion homeostasis as part of the SloR regulon.

A structural overview of the zinc transporters in the cation diffusion facilitator family.

The cation diffusion facilitators (CDFs) are a family of membrane-bound proteins that maintain cellular homeostasis of essential metal ions. In humans, the zinc-transporter CDF family members (ZnTs) play important roles in zinc homeostasis. They do this by facilitating zinc efflux from the cytoplasm to the extracellular space across the plasma membrane or into intracellular organelles. Several ZnTs have been implicated in human health owing to their association with type 2 diabetes and neurodegenerative diseases. Although the structure determination of CDF family members is not trivial, recent advances in membrane-protein structural biology have resulted in two structures of bacterial YiiPs and several structures of their soluble C-terminal domains. These data reveal new insights into the molecular mechanism of ZnT proteins, suggesting a unique rocking-bundle mechanism that provides alternating access to the metal-binding site.

Cyamopsis tetragonoloba type 1 metallothionein (CtMT1) gene is upregulated under drought stress and its protein product has an additional C-X-C motif and unique metal binding pattern

Metallothioneins (MTs) are involved in cellular homeostasis of essential metal ions and detoxification of nonessential metal ions. We report here the identification of four MT genes, CtMT1, CtMT2, CtMT3 and CtMT4, encoding CtMT1, CtMT2, CtMT3 and CtMT4 proteins, respectively, from the industrial guar crop. The primary structures of last three proteins were similar to those of respective MT proteins of other plants but the CtMT1 protein primary structure was different from the other plant MT1 proteins in having an additional C-X-C motif. The four MT genes showed tissue specific expression patterns suggesting their specific roles in different tissues. High expression of CtMT1 gene was observed in roots and nodules whereas CtMT2 and CtMT3 genes showed high expression in leaves. The expression of CtMT4 gene was high in seeds. The qRT-PCR studies revealed upregulation in expression of CtMT1 gene under drought stress. Recombinant CtMT1 protein was produced in E. coli Rosetta cells and purified by metal affinity chromatography. The purified protein showed antioxidant property and the order of its metal ion binding affinities was Cu2+ > Zn2+ > Fe2+ > Cd2+. This information about CtMT1 protein is expected to be useful in understanding its role in drought tolerance and other physiological processes of guar.

Autoregulation of the Streptococcus mutans SloR Metalloregulator Is Constitutive and Driven by an Independent Promoter.

Streptococcus mutans, one of ∼600 bacterial species in the human oral cavity, is among the most acidogenic constituents of the plaque biofilm. Considered to be the primary causative agent of dental caries, S. mutans harbors a 25-kDa SloR metalloregulatory protein which controls metal ion transport across the bacterial cell membrane to maintain essential metal ion homeostasis. The expression of SloR derives in part from transcriptional readthrough of the sloABC operon, which encodes a Mn2+/Fe2+ ABC transport system. Here we describe the details of the sloABC promoter that drives this transcription as well as those for a novel independent promoter in an intergenic region (IGR) that contributes to downstream sloR expression. Reverse transcriptase PCR (RT-PCR) studies support the occurrence of sloR transcription that is independent of sloABC expression, and the results of 5' rapid amplification of cDNA ends (5' RACE) revealed a sloR transcription start site in the IGR, from which the -10 and -35 promoter regions were predicted. The results of gel mobility shift assays support direct SloR binding to the IGR, albeit with a lower affinity than that for SloR binding to the sloABCR promoter. The function of the sloR promoter was validated by semiquantitative real-time PCR (qRT-PCR) experiments. Interestingly, sloR expression was not significantly affected when bacteria were grown in the presence of a high manganese concentration, whereas expression of the sloABC operon was repressed under these conditions. The results of in vitro transcription studies support the occurrence of SloR-mediated transcriptional activation of sloR and repression of sloABC Taken together, these findings implicate SloR as a bifunctional regulator that represses sloABC promoter activity and encourages sloR transcription from an independent promoter.IMPORTANCE Tooth decay is a ubiquitous infectious disease that is especially pervasive in underserved communities worldwide. S. mutans-induced carious lesions cause functional, physical, and/or esthetic impairment in the vast majority of adults and in 60 to 90% of schoolchildren in industrialized countries. Billions of dollars are spent annually on caries treatment, and productivity losses due to absenteeism from the workplace are significant. Research aimed at alleviating S. mutans-induced tooth decay is important because it can address the socioeconomic disparity that is associated with dental cavities and improve overall general health, which is inextricably linked to oral health. Research focused on the S. mutans SloR metalloregulatory protein can guide the development of novel therapeutics and thus alleviate the burden of dental cavities.

A Speciation Study on the Perturbing Effects of Iron Chelators on the Homeostasis of Essential Metal Ions.

A number of reports have appeared in literature calling attention to the depletion of essential metal ions during chelation therapy on β-thalassaemia patients. We present a speciation study to determine how the iron chelators used in therapy interfere with the homeostatic equilibria of essential metal ions. This work includes a thorough analysis of the pharmacokinetic properties of the chelating agents currently in clinical use, of the amounts of iron, copper and zinc available in plasma for chelation, and of all the implied complex formation constants. The results of the study show that a significant amount of essential metal ions is complexed whenever the chelating agent concentration exceeds the amount necessary to coordinate all disposable iron —a frequently occurring situation during chelation therapy. On the contrary, copper and zinc do not interfere with iron chelation, except for a possible influence of copper on iron speciation during deferiprone treatment.

Alfred Werner Lectures

At Eurobic11 in Granada we presented a Keynote Lecture on chelation therapy, a consolidated medical procedure used primarily to hinder the effects of toxic metal ions on human tissues. Its application spans a broad spectrum of serious disorders, ranging from acute metal intoxication to genetic metal-overload. The use of chelating agents is compromised by a number of serious side effects, mainly attributable to perturbed equilibrium of essential metal ion homeostasis and dislocation of complexed metal ions to dangerous body sites. For this reason, chelation therapy has been limited to specific critical and otherwise untreatable conditions and it needs to be monitored within an appropriate clinical context. In this meeting we want warn against the widespread fraudulent use of the term ‘‘chelation therapy’’ to take advantage of and make profit from people with tragic health problems. We believe that scientists working in this field have the corollary obligation to deter these frauds and to inform the scientific community of the possible side effects and complications of chelation therapy. This duty is all the more important if we consider the detrimental and even life threatening consequences that can occur in subjects with no clear clinical and laboratory evidence of metal intoxication. The aim of this communication is to present how this ‘‘false chelation therapy’’ developed and in which diseases it is currently applied. This research was supported by the Regione Autonoma Sardegna [CRP-27564].

Barley metallothioneins differ in ontogenetic pattern and response to metals

The barley genome encodes a family of 10 metallothioneins (MTs) that have not previously been subject to extensive gene expression profiling. We show here that expression of MT1a, MT2b1, MT2b2 and MT3 in barley leaves increased more than 50-fold during the first 10 d after germination. Concurrently, the root-specific gene MT1b1 was 1000-fold up-regulated. Immunolocalizations provided the first evidence for accumulation of MT1a and MT2a proteins in planta, with correlation to transcript levels. In developing grains, MT2a and MT4 expression increased 4- and 300-fold over a 28-day-period after pollination. However, among the MT grain transcripts MT2c was the most abundant, whereas MT4 was the least abundant. Excess Cu up-regulated three out of the six MTs expressed in leaves of young barley plants. In contrast, most MTs were down-regulated by excess Zn or Cd. Zn starvation led to up-regulation of MT1a, whereas Cu starvation up-regulated MT2a, which has two copper-responsive elements in the promoter. Arabidopsis lines constitutively overexpressing barley MT2a showed increased sensitivity to excess Cd and Zn but no Cu-induced response. We suggest that barley MTs are differentially involved in intracellular homeostasis of essential metal ions and that a subset of barley MTs is specifically involved in Cu detoxification.

Phylogenomic analysis of Cation Diffusion Facilitator proteins uncovers Ni2+/Co2+ transporters

The ubiquitous Cation Diffusion Facilitator proteins (CDF) play a key role in maintaining the cellular homeostasis of essential metal ions. Previous neighbor-joining phylogenetic analysis classified CDF proteins into three substrate-defined groups: Zn(2+), Fe(2+)/Zn(2+) and Mn(2+). These studies were unable to discern substrate-defined clades for Ni(2+), Co(2+), Cd(2+) and Cu(2+) transporters, despite their existence in this family. In this study we improved the accuracy of this previous functional classification using a phylogenomic approach based on a thorough maximum-likelihood phylogeny and the inclusion of recently characterized CDF transporters. The inference of CDF protein function predicted novel clades for Zn(2+), Fe(2+), Cd(2+) and Mn(2+). The Ni(2+)/Co(2+) and Co(2+) substrate specificities of two clades containing uncharacterized proteins were defined through the functional characterization of nepA and cepA metal inducible genes which independently conferred Ni(2+) and Co(2+) resistances to Rhizobium etli CFN42 and increased, respectively, Ni(2+)/Co(2+) and Co(2+) resistances to Escherichia coli. Neither NepA nor CepA confer Zn(2+), Fe(2+) and Mn(2+) resistances. The ability of NepA to confer Ni(2+)/Co(2+) resistance is dependent on clade-specific residues Asn(88) and Arg(197) whose mutations produce a non-functional protein.

Ni2+-uptake inPseudomonas putida strain S4: a possible role of Mg2+-uptake pump

Essential metal ion homeostasis is based on regulated uptake of metal ions, both during its scarcity and abundance. Pseudomonas putida strain S4, a multimetal resistant bacterium, was employed to investigate Ni(2+) entry into cells. It was observed that Mg(2+) regulates the entry of Ni(2+) and by this plays a protective role to minimize Ni(2+) toxicity in this strain. This protection was evident in both growth as well as viability. Intracellular accumulation of Ni(2+) varied in accordance with Mg(2+) concentrations in the medium. It was hypothesized that Ni(2+) enters the cell using a broad Mg(2+) pump, i.e. the CorA system, as the CorA inhibitor, i.e. Co(III) Hex, also inhibits Ni 2+ uptake. This led to the inference that Mg(2+)-based protection was basically due to competitive inhibition of Ni(2+) uptake. We also show that Zn(2+) can further regulate the entry of Ni(2+).

Lead exposure alters the drug metabolic activity and the homeostasis of essential metal ions in the lenticular system of the rat

Potential lead exposure to the eyes as a result of the use of traditional cosmetic Kohl in Asia, Africa and the Middle East has been a subject of recent debate to the scientific community. In continuation of our earlier work we therefore examine in the present study, the drug metabolic activity and the homeostasis of essential metal ions in the lenticular system of adult rats exposed to long term low level lead (lead acetate 0.1% w/v). The results of our investigation demonstrate that long term low level lead exposure impaired the phase I & phase II metabolic activity of the lenticular system when assessed by aminopyrine demethylase, benzo[a]pyrene hydroxylase, aniline hydroxylase and UDP glucuronyl transferase (UDPGT), glutathione S-transferase (GST), respectively. A more pronounced decrease (55%) in GST was noticed compared to UDPGT, aminopyrene demethylase, benzo[a]-pyrene hydroxylase and aniline hydroxylase (20–30%). Increased lead concentration in the lenticular system of the rats as monitored by atomic absorption spectroscopy resulted in a significant decrease (15–35%) in the levels of Ca, Cu, Zn and Fe, along with a progressive loss in body weight. Respective increase in blood lead level was also monitored parallel to increase in lenticular lead concentration at different time points in lead treated rats. The present investigation, therefore, demonstrates that long term low level lead exposure to rats results in a profound impairment in the homeostasis of essential metal ions, lenticular drug metabolizing enzymatic activity and significant loss in body weight when compared to untreated control rats. Whether such a decrease in these functions reflects an inhibition of protein synthesis at transcriptional/post transcriptional levels or gene regulation at molecular level remains to be established.