Significance of the Disulfide Bridge in the Structure and Stability of Metalloprotein Azurin.

Metalloproteins make up a class of proteins that incorporate metal ions into their structures, enabling them to perform essential functions in biological systems, such as catalysis and electron transport. Azurin is one such metalloprotein with copper cofactor, having a ß-barrel structure with exceptional thermal stability. The copper metal ion is coordinated at one end of the ß-barrel structure, and there is a disulfide bond at the opposite end. In this study, we explore the effect of this disulfide bond in the high thermal stability of azurin by analyzing both the native S-S bonded and S-S nonbonded (S-S open) forms using temperature replica exchange molecular dynamics (REMD). Similar to experimental observations, we find a 35 K decrease in denaturation temperature for S-S open azurin compared to that of the native holo form (420 K). As observed in the case of native holo azurin, the unfolding process of the S-S open form also started with disruptions of the α-helix. The free energy surfaces of the unfolding process revealed that the denaturation event of the S-S open form progresses through different sets of conformational ensembles. Subsequently, we compared the stabilities of individual ß-sheet strands of both the S-S bonded and the S-S nonbonded forms of azurin. Further, we examined the contacts between individual residues for the central structures from the free energy surfaces of the S-S nonbonded form. The microscopic origin of the lowering in the denaturation temperature is further supplemented by thermodynamic analysis.

MTM1 displays a new function in the regulation of nickel resistance in Saccharomyces cerevisiae.

Nickel (Ni) is an essential yet toxic trace element. Although a cofactor for many metalloenzymes, nickel function and metabolism is not fully explored in eukaryotes. Molecular biology and metallomic methods were utilized to explore the new physiological functions of nickel in Saccharomyces cerevisiae. Here we showed that MTM1 knockout cells displayed much stronger nickel tolerance than wild-type cells and mitochondrial accumulations of Ni and Fe of mtm1Δ cells dramatically decreased compared to wild-type cells when exposed to excess nickel. Superoxide dismutase 2 (Sod2p) activity in mtm1Δ cells was severely attenuated and restored through Ni supplementation in media or total protein. SOD2 mRNA level of mtm1Δ cells was significantly higher than that in the wild-type strain but was decreased by Ni supplementation. MTM1 knockout afforded resistance to excess nickel mediated through reactive oxygen species levels. Meanwhile, additional Ni showed no significant effect on the localization of Mtm1p. Our study reveals the MTM1 gene plays an important role in nickel homeostasis and identifies a novel function of nickel in promoting Sod2p activity in yeast cells.

Nitric oxide biosensor uncovers diminished ferrous iron-dependency of cultured cells adapted to physiological oxygen levels.

Iron is an essential metal for cellular metabolism and signaling, but it has adverse effects in excess. The physiological consequences of iron deficiency are well established, yet the relationship between iron supplementation and pericellular oxygen levels in cultured cells and their downstream effects on metalloproteins has been less explored. This study exploits the metalloprotein geNOps in cultured HEK293T epithelial and EA.hy926 endothelial cells to test the iron-dependency in cells adapted to standard room air (18 kPa O2) or physiological normoxia (5 kPa O2). We show that cells in culture require iron supplementation to activate the metalloprotein geNOps and demonstrate for the first time that cells adapted to physiological normoxia require significantly lower iron compared to cells adapted to hyperoxia. This study establishes an essential role for recapitulating oxygen levels in vivo and uncovers a previously unrecognized requirement for ferrous iron supplementation under standard cell culture conditions to achieve geNOps functionality.

In Vivo Assembly of Artificial Metalloenzymes and Application in Whole-Cell Biocatalysis*.

We report the supramolecular assembly of artificial metalloenzymes (ArMs), based on the Lactococcal multidrug resistance regulator (LmrR) and an exogeneous copper(II)-phenanthroline complex, in the cytoplasm of E. coli cells. A combination of catalysis, cell-fractionation, and inhibitor experiments, supplemented with in-cell solid-state NMR spectroscopy, confirmed the in-cell assembly. The ArM-containing whole cells were active in the catalysis of the enantioselective Friedel-Crafts alkylation of indoles and the Diels-Alder reaction of azachalcone with cyclopentadiene. Directed evolution resulted in two different improved mutants for both reactions, LmrR_A92E_M8D and LmrR_A92E_V15A, respectively. The whole-cell ArM system required no engineering of the microbial host, the protein scaffold, or the cofactor to achieve ArM assembly and catalysis. We consider this a key step towards integrating abiological catalysis with biosynthesis to generate a hybrid metabolism.

Site-Directed Mutagenesis at the Molybdenum Pterin Cofactor Site of the Human Aldehyde Oxidase: Interrogating the Kinetic Differences Between Human and Cynomolgus Monkey.

The estimation of the drug clearance by aldehyde oxidase (AO) has been complicated because of this enzyme's atypical kinetics and species and substrate specificity. Since human AO (hAO) and cynomolgus monkey AO (mAO) have a 95.1% sequence identity, cynomolgus monkeys may be the best species for estimating AO clearance in humans. Here, O6-benzylguanine (O6BG) and dantrolene were used under anaerobic conditions, as oxidative and reductive substrates of AO, respectively, to compare and contrast the kinetics of these two species through numerical modeling. Whereas dantrolene reduction followed the same linear kinetics in both species, the oxidation rate of O6BG was also linear in mAO and did not follow the already established biphasic kinetics of hAO. In an attempt to determine why hAO and mAO are kinetically distinct, we have altered the hAO V811 and F885 amino acids at the oxidation site adjacent to the molybdenum pterin cofactor to the corresponding alanine and leucine in mAO, respectively. Although some shift to a more monkey-like kinetics was observed for the V811A mutant, five more mutations around the AO cofactors still need to be investigated for this purpose. In comparing the oxidative and reductive rates of metabolism under anaerobic conditions, we have come to the conclusion that despite having similar rates of reduction (4-fold difference), the oxidation rate in mAO is more than 50-fold slower than hAO. This finding implies that the presence of nonlinearity in AO kinetics is dependent upon the degree of imbalance between the rates of oxidation and reduction in this enzyme. SIGNIFICANCE STATEMENT: Although they have as much as 95.1% sequence identity, human and cynomolgus monkey aldehyde oxidase are kinetically distinct. Therefore, monkeys may not be good estimators of drug clearance in humans.

Comparative Assessment of Seven Docking Programs on a Nonredundant Metalloprotein Subset of the PDBbind Refined.

Extensive usage of molecular docking for computer-aided drug discovery resulted in development of numerous programs with versatile scoring and posing algorithms. Selection of the docking program among these vast number of options is central to the outcome of drug discovery. To this end, comparative assessment studies of docking offer valuable insights into the selection of the optimal tool. Despite the availability of various docking assessment studies, the performance difference of docking programs has not been well addressed on metalloproteins which comprise a substantial portion of the human proteome and have been increasingly targeted for treatment of a wide variety of diseases. This study reports comparative assessment of seven docking programs on a diverse metalloprotein set which was compiled for this study. The refined set of the PDBbind (2017) was screened to gather 710 complexes with metal ion(s) closely located to the ligands (<4 Å). The redundancy was eliminated by clustering and overall 213 complexes were compiled as the nonredundant metalloprotein subset of the PDBbind refined. The scoring, ranking, and posing powers of seven noncommercial docking programs, namely, AutoDock4, AutoDock4Zn, AutoDock Vina, Quick Vina 2, LeDock, PLANTS, and UCSF DOCK6, were comprehensively evaluated on this nonredundant set. Results indicated that PLANTS (80%) followed by LeDock (77%), QVina (76%), and Vina (73%) had the most accurate posing algorithms while AutoDock4 (48%) and DOCK6 (56%) were the least successful in posing. Contrary to their moderate-to-high level of posing success, none of the programs was successful in scoring or ranking of the binding affinities (r2 ≈ 0). Screening power was further evaluated by using active-decoy ligand sets for a large compilation of metalloprotein targets. PLANTS stood out among other programs to be able to enrich the active ligand for every target, underscoring its robustness for screening of metalloprotein inhibitors. This study provides useful information for drug discovery studies targeting metalloproteins.

Intersection of Iron and Copper Metabolism in the Mammalian Intestine and Liver.

Iron and copper have similar physiochemical properties; thus, physiologically relevant interactions seem likely. Indeed, points of intersection between these two essential trace minerals have been recognized for many decades, but mechanistic details have been lacking. Investigations in recent years have revealed that copper may positively influence iron homeostasis, and also that iron may antagonize copper metabolism. For example, when body iron stores are low, copper is apparently redistributed to tissues important for regulating iron balance, including enterocytes of upper small bowel, the liver, and blood. Copper in enterocytes may positively influence iron transport, and hepatic copper may enhance biosynthesis of a circulating ferroxidase, ceruloplasmin, which potentiates iron release from stores. Moreover, many intestinal genes related to iron absorption are transactivated by a hypoxia-inducible transcription factor, hypoxia-inducible factor-2α (HIF2α), during iron deficiency. Interestingly, copper influences the DNA-binding activity of the HIF factors, thus further exemplifying how copper may modulate intestinal iron homeostasis. Copper may also alter the activity of the iron-regulatory hormone hepcidin. Furthermore, copper depletion has been noted in iron-loading disorders, such as hereditary hemochromatosis. Copper depletion may also be caused by high-dose iron supplementation, raising concerns particularly in pregnancy when iron supplementation is widely recommended. This review will cover the basic physiology of intestinal iron and copper absorption as well as the metabolism of these minerals in the liver. Also considered in detail will be current experimental work in this field, with a focus on molecular aspects of intestinal and hepatic iron-copper interplay and how this relates to various disease states. © 2018 American Physiological Society. Compr Physiol 8:1433-1461, 2018.

Evolutionary relationship between the cysteine and histidine rich domains (CHORDs) and Btk-type zinc fingers.

Summary: Cysteine and histidine rich domains (CHORDs), implicated in immunity and disease resistance signaling in plants, and in development and signal transduction in muscles and tumorigenesis in animals, are seen to have a cylindrical three-dimensional structure stabilized by the tetrahedral chelation of two zinc ions. CHORDs are regarded as novel zinc-binding domains and classified independently in Pfam and ECOD. Our sequence and structure analysis reveals that both the zinc-binding sites in CHORD possess a zinc ribbon fold and are likely related to each other by duplication and circular permutation. Interestingly, we also detect an evolutionary relationship between each of the CHORD zinc fingers (ZFs) and the Bruton's tyrosine kinase (Btk)-type ZF of the zinc ribbon fold group. Btk_ZF is found in eukaryotic Tec kinase family proteins that are also implicated in signaling pathways in several lineages of hematopoietic cells involved in mammalian immunity. Our analysis suggests that the unique zinc-stabilized fold seen only in the CHORD and Btk_ZFs likely emerged specifically in eukaryotes to mediate diverse signaling pathways. Supplementary information: Supplementary data are available at Bioinformatics online.

Effect of tea saponins on milk performance, milk fatty acids, and immune function in dairy cow.

This study investigated the effects of tea saponins (TSP) on milk performance, milk fatty acids, and blood immune function in dairy cows. A total of 20 early-lactation Holstein cows (days in milk = 66.4 ± 16.8 d; parity = 1.75 ± 0.91; and milk yield = 36.3 ± 7.32 kg/d; mean ± standard deviation) were randomly divided into 4 homogeneous treatment groups, with TSP added at 0, 20, 30, and 40 g/d per head, respectively. All cows had 2 wk of adaptation and 6 wk of treatments. Feed, milk, and blood were sampled and analyzed weekly. At the end of the experimental period (wk 6), the dry matter intake and yields of energy-corrected milk, milk, and milk protein, fat, and lactose in the cows fed TSP showed a quadratic response, with the lowest values in cows fed TSP at 40 g/d. The milk fat content of cows fed TSP increased linearly. Significant interactions for treatment by week were found in milk C16:1 cis-9 and C18:1 cis-9, with the highest values at wk 2, 3, and 4 in the cows fed TSP at 40 g/d. The levels declined quickly after 4 wk of feeding to values similar to those for other TSP treatments and the control at wk 5 and 6. Plasma malondialdehyde concentration decreased as the supplement level of TSP increased. The concentration of superoxide dismutase increased as the supplement level of TSP increased. The plasma concentration of tumor necrosis factor-α increased as the supplement level of TSP increased. In summary, this study showed that an intermediate dose of TSP (20 and 30 g/d) had no significant effect on feed intake, but the supplementation of 40 g/d TSP decreased feed intake, resulting in a lower milk yield. The energy-corrected milk of cows fed 40 g/d TSP declined at first but increased after 3 wk of feeding, indicating the potential adaptation to high doses of TSP supplements in dairy cows. The supplementation of TSP could reduce oxidative stress in cows and improve the immunity of dairy cows during 6 wk of feeding.

In vivo identification of potential uranium protein targets in zebrafish ovaries after chronic waterborne exposure.

Ecotoxicological studies have indicated the reprotoxicity of uranium (U) in zebrafish, but its molecular mechanisms remain unclear. Due to the non-covalent nature of U-protein complexes, canonical proteomics approaches are often not relevant as they usually use denaturating reagents or solvents. In this study, non-denaturating (ND) methods were used to obtain insight into the nature of U potential targets in ovaries of reproduced and non-reproduced zebrafish after 20 days of exposure to an environmentally relevant U concentration (20 µg L-1). After the ND sample preparation, 1-dimensional (SEC) and 2-dimensional (OGE × SEC) separations followed by ICP-sector-field MS measurements (U, P, Fe, Cu, and Zn) enabled the determination of chemical characteristics (MW, pI) of the metal-protein complexes. Phosphorus and U coelution confirmed the affinity of U for P-containing proteins. In addition, 2D separation allowed the discrimination of Fe-metalloproteins as potential U targets. Finally, 20 protein candidates for U complexation were identified after tryptic digestion conditions by LC-ESI FT MS and a database search. Potential U targets were mainly involved in three biological processes: oxidative stress regulation (SOD, GST), cytoskeleton structure (actin) and embryo early development (vtg, initiation factor).

Enrichment, Distribution of Vanadium-Containing Protein in Vanadium-Enriched Sea Cucumber Apostichopus japonicus and the Ameliorative Effect on Insulin Resistance.

Sea cucumbers are a potential source of natural organic vanadium that may improve insulin resistance. In this work, vanadium was accumulated rapidly in blood, body wall, and intestine by sea cucumber Apostichopus japonicus. Furthermore, water-soluble vanadium-containing proteins, the main form of the organic vanadium, were tentatively accumulated and isolated by a bioaccumulation experiment. It was also designed to evaluate the beneficial effect of vanadium-containing proteins (VCPs) from sea cucumber rich in vanadium on the development of hyperglycemia and insulin resistance in C57BL/6J mice fed with a high-fat high-sucrose diet (HFSD). HFSD mice treated with VCPs significantly decreased fasting blood glucose, serum insulin, and HOMA-IR values as compared to HFSD mice, respectively. Serum adiponectin, resistin, TNF-α, and leptin levels in insulin-resistant mice were dramatically reduced by a VCP supplement. These results show an ameliorative effect on insulin resistance by treatment with VCPs. Such compound seems to be a valuable therapy to achieve and/or maintain glycemic control and therapeutic agents in the treatment arsenal for insulin resistance and type 2 diabetes.

Ab initio coordination chemistry for nickel chelation motifs.

Chelation therapy is one of the most appreciated methods in the treatment of metal induced disease predisposition. Coordination chemistry provides a way to understand metal association in biological structures. In this work we have implemented coordination chemistry to study nickel coordination due to its high impact in industrial usage and thereby health consequences. This paper reports the analysis of nickel coordination from a large dataset of nickel bound structures and sequences. Coordination patterns predicted from the structures are reported in terms of donors, chelate length, coordination number, chelate geometry, structural fold and architecture. The analysis revealed histidine as the most favored residue in nickel coordination. The most common chelates identified were histidine based namely HHH, HDH, HEH and HH spaced at specific intervals. Though a maximum coordination number of 8 was observed, the presence of a single protein donor was noted to be mandatory in nickel coordination. The coordination pattern did not reveal any specific fold, nevertheless we report preferable residue spacing for specific structural architecture. In contrast, the analysis of nickel binding proteins from bacterial and archeal species revealed no common coordination patterns. Nickel binding sequence motifs were noted to be organism specific and protein class specific. As a result we identified about 13 signatures derived from 13 classes of nickel binding proteins. The specifications on nickel coordination presented in this paper will prove beneficial for developing better chelation strategies.

Cloning and functional validation of molybdenum cofactor sulfurase gene from Ammopiptanthus nanus.

KEY MESSAGE: The molybdenum cofactor sulfurase gene ( AnMCSU ) was cloned from xerophytic desert plant Ammopiptanthus nanus and validated for its function of tolerance toward abiotic stresses by heterologous expression in Arabidopsis thaliana. Molybdenum cofactor sulfurase participates in catalyzing biosynthesis of abscisic acid, which plays a crucial role in the response of plants to abiotic stresses. In this study, we cloned molybdenum cofactor sulfurase gene (AnMCSU) from a super-xerophytic desert plant, Ammopiptanthus nanus, by using rapid amplification of cDNA ends method. This gene has a total length of 2544 bp, with a 5'- and a 3'-untranslated region of 167 and 88 bp, and an open reading frame of 2289 bp, which encodes an 84.85 kDa protein of 762 amino acids. The putative amino acid sequence shares high homology and conserved amino acid residues crucial for the function of molybdenum cofactor sulfurases with other leguminous species. The encoded protein of the AnMCSU gene was located in the cytoplasm by transient expression in Nicotiana benthamiana. The result of real-time quantitative PCR showed that the expression of the AnMCSU gene was induced by heat, dehydration, high salt stresses, and ABA induction, and inhibited by cold stress. The heterologous expression of the AnMCSU gene significantly enhanced the tolerance of Arabidopsis thaliana to high salt, cold, osmotic stresses, and abscisic acid induction. All these results suggest that the AnMCSU gene might play a crucial role in the adaptation of A. nanus to abiotic stress and has potential to be applied to transgenic improvement of commercial crops.

Copper and copper proteins in Parkinson's disease.

Copper is a transition metal that has been linked to pathological and beneficial effects in neurodegenerative diseases. In Parkinson's disease, free copper is related to increased oxidative stress, alpha-synuclein oligomerization, and Lewy body formation. Decreased copper along with increased iron has been found in substantia nigra and caudate nucleus of Parkinson's disease patients. Copper influences iron content in the brain through ferroxidase ceruloplasmin activity; therefore decreased protein-bound copper in brain may enhance iron accumulation and the associated oxidative stress. The function of other copper-binding proteins such as Cu/Zn-SOD and metallothioneins is also beneficial to prevent neurodegeneration. Copper may regulate neurotransmission since it is released after neuronal stimulus and the metal is able to modulate the function of NMDA and GABA A receptors. Some of the proteins involved in copper transport are the transporters CTR1, ATP7A, and ATP7B and the chaperone ATOX1. There is limited information about the role of those biomolecules in the pathophysiology of Parkinson's disease; for instance, it is known that CTR1 is decreased in substantia nigra pars compacta in Parkinson's disease and that a mutation in ATP7B could be associated with Parkinson's disease. Regarding copper-related therapies, copper supplementation can represent a plausible alternative, while copper chelation may even aggravate the pathology.

Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2.

Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b5, and NADH-dependent cytochrome b5 reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar Kcat and Vmax values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.

Phosphorus binding sites in proteins: structural preorganization and coordination.

Phosphorus is a ubiquitous element of the cell, which is found throughout numerous key molecules related to cell structure, energy and information storage and transfer, and a diverse array of other cellular functions. In this work, we adopt an approach often used for characterizing metal binding and selectivity of metalloproteins in terms of interactions in a first shell (direct residue interactions with the metal) and a second shell (residue interactions with first shell residues) and use it to characterize binding of phosphorus compounds. Similar analyses of binding have previously been limited to individual structures that bind to phosphate groups; here, we investigate a total of 8307 structures obtained from the RCSB Protein Data Bank (PDB). An analysis of the binding site amino acid propensities reveals very characteristic first shell residue distributions, which are found to be influenced by the characteristics of the phosphorus compound and by the presence of cobound cations. The second shell, which supports the coordinating residues in the first shell, is found to consist mainly of protein backbone groups. Our results show how the second shell residue distribution is dictated mainly by the first shell of the binding site, especially by cobound cations and that the main function of the second shell is to stabilize the first shell residues.

Iron-sulphur clusters, their biosynthesis, and biological functions in protozoan parasites.

Fe-S clusters are ensembles of sulphide-linked di-, tri-, and tetra-iron centres of a variety of metalloproteins that play important roles in reduction and oxidation of mitochondrial electron transport, energy metabolism, regulation of gene expression, cell survival, nitrogen fixation, and numerous other metabolic pathways. The Fe-S clusters are assembled by one of four distinct systems: NIF, SUF, ISC, and CIA machineries. The ISC machinery is a house-keeping system conserved widely from prokaryotes to higher eukaryotes, while the other systems are present in a limited range of organisms and play supplementary roles under certain conditions such as stress. Fe-S cluster-containing proteins and the components required for Fe-S cluster biosynthesis are modulated under stress conditions, drug resistance, and developmental stages. It is also known that a defect in Fe-S proteins and Fe-S cluster biogenesis leads to many genetic disorders in humans, which indicates the importance of the systems. In this review, we describe the biological and physiological significance of Fe-S cluster-containing proteins and their biosynthesis in parasitic protozoa including Plasmodium, Trypanosoma, Leishmania, Giardia, Trichomonas, Entamoeba, Cryptosporidium, Blastocystis, and microsporidia. We also discuss the roles of Fe-S cluster biosynthesis in proliferation, differentiation, and stress response in protozoan parasites. The heterogeneity of the systems and the compartmentalization of Fe-S cluster biogenesis in the protozoan parasites likely reflect divergent evolution under highly diverse environmental niches, and influence their parasitic lifestyle and pathogenesis. Finally, both Fe-S cluster-containing proteins and their biosynthetic machinery in protozoan parasites are remarkably different from those in their mammalian hosts. Thus, they represent a rational target for the development of novel chemotherapeutic and prophylactic agents against protozoan infections.

Clawing back: broadening the notion of metal chelators in medicine.

The traditional notion of chelation therapy is the administration of a chemical agent to remove metals from the body. But formation of a metal-chelate can have biological ramifications that are much broader than metal elimination. Exploring these other possibilities could lead to pharmacological interventions that alter the concentration, distribution, or reactivity of metals in targeted ways for therapeutic benefit. This review highlights recent examples that showcase four general strategies of using principles of metal chelation in medicinal contexts beyond the traditional notion of chelation therapy. These strategies include altering metal biodistribution, inhibiting specific metalloenzymes associated with disease, enhancing the reactivity of a metal complex to promote cytotoxicity, and conversely, passivating the reactivity of metals by site-activated chelation to prevent cytotoxicity.

Zinc and human disease.

The vast knowledge of the physiologic functions of zinc in at least 3000 proteins and the recent recognition of fundamental regulatory functions of zinc(II) ions released from cells or within cells links this nutritionally essential metal ion to numerous diseases. However, this knowledge so far has had remarkably limited impact on diagnosing, preventing, and treating human diseases. One major roadblock is a lack of suitable biomarkers that would detect changes in cellular zinc metabolism and relate them to specific disease outcomes. It is not only the right amount of zinc in the diet that maintains health. At least as important is the proper functioning of the dozens of proteins that control cellular zinc homeostasis, regulate intracellular traffic of zinc between the cytosol and vesicles/organelles, and determine the fluctuations of signaling zinc(II) ions. Cellular zinc deficiencies or overloads, a term referring to zinc concentrations exceeding the cellular zinc buffering capacity, compromise the redox balance. Zinc supplementation may not readily remedy zinc deficiency if other factors limit the capability of a cell to control zinc. The role of zinc in human diseases requires a general understanding of the wide spectrum of functions of zinc, how zinc is controlled, how it interacts with the metabolism of other metal ions, in particular copper and iron, and how perturbation of specific zinc-dependent molecular processes causes disease and influences the progression of disease.

Molecular cloning, sequencing, and expression analysis of cDNA encoding metalloprotein II (MP II) induced by single and combined metals (Cu(II), Cd(II)) in polychaeta Perinereis aibuhitensis.

We amplified and analyzed the complete cDNA of metalloprotein II (MP II) from the somatic muscle of the polychaete Perinereis aibuhitensis, the full length cDNA is 904 bp encoding 119 amino acids. The MP II cDNA sequence was subjected to BLAST searching in NCBI and was found to share high homology with hemerythrin of other worms. MP II expression of P. aibuhitensis exposed to single and combined metals (Cu(II), Cd(II)) was analyzed using real time-PCR. MP II mRNA expression increased at the start of Cu(II) exposure, then decreased and finally return to the normal level. Expression pattern of MP II under Cd(II) exposure was time- and dose-dependent. MP II expression induced by a combination of Cd(II) and Cu(II) was similar to that induced by Cd(II) alone.