The role of metallothionein interactions with other proteins.

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Proteomics

The role of metallothionein interactions with other proteins

Marta Zalewska1, Jagoda Trefon2, Halina Milnerowicz1

1

Department of Biomedical and Environmental Analysis, Faculty of Pharmacy, Wroclaw

Medical University, Poland 2

Students Scientific Association, Department of Biomedical and Environmental Analysis,

Faculty of Pharmacy, Wroclaw Medical University, Poland

Keywords: Metallothionein / Protein-protein interaction / Ferritin / Transcription factors / Low density lipoprotein receptor / Glutathione / Zn transfer / P53

Abbreviations: ALAD, δ-aminolevulinic dehydratase; ER, estrogen receptor; Ft, ferritin; GSH, glutathione reduced; GSSG, glutathione oxidized; LDLR, low density lipoprotein receptor; MMP-9, matrix metalloproteinase-9; MT, metallothionein; NF-κB, nuclear factorκB; Sp1, specificity protein 1; TFIIIA, transcription factor IIIA; TTK, tramtrack

Correspondance: Marta Zalewska, Department of Biomedical and Environmental Analysis, Faculty of Pharmacy, Wroclaw Medical University, Poland E-mail: [email protected] Tel: 48 71 7840173

Received: 09-Nov-2013; Revised: 20-Feb-2014; Accepted: 06-Mar-2014 This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/pmic.201300496. This article is protected by copyright. All rights reserved.


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Abstract Metallothionein (MT) is a protein involved in numerous key processes, and the most important include zinc ion homeostasis, detoxification of heavy metals, and protection against oxidative stress. MT by interaction with other proteins fulfill its function, resulting in different effects in the body. Interaction of MT with Ft, that cause a redox reaction, resulting in the reduction of Fe3+ stored in Ft and a release of harmful Fe2+ was observed. Referring to the redox function of MT, has been shown that the pair of GSH/GSSG modulates transfer of Zn between MT and Zn-binding proteins. Furthermore, it was shown that GSSG, in the presence of GSH, interacts directly with MT. Apo-MT can retrieve Zn from the transcription factors or Zn-containing enzymes. Apo-MT by taking Zn can deactivate metal-dependent enzymes while Zn-MT has the opposite effect. As the effect of MT interaction with LDL receptors - megalin and LRP1, the uptake of Cd-MT occurs and results in the disruption of many functions of proximal tubules. MT is involved in numerous processes and many of them are regulated by proteinprotein interactions. Possibly in the future MT will become a therapeutic agent, which will result

in

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This article is protected by copyright. All rights reserved.

field

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medicine.


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1 Introduction Metallothioneins (MTs) belong to a family of small molecular weight (approx. 7 kDa) proteins that was called MT due to a number of sulfhydryl groups and the presence of associated metals [1]. MT was first isolated from horse kidney cortex, as a protein binding large amounts of cadmium [2]. MTs are involved in the transport, storage, and regulation of essential metal ions concentration, detoxification of heavy metals, anti-stress function and oxidative stress [3-10]. In this context, it is interesting to know, how Cu, Zn - metals essential for homeostasis - are transported by MT to other proteins, as well as to know which proteins take part in this process. Therefore, we would like to summarize proteins, which interact with MT and thus to know signaling pathways and processes, in which MT participates. Do the proteins recognize the apo-MT or its complex with the metals? The knowledge of how MT interacts with metals, and what is the mechanism of its interactions with other macromolecules, is an important aspect of cognition. 2 Structure, expression and function of metallothionein Human MTs are encoded by 17 genes located on chromosome 16q13 [9, 11]. They consist of 61-68 amino acid residues, wherein the cysteine content reaches 25-30% of all amino acids and they have no aromatic amino acids [1, 12, 13]. MTs are divided into 4 isoforms. MT-2, MT-3 and MT-4 are encoded by a single gene, but the MT-1 has many subtypes (MT-1A,-B,-E,-F,-G,-H,-M,-X) [11, 14]. Differences between isoforms arise mainly from changes in the amino acid sequence [15, 16]. MT-1 and MT-2 are similar; the MT-3 contains additional threonine-element in the N-terminal part and acidic hexapeptide in the Cterminal region (Fig. 1). In addition, MT-3 contains the Cys(6)-Pro-Cys-Pro(9) motif, which does not exist in other MTs. These differences between isoforms may be crucial for the role



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of each of them and their divers presence in tissues [16]. The induction of MT synthesis at the transcriptional level is regulated by specific regulatory sequences present within the promoter. These include: metal response element, which is activated by the metal-responsive transcription factor (MTF-1), glucocorticoid response element, signal transducer and activator of transcription and antioxidant response element activated by the redox status [15]. MT-1 and MT-2 are present in any type of tissue, its synthesis is induced by many factors such as metal ions, glucocorticoids, cytokines and oxidative stress [1, 11, 12]. Therefore, these two isoforms are often described together as MT-1/2. MT-3 was detected mainly in the central nervous system (CNS) (neurons, astrocytes in the cortex, hippocampus), but also in the heart, kidneys and reproductive organs. MT-4 can be found in the cells of squamous epithelium (mouth, upper gastrointestinal track, skin) [1, 9, 16]. The presence of MTs in all types of mammalian cells suggests that they play an important role as an intracellular protein as well as extracellularly active molecules [9]. It is known that the extracellular MT can be detected in the various body fluids: plasma, urine, pancreatic and amniotic fluid or milk [7, 8, 17, 18]. Inside cells, MTs have been detected in the cytoplasm, mitochondria, nucleus and in the lysosome. It was found that MT, according to the life cycle of the cell and oxidative stress, can enter the cell nucleus, beside it is present in the cytoplasm and mitochondria [6, 19]. MTs bind monovalent metals of 11th group and divalent of 12th group. The interaction of metal-cysteine dominates secondary structure of the protein [1]. MTs are composed of two domains: α and β, each of which has a different ability to bind metals. Beta domain by the 9 cysteine residues binds 3 Cd2+ ions or Zn2+, or 6 Cu+ ions. Alpha domain binds 4 Zn2+ or Cd2+, or 6 Cu+ ions by 11 cysteine residues [1]. It is known that in the physiological state, MTs are associated mostly with the Zn2+ ions, but may be dissociated and replaced by another metal that has a higher affinity for MT e.g. Cd, Hg and Cu [9]. The effect of metal-



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binding by MT is the reduction of pKa of the cysteines of up to 6 orders of magnitude. In the metal-bound form, MTs are more stable, and the value of the dissociation constant for the clusters MTs is low (e.g. for Zn7-MT Kdys= 3.2×10-13 mol/l, pH=7.4) [20]. MTs are also present in the cell as apothioneins (apo-MT, thionein) that are not associated with the metals. Apo-MT is more susceptible to proteolytic enzymes action [21] and their quantity in the cell is less than MT. Apo-MT tends to be disordered, but upon binding of metal ions, it takes up three dimensional structures. After filling four places in the α domain, metals bind to the β domain. The metal ions bind weaker to the β domain and are easier released from it. Recently, it has been demonstrated that human MT-1A binds seven ions of Zn2+ or Cd2+ and then it binds eighth structurally significant ion - Cd2+, which leads to the creation of supermetalated form Cd8-βα-rhMT-1a, with a structure forming a single domain [13]. Creation of Cd8-MT results in the loss of the known α and β domains (Fig. 2). Possible consequences of this additional metalation of MT remain to be discussed, due to the role of MT in the metal homeostasis, as well as changes in the protein structure so important in the recognition of other proteins [13, 22]. MTs because of their active sulfhydryl groups (-SH) in molecule may interact with a number of substances, including proteins. Numerous studies on the interaction between MT with other proteins were conducted, and the results of these experiments are sometimes surprising [16]. The -SH groups of MT bind Zn with high affinity and can pass it to the other Zn binding proteins and receive it from them [23]. MT therefore plays an important role as a donor and acceptor of metals ions, to and from a variety of enzymes and transcription factors. However, if the processes of Zn exchange require the direct interaction between the proteins, is still in doubt, and it needs further studies [16]. The role of MTs as the main Zn2+ buffering proteins in the organism is emerging [19, 24]. Transfer of zinc from MT to Zn-binding proteins may contribute to the regulation of gene expression, affect apoptosis, proliferation or



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cell differentiation [9, 25, 26]. It should be noted that most of these functions MT performs by interacting with other proteins, as it will be explained in the following sections. 3 Dangerous liaison of MT with ferritin Iron is essential for life, it plays an important role in various biological processes. However, the excess of iron is toxic, particularly Fe2+. It reacts with oxygen and reactive oxygen species (ROS) are formed. ROS are very strong oxidants, and cause damage to the cells. Organisms have developed some mechanisms to isolate iron in a non-toxic form. One such is ferritin (Ft), the main protein responsible for the iron storage [27]. Ft stores iron in the Fe3+ non-toxic form, and thus plays a vital role in protecting cells against the harmful Fe2+ [27]. Control of iron metabolism has paramount importance to aerobic cells, as free iron atoms together with superoxide and hydrogen peroxides are deadly combination, capable of producing hydroxyl radicals in the Haber-Weiss reaction catalyzed by iron ions. The hydroxyl radical is highly reactive, causes lipid peroxidation, DNA strand breaks, leading to chronic degenerative and vascular diseases [28]. Ft expression increases both transcriptionally and post-transcriptionally in diseases associated with inflammation and oxidative damage [27]. Ft is present predominantly in the cytoplasm and the main place of Fe storage is the liver, bone marrow, spleen and muscles [29]. Its molecular weight is about 480-500 kDa. This protein is formed in the shape of a hollow sphere and is capable of storing up to 4,500 atoms of iron Fe3+ as an inorganic compound, namely ferrihydrite (Fe2O3) [28]. Ft is also present in small amounts in the plasma, where its concentration ranges from less than 10 μg/l to 1-10 mg/l, depending on the clinical status. The role of plasma Ft is not clear, but its concentration there, is an important biological marker of the iron content in the organism and it is therefore often determined for this purpose [28]. Some research have shown that Ft is also capable of binding other metals e.g. Al, Cd, Zn, and Be in vitro [27, 30, 31]. Moreover, in recent publications have been



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shown that Ft present in the plasma contains a lot of aluminum atoms, and zinc with high levels of Al/Fe and Al/Zn. Therefore, the hypothesis was put forward on the function of Ft, that it controls potentially toxic metals, such as Fe and Al [28]. Ft plays an important role in the Fe metabolism. It is both its reservoir and plays a role in the detoxification of this metal [29]. Fe deficiency increases absorption of Cd, Pb, and Al. Pb replaces Zn on heme enzymes and Cd replaces Zn on MT [32]. Healthy organisms can safely release iron from Ft to activate other Fe-proteins. In conditions associated with oxidative damage, this protective pathway fails and iron reacts to form ROS. Therefore, it is essential to understand the nature of molecule/s involved in the controlled release of iron from Ft to develop treatments for oxidative damage diseases. Cys residues of MT are capable of donating electrons to reduce the Fe3+ to Fe2+ in mineral core of Ft and facilitate iron release. Concomitantly, the MT thiolates become fully oxidized to disulfides with the release of Zn2+. The resulting Fe2+ did not bind to the oxidized MT. Excess of iron is toxic and Fe2+ reacts with oxygen to produce ROS, which are extremely powerful oxidizing agents capable of causing cell damage [27]. The dissociation constant and the kinetics of binding MT to Ft is unknown. The most likely model, of iron release from Ft, is that the reduction of Fe3+ ions is due to the mobilization of Fe2+. In the case of small reductors, which are able to pass through the channels of Ft, the reduction takes place inside the protein. In the case of the larger particles, reduction is by the electrons tunneling [27]. It is known, that MTs are able to catalyze the redox reaction and they have binding sites for metals. In the model system, MTs-Zn complexes of mammalian MT-1, MT-2 and MT-3 have been identified to interact with Ft derived from horse spleen. Certain forms of apo-MT are able to coordinate Fe2+, however MT is present in the organism bound in a complex with Zn and/or Cu. Further investigation



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revealed that Fe2+ binds to the Zn-MT-2 in the presence of nitric oxide, that is a factor inducing the release of Zn2+ from the complex of Zn-MT [27]. Firstly, binding ability of Fe2+ and Fe3+ by Zn-MTs isoforms (MT-1, MT-2 and MT-3) was tested. Fe2+ reaction with Zn-MTs has shown that Fe2+ is unable to substitute Zn2+ and binds with MTs. This is consistent with the affinity of the metal ion binding to thiolate groups. The ability of Zn-MT complexes to reduce Fe3+ was verified and it was concluded that the cysteine residues may be electron donors for Fe3+. During the oxidation reaction, Zn2+ was released from the Zn-MTs complex, but the resulting Fe2+ ions do not associated with MT [27]. For determining the amount of Fe2+ released from Ft, ferrozine (FZ) as an external Fe2+ chelating agent, was used. The study of interaction between Ft and Zn-MT-1, Zn-MT-2, Zn-MT-3 were performed under anaerobic conditions using FZ and the total amount of iron released in 24 hours was calculated [27]. Considering the total initial concentration of iron in Ft, it was found that 55.6%, 23.9% and 55.9% of the iron was removed from the Ft by the MT-1, MT-2, MT-3 respectively. It was found that the interaction of Ft-Zn-MT causes the complete oxidation of the 20 Cys of MTs. The use of anaerobic conditions excluded the possibility of MT oxidation by oxygen. Additionally, to exclude distortions of the quaternary structure of Ft and of the spontaneous release of iron during the interaction of Zn-MT-Ft, polyacrylamide gel electrophoresis was performed. No difference was detected in the mobility of Ft [27]. The reaction was also performed with Tiron (1,2dihydroxy-3,5-benzenedisulfonate), which has a high affinity for Fe3+. Ft incubation with Tiron in the presence of Zn-MT showed no absorption band in UV-vis spectrum. This reflects the lack of Fe3+ release from Ft in the presence of Zn-MT. In conclusion, the complexes of Zn-MT may be electron donors for the reduction of Fe3+, and may facilitate the release of iron. At the same time, MT thiolates are oxidized to disulfides, releasing Zn2+. The lowest yield of MT-2 for iron release from Ft reveals the



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existence of a correlation between the reduction process and the structure of MT. This may be the subject of further studies [27]. Probably, the redox reaction that occurs during that interaction causes transfer of electrons through Ft, as complexes of Zn-MT are too large to pass through the Ft channels [27]. Interaction of these two proteins may be potentially harmful process because the release of Fe2+ ions may cause toxicity in the cells. This process can be particularly important in the brain, where disorders of metals metabolism are the cause of neurological disorders. During redox reaction, Fe2+ ions are formed and diffuse through the channels of Ft to the intracellular environment, where they may participate in the reactions producing free radicals, that is, undesirable process, bringing harmful effects [27].

4 Role of glutathione in the redox cycle of metallothionein Glutathione, γ-glutamylocysteinyloglycine, occurs in two forms: GSSG – oxidized and GSH - reduced. In the cells, the reduced form of glutathione dominates, about 90% is present in the cytosol, 10% in the mitochondria, while a small percentage in the endoplasmic reticulum. GSH synthesis takes place in the liver, where it is present in the highest concentration. Antioxidant defense is one of the most important functions of glutathione. It maintains redox balance in cells and counteracts the effects of oxidative stress. It removes free radicals and protects against toxic effects of many substances by the reaction with xenobiotics or their metabolites [33, 34]. MT cooperates with GSH in maintaining the cellular redox state. It may function as a secondary antioxidant in the cellular protection system that exerts its antioxidant action only under extreme conditions of oxidative stress. When GSH synthesis was blocked, the enhanced role of MTs was found. Pre-induction of MT synthesis leads to the significant inhibition of oxidative-stress induced lipid peroxidation [26].



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MTs binding to zinc is thermodynamically stable and physiologically important [35]. Each Zn atom is linked tetrahedrally with four cysteines of MT, to form clusters of thiol groups with zinc between 7 Zn atoms and 20 cysteine residues [36]. The presence of such a bond between the zinc and sulfur allows the redox reaction to take place, where the SH groups are oxidized, at the same time, the zinc is released. In vitro studies showed that the GSH/GSSG redox couple modulates zinc transfer between MT and zinc-binding proteins. On this basis, it is suggested that redox state acts as signal and motive power of zinc distribution from MT – cell reservoir, at the time of it demand [36, 37]. Zn associated with MT under physiological conditions, is released during the oxidation of SH-groups in MT molecule. MT oxidation leads to the formation of different intermolecular or intramolecular disulfide bonds or to the formation of thionin (all the 7 Zn atoms are removed and 20 thiol groups are oxidized) [36, 38]. While the oxidized environment is reduced, for example the concentration ratio of GSH/GSSG is increased, then MT-disulfide or thionein will be reduced to MT-thiol (thiol groups bound or not with zinc) [35, 38] (Fig. 3). The process of reduction is accelerated in the presence of a catalyst selenium. Reduction of oxidized MT restores its ability to bind zinc, and thus complements its reservoir in the form of MT [35]. These processes establish a redox cycle of MT, which plays a role in metal homeostasis, protection against oxidative stress and detoxification of toxic metals [35]. It should be noted that zinc release from MT is modulated by both the GSH and GSSG. GSH inhibits the release of zinc in the absence of GSSG, indicating that MT is stabilized at a relatively high concentration of GSH. The presence of GSSG causes the release of zinc. Rate of release depends linearly on the amount of GSSG - the higher oxidation, the more efficient release of Zn from MT [35]. Mobilization of Zn from MT by the reactions of oxidation can be the main path of Zn release into cells, when Zn is needed in the antioxidant defense system



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[35]. It is worth to note, that the release of metal from MT in the presence of GSSG, under conditions of oxidative stress can cause interference in its metabolism. This may result in the progression of the diseases such as Alzheimer or Parkinson, where oxidative stress occurs in the tissues affected by the disease. Effect of the increased amount of free metal must be carefully tested before starting the pharmacological treatment of CNS disorders by MT [39]. Jiang et al studied the role of glutathione during the release of zinc in the presence of Zn acceptor - sorbitol dehydrogenase (SDH) [40]. It has been shown, that only one of seven Zn ions is transferred from the MT to this enzyme in the absence of glutathione, and that Zn transfer is regulated by interaction between the MT and the GSH/GSSG. GSSG, which causes the release of Zn from MT, accelerates the activation of the apo-SDH [40]. In the presence of GSSG, GSH stimulates the exchange of zinc in the apo-SDH/MT/GSSG system, showing the MT control during the transfer of Zn by the redox couple of GSH/GSSG [40]. Three possible molecular mechanisms are assumed, in which GSH and GSSG can: bind to SDH and affect its activity; control the amount of free zinc ions released from MT; bind to MT and affect its conformation and the binding of Zn. The most probable is the last assumption, because molecular modeling studies have shown that GSH binds to the β domain niche and its thiol group displaces ligand from Cys26 of MT. Then, exposed thiol groups can change into disulfides during the exchange with GSSG, which explains why GSH stimulates the reaction between MT and GSSG. The interaction between GSH and MT can protect MT against the release of Zn. Distribution of Zn seems to depend on the interaction between glutathione and MT and GSH/GSSG cellular redox state [40]. The quantity of released Zn from MT depends on the state of cellular GSH/GSSG [35]. The transfer of Zn in the cell is controlled and the amount of free Zn in the cell is limited. Intracellular Zn is a component of the cytoskeleton structure, transcription factors and metalloenzymes. Summarizing, MT



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transmits zinc in vivo to other proteins, and some of these processes require a direct interaction between the MT and Zn acceptor proteins [35].

5 Metallothionein as a metals transporter Owing to the significant number of cysteine residues in the sequence (~30% of all amino acids), the functions of MT have been postulated to include toxic metal detoxification, protection against oxidative stress and regulation of the metal ion homeostasis of essential Zn and Cu – MT as a metallochaperone. Some of these functions are based on MT homeostatic buffering role, i.e. maintaining the free intracellular concentration of Zn2+ at a low level, while others depend on supplying Zn2+ to various protein targets, e.g. Zn2+-dependent enzymes, zinc-finger-dependent transcription factors and to pre-synaptic vesicles in zinccontaining neurons. Fundamental issues in zinc biology are how proteins control the concentration of free Zn2+ ions and how tightly they bind them. With the sequencing of genome, predictions were made that at least 10% (about 2800 gene products) of the human genome encodes for zinc-binding proteins. These include 397 hydrolases; 302 ligases; 167 transferases; 43 oxidoreductases and 24 lyases/isomerases; 957 transcription factors; 221 signaling proteins; 141 transport/storage proteins; 53 proteins with structural metal sites; 19 proteins involved in DNA repair, replication, and translation; 427 zinc finger proteins of unknown function; and 456 proteins of unknown function [24]. Almost all of the intracellular Zn2+ is bound to the metal-binding proteins, thereby limiting the amount of free Zn2+ in the cytoplasm. MTs are the family of proteins, that shuttles metal ions to specific intracellular locations, and therefore metalloenzymes bind these metals and use them as cofactors to carry out essential biochemical reactions. In Zn-MT complex, the protein plays a role in the biological function of zinc, a paradigm quite different from that in most other zinc proteins, where zinc plays a role in the biological function of the protein. The tight binding of zinc to



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MT raises questions of how it is released and whether or not the release is controlled. If the protein functions as a metal delivery carrier, there must be a biological mechanism to facilitate metal release. One means to achieve, this would be a labilization of the metals by interaction of MT with an appropriate cellular ligand. It is thought to proceed through ligandexchange processes involving direct molecular contact between the reactants. While the molecular mechanism for metal release is unknown, it does appear to be controlled by protein-protein interaction. Studies with As-MT showed, that the transfer of As3+ between the full-MT protein and its domain occurs by protein-protein interactions, not spontaneously by the dissociation-association [41]. As3+ bound to human MT-1 (hMT-1) is stable at pH 7 and metal translocates via protein–protein interaction to other protein. The transfer of As3+ under conditions in which the free As3+ ion is not stable (pH 7), provides evidence that Cd2+ and Zn2+ transfer may also take place through protein-protein interactions and that partially metallated Cd-MT and Zn-MT would be stable. As-MT species are stable at pH 7, and metal translocation takes place from the As6-β α-hMT-1 directly to the isolated apo-α and apo-β fragments resulting in stable, partially metallated species. Therefore, it is very interesting, with which proteins MT interacts and transfers metals.

5.1 Regulation of gene expression by MT Zinc is a part of many proteins. Among Zn-binding proteins, there are those which contain zinc fingers - domains consisting essentially of cysteine and histidine residues that coordinate Zn2+ atom tetrahedrally [16, 42]. Their function is to identify and control DNA transcription processes [16]. The addition of chelating agents to transcription factors causes Zn release, which is associated with reversible loss of DNA-binding function and disorder regulation of transcription process in vitro [43]. Transcription factors such as p53 protein, nuclear factor-κB (NF-κB), specificity protein 1 (Sp1), transcription factor IIIA (TFIIIA),



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estrogen receptor (ER), Gal4 and tramtrack (TTK) can interact with MT and change their function.

5.1.1 Protein p53 A tumor suppressor, protein p53 (also known as tumor protein, TP53) is a transcription factor whose primary role is to maintain the integrity of genome, regulation of the cell cycle and apoptosis [44]. The molecule of the human p53 consists of 393 amino acids and its molecular weight is 53 kDa [45]. In human cancers, mutations in this protein are present, typically in a central DNA binding domain, which is responsible for the sequence-dependent binding of p53 to DNA [46]. In a latent form, which is present in all normal cells, p53 is associated with the murine double minute 2 (MDM2) and it has a low affinity for specific DNA sequences, however, under the influence of stressors, it passes into an active form, which has a high DNA binding capacity [45]. The central domain of p53 has a zinc finger motif, wherein one Zn atom is tetrahedrally coordinated to three cysteines and one histidine. The zinc ion is required for the maintenance of normal p53 protein conformation, stability and DNA binding capacity [46]. P53 plays an important role in preventing the growth of cancer cells at different points, what explains why it has a large influence on carcinogenesis. Activation of p53 induces a response of cells including differentiation and aging, DNA repair, inhibition of angiogenesis, cell cycle arrest and apoptosis [47]. The main feature that can be attributed to p53 is the regulation of gene expression. It has been shown, that the process of apoptosis can be regulated by transactivation, therefore the direct interaction of the p53 on genes or through interactions with other proteins of the cell. Active p53 regulates the transcription of p21 gene [48]. P21 protein mediates cells growth inhibition that is induced by p53 e.g. in response to



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DNA damage. P21 inhibits the activity of cyclin-dependent kinases (CDKs). CDK inhibitor thus regulates the cell cycle by modulating the activity of cyclin/CDK complexes in response to various extra- and intra-cellular signals [49]. It was demonstrated that MT can alter the DNA binding activity of p53 by regulating the availability of Zn [44]. Zn stabilizes the DNA binding domain of p53 protein and is an important cofactor for the DNA binding activity of p53. Most evidences suggest that metal chelators can remove Zn from p53, and transform it into an inactive form with no ability to bind DNA [50]. High correlation between the expression of p53 and MT was shown, since high expression of MT in tumors is associated with the presence of mutant p53 and a higher degree of tumor. As previously mentioned, the Zn ions are necessary for maintaining conformation of p53 protein, its stabilization and maintaining its affinity for specific DNA sequence in the transcriptional activity. Thionein can remove zinc from zinc fingers what results in a decrease of p53 transcriptional activity [48, 50]. The complex between the MT and p53 was detected in the epithelial cells of breast cancer. In the study conducted by Ostrakhovitch et al., epithelial human breast cells line MN1, which contains active p53 and MDD2 cell line, a variant derived from the Michigan Cancer Foundation-7 (MCF-7) cells after transfection with inactive p53, were used [44]. In MN-1 cells, treated with Zn, a rise in the level of p21 protein was observed, which was primarily due to the stabilization and accumulation of functionally active p53. In addition, there was an increase of MT concentration after the addition of Zn. It should be noted that the initial concentration of MT and p21 were low, in contrast to MDD2 cells with inactive p53, hence the addition of Zn did not cause a significant change in the level of MT or p21, which suggests a relationship between the expression of p53 and MT in cells. To confirm the interaction between p53 and MT, co-precipitation was performed. Anti-MT and anti-p53 antibody were used and simultaneous precipitation of MT and p53 was observed in both



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cases and in both cell lines (MN-1 and MDD2). P53 and MT form complexes in the epithelial cells of breast cancer, regardless of the p53 status (active, inactive). To determine the type of this interaction, GST pull-down test in vitro, using Zn, Cd-MT and apo-MT, was performed. It has been shown that only the apo-MT interacts directly with p53 [44]. Presented studies indicate the existence of MT-p53 complexes in cells. Moreover, the apo-MT can retrive Zn from metalloproteins, including p53, through competition about Zn with these proteins. Thionein can disrupt the DNA binding capacity by chelation of Zn. It was also shown that only the apo-MT interacts with p53. The complex can be formed by the interaction between the sulfhydryl groups of apo-MT and zinc ion of p53 which would prevent the binding of p53 to DNA and at the same time inhibit the degradation of apo-MT, which in the metal free form is less stable. Further studies conducted by Xia et al. were carried out using surface plasmon resonance (SPR) method [46]. It has been shown that there is an interaction of apo-MT with recombinant p53, whereas it has not been observed with MT. Results were similar as in the previous studies. Additionally, the binding ability of p53 to DNA upon interaction with apoMT was tested. It was found that p53 protein had lost its activity of binding to DNA [46]. In summary, numerous studies indicate that apothionein can take zinc from the zinc finger transcription factor that is p53, with the loss of its DNA binding activity [15]. It was suggested that apo-MT may be involved in the regulation of the activity of p53 protein, and thus, in the regulation of gene transcription [44].



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5.1.2 Nuclear transcription factor κB The one of the most important function of interactions of MT with proteins is the regulation of the activity of a nuclear transcription factor κB (NF-κB). NF-κB is involved in the regulation of cell death [16, 51]. It plays an anti-apoptotic role [52]. Active, the most widespread in the body form, is a heterodimer composed of p50 and RelA protein. In such form, this factor is capable of activating gene transcription. To confirm, the MT effect on NF-κB activation, MCF-7 cell line was stimulated by zinc and 17β-estradiol to stimulate the proliferation. As a result, the induction of MT expression and the activation of NF-κB was observed. To prove that MT can transactivate NF-κB, tests without other stimulating factor were conducted. Activation of NF-κB only in the presence of MT was proved. In addition, to confirm the interaction between these two proteins anti-MT antibodies were used and the reduction in the amount of active NF-κB was observed. By gel mobility shift assay, was demonstrated specific interaction between the p50 subunit of NF-κB and MT. The increase in binding strength to the p50 subunit binding sites in the presence of MT suggests that MT interacts specifically with p50 subunit of the dimer p50/RelA, so MT may be required to stabilize the formation of the complex p50 with DNA [52]. In summary, MT can cause transactivation of NF-κB and was proved that it interacts directly with the p50 subunit of this protein. Both proteins are translocated to the nucleus, and the activation of NF-κB and induction of MT gene expression occurs during rapid tissue regeneration after partial hepatectomy. Additionally, MT and NF-κB have anti-apoptotic properties and in the future, inhibitors of these two proteins could be a potential therapeutic agent in the induction of apoptosis in cancer cells [52].



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5.1.3 Transcription factors: Sp1, TFIIIA, ER, Gal4, TTK By zinc transfer, MTs influence the binding of transcription factors to DNA and thus regulate transcription [53]. Transcription factors as: Sp1, TFIIIA, ER, Gal4 and TTK due to similar mechanism of interaction with MT will be discussed. Specificity protein 1 (Sp1), a transcription factor identified in all vertebrates, has a DNA binding domain and it comprises three zinc fingers. The removal of zinc from zinc finger of Sp1 abolishes DNA binding, thereby transcription of Sp1-dependent genes. It has been shown that thionein was able to take Zn from Sp1 and this was an irreversible reaction. After Zn sequestration from Sp1 and inhibition its ability to activate genes, the addition of Zn-MT did not restore the function of Sp1. Thus, the interaction between Sp1 and MT cause irreversible sequestration of Zn by apo-MT. In vitro studies suggest that changes in thionein concentration affect the concentration of Zn in vivo, the ratio of Zn7-MT/thionein defines the concentration of free Zn. Thus the couple donor - acceptor of Zn contributes to the maintenance of cellular Zn homeostasis [43, 54]. Posewitz and Wilcox showed that a single Zn-finger from Sp1 reacted with the α-domain of MT through a ligand-substitution process [55]. Metal binding competition between Sp1 and the α domain of human MT-2 (α-hMT-2) indicates a similar affinity for Zn2+. However, α-hMT-2 has a higher affinity for Ni2+, suggesting that MT may play a protective role by ensuring Zn2+, rather than Ni2+, coordination to zinc finger sequences of transcription factors [55]. In contrast to Sp1, where thionein removes the zinc and MT cannot deliver it again, reversible metal exchange between MT and transcription factor IIIA (TFIIIA) was observed. By removing Zn, it suppresses the binding of TFIIIA zinc finger to 5S RNA and to the 5S RNA gene and abrogates the capacity of TFIIIA to initiate the RNA polymerase III-catalyzed synthesis of 5S RNA. The way of Zn release from TFIIIA is unknown. It can rely on the Zn release to the free pool of Zn and then taking by apo-MT, or by transfer from TFIIIA to



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thionein due to collision or the direct exchange between proteins [56]. The inhibition of TFIIIA dependent transcription by apo-MT has also been observed in vivo. Injections of thionein to Xenopus laevis oocytes effectively blocked the synthesis of 5S RNA TFIIIAdependent. Thus, studies have found that apo-MT competes for zinc in both conditions in vitro and in vivo. Furthermore, it was shown that the loss of transcriptional activity by TFIIIA may be reversed by the addition of zinc [56]. It was shown that both Cd2+ and Pb2+ can displace Zn2+ from TFIIIA. Cd-TFIIIA reacts with apo-MT to form Cd-MT and apo-TFIIIA. Similarly, Cd2+ and Zn2+ can be exchanged in the reaction of Cd-TFIIIA with Zn-MT [57]. Under near stoichiometric conditions, apo-MT was able to remove most if not all of the Zn ions from TFIIIA, whether or not the TFIIIA was bound to the 5S DNA, and concomitantly inhibit its DNA-binding activity. Similar observations were made for the reaction between apo-MT and Cd-substituted TFIIIA, which proceeded without observable intermediates. A very slow metal ion exchange occurred between Cd-TFIIIA and Zn-MT, but not between CdMT and Zn-TFIIIA [58]. Thionein also removes zinc from estrogen receptor (ER) which is also a transcription factor containing zinc fingers. ER binding to specific DNA sequences - estrogen response element (ERE) is inhibited by thionein. The ability of ER to bind to the ERE can be restored by the addition of Zn or Zn-MT. MT is therefore metal donor to metal dependent ER and allows for re-binding to DNA. Reversible zinc exchange indicates that MT may be a regulator of Zn and it maintains the balance between storage and free cytosolic pool of Zn ions in cell and other proteins, in which Zn plays a structural or functional role [59]. It should be noted that the ability of thionein to take Zn from 4 cysteine residues of the ER zinc finger is consistent with the way of zinc loss by the 2Cys-2His zinc fingers of TFIIIA and SP1. Studies have shown that 20-fold less of thionein was needed to stop the transcription factors Sp1 and TFIIIA, than for ER. These differences may reflect structural differences that can



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cause a different affinity for Zn. Zinc-binding motifs in the ER are not classical zinc fingers, but the Zn twist. However, the observed reversibility of Zn exchange between MT and ER suggested that Zn ions have similar affinity to the ER zinc fingers as to MT. It is known, that in the case of Sp1, MT was not able to retake Zn if Sp1 was inactivated by apo-MT. Alternatively, differences in the affinity of thionein to inhibit these transcription factors may result from overexpression of ER in MCF-7 cells where the test was conducted as compared to TFIIIA, where the test was carried out in X. laevis oocytes [56, 59]. Reversible Zn exchange has been described between MT and ER zinc finger. Thus, MTs may also have a strong affinity to the Zn rich progesterone receptor binding Zn fingers, representing still another potential control factor for the down-regulation of the steroid receptor. MTs being cystein rich, Zn and Cu binding, have shown to be hormone dependent and expression of MTs in the secretory transformed human endometrium and in other species under influence of progesterone, have been described [59]. Another transcription factor, that interacts with MT is the yeast Gal4. In its structure, there is a cluster of six cysteine residues and two zinc atoms [20]. It regulates the expression of genes encoding enzymes that metabolize galactose [53]. An exchange of Zn between MT and Gal4 cluster was found. 20 cysteine residues bind 7 atoms of zinc. Fast replacement of zinc (k~ 800 min-1·M-1, pH=8.25ºC) between MT and Gal4 suggests that this process is controlled by direct contact between these two molecules [20]. MT ability to modulate DNA binding by tramtrack (TTK) was investigated using two forms of rabbit MT-2: apo-form and the form associated with the zinc. TTK is a transcription factor that inhibits the genes involved in the regulation of growth and differentiation in Drosophila. Zn atom is tetrahedrally coordinated in TTK to two cysteine and two histidine residues. In the study conducted by Roesijadi et al., after adding thionein, was observed inhibition of TTK-DNA complex formation and this capacity was restored after the addition



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of Zn-MT [60]. It should also be noted that in this study, to inhibit DNA binding by TTK, excess of thionein was used, which suggests a higher affinity of TTK to Zn compared to MT. These observations do not exclude the direct interaction between TTK and MT in the metal exchange process, which would be consistent with the role of MT in the regulation of the availability of free zinc [60]. In addition, it was examined whether the exchange of Zn-Cd between Zn-MT and Cd-TTK could be the mechanism of cadmium detoxification as Cd-TTK complex resulting in reduced DNA binding by TTK. Zinc fingers, in which Zn is replaced with Cd, exhibit lower affinity to DNA as a result of a different conformation of DNAbinding domains [61]. In the presence of Zn-MT this activity has been restored. This was probably due to the exchange of Zn-Cd and restoration of the TTK structure. This demonstrates the probability of the active role of Zn-MT in the detoxification of metals by metal-metal exchange. This makes it possible to change the structures of other proteins by MT, by binding toxic metals that have a greater affinity for the MT than Zn [60]. Cd-TTK has at least a 10-fold lower affinity for its cognate DNA base sequence than Zn-TTK because it has lost much of the α-helical structure of the native protein [60]. In contrast, the substitution of Cd2+ for Zn2+ in Sp1 does not alter qualitatively its DNA binding affinity [62]. Exposure to Cd2+ inhibits the binding of transcription factors such as TFIIIA and MTF-1 to their cognate DNA sites. In contrast, another Cd-substituted zinc finger protein, Sp1, retains its capacity to bind specifically to DNA [58].

5.2 Metallothionein as a source of zinc and cooper for metalloenzymes MT plays a role in maintaining homeostasis of metals and in their storage. MT is an intracellular

source

of

zinc

for

various

metalloenzymes,

for

example,

matrix

metalloproteinase-9 (MMP-9) δ-aminolevulinic dehydratase (ALAD) and cooper for superoxide dismutases (SOD) [63-65]. Zn transfer from MT to carbonic anhydrase, sorbitol



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dehydrogenase and alkaline phosphatase was also observed. Moreover, there is also Zn transfer from MT to other proteins in vivo [35]. It was found that mitochondrial aconitase (maconitase) is among the proteins which accepts Zn directly from the Zn-MT [66]. Cu-thionein donates Cu to apoceruloplasmin after oxidation of the Cu-thiolate centers of MT. Moreover, Cu-MT donates Cu to the apo form of dopamine β-monooxygenase (DBH), resulting in complete restoration of enzymatic activity, but also apothionein readily extracts Cu from the holoenzyme, causing an inhibition of enzymatic activity [67]. MT interaction with enzymes will be presented on the example of MMP-9, ALAD and Cu/Zn SOD.

5.2.1 Activation of matrix metalloproteinase-9 by MT MMPs are proteolytic enzymes, multidomain zinc endopeptidases [68]. It was identified 21 such enzymes, which are indicated by numbers of MMP-1 to MMP-28 [64]. The catalytic domain, in an active site, contains zinc ion coordinated by three histidine residues. MMPs are synthesized as inactive enzymes (proenzymes, zymogens, proMMPs) and in such form are secreted outside the cell or are associated with cell membranes. The main physiological function of MMPs is the degradation of proteins such as collagen and elastin [68]. MMPs are also involved in angiogenesis and tissue remodeling [64, 68]. What is more, they participate in numerous pathological processes such as Alzheimer's disease, arthritis, atherosclerosis, and stomach ulcers. MMPs serve as a markers of certain cancers, such as colorectal, thyroid, or breast [64]. MMP-9 is gelatinase, due to the high specificity for denatured gelatin and collagen. The principal known function of MMP-9 is the degradation of collagen type IV, which is a component of the vascular basement membrane. The result of this degradation is an elimination of physical barriers of the cells, and in inflammation



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conditions leukocytes can migrate freely [68]. Knowledge about its activity in pathological conditions leads to numerous studies, especially, inhibitors for MMPs are sought [68]. To determine whether there is an interaction between proteins, MMP-9 activation in the presence of MT was tested. The experiment was carried out by an electrochemical method using voltameter. The current changes depending on the potential peak, which was responsible for the availability of groups (-SH,-NH2) of electroactive protein, called Cat1 gradually increased during the incubation of MMP-9 and collagen. There is the degradation of collagen by MMP-9, and therefore the amount of electroactive groups capable to electrochemical exchange on the surface of the working electrode increases. In the case of the mixture of collagen, MMP-9 and MT, signal Cat1 increased during the incubation (after 7 and 8 hours) [65]. It is likely that the increase in peak Cat1 pointed to the degradation of collagen. The result is that the activity of MMP-9 is increased in the presence of MT. Most likely, Zn2+ ions present in the MT molecule, activate MMP-9, which then degrades collagen. The presence of small fragments of degraded collagen, which has then more content free groups, give rise of the peak responsible for the availability of SH and NH2 groups [65]. 5.2.2 MT interaction with δ-aminolevulinic acid dehydratase ALAD is a zinc-containing enzyme, present in the cytoplasm, which catalyzes the second step of heme synthesis, namely the condensation of two molecules of δaminolevulinic acid into one molecule of porphobilinogen. It is also the most sensitive indicator of exposure and toxicity of the Pb, as it is blocked by this metal [63]. ALAD requires Zn as a cofactor and may be activated with an excess of Zn in vitro and in vivo. Addition of Zn eliminates the Pb-induced inhibition of the erythrocyte ALAD activity and the degree of the elimination seems to be dependent on the molar ratio of Zn/Pb concentrations [69]. Zn-MT has the ability to transfer Zn to ALAD resulting in the activation of this enzyme.



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It should be noted that the Zn-MT is not able to release all Zn ions during its delivery to metalloenzymes. This is probably related to the different affinity of Zn to the MT clusters. Gel chromatography analysis was performed to confirm that Zn from Zn-MT is transferred to ALAD. It is interesting that in the control, 70% of Zn was in the form of Zn-MT, and in the presence of ALAD only 20%. ALAD bound 35% of Zn. It is believed that free Zn (45%) is the result of dissociation from ALAD. The effect of thionein (MT form without metal) on ALAD activity was also verified. In the presence of thionein, ALAD activity, which is a metal dependent enzyme, decreased to 15% [63]. Whether the mechanism of regulation the availability of Zn requires direct interaction between these two proteins is the subject of further research [63]. In conclusion, Zn-MT by providing Zn can activate metal-dependent enzymes while thionein has the opposite effect. 5.2.3 MT interaction with Cu/Zn superoxide dismutase Copper (Cu) is an essential trace element in all living organisms and it forms the active center of cuproenzymes, such as Cu, Zn-superoxide dismutase (Cu/Zn-SOD, SOD1) [70]. SOD catalyses the decomposition of superoxide to hydrogen peroxide and MT scavenges a wide range of ROS, including superoxide, hydrogen peroxide, hydroxyl radical, and nitric oxide more efficiently than other antioxidants [71]. MT binds Cu via Cu-thiolate clusters [70]. As the binding of Cu by MT is thermodynamically and kinetically stable, excess of Cu is sequestered by MT to mask Cu toxicity. It has been revealed that MT is able to directly donate Cu to SOD1 in vitro [72]. Moreover, Cu released from MT and incorporated into SOD, caused the increase of its activity. Increased Cu/Zn-SOD activity can be induced by oxidative stress in the absence of transcriptional activation, suggesting that redox-induced transfer of Cu to apo-SOD could account for acute regulation of SOD enzyme activity. The transfer of Cu from Cu-MT to



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apo/Zn-SOD was possible only in the presence of a nitric oxide donor [65] and it was shown that MT behaves as an activator of SOD, but apo-MT behaves as an inhibitor [73]. The combination of MT and SOD protects pancreatic β cells from oxidative damage [71]. Cu/Zn SOD activity and MT concentration were both changed in the inflammatory processes in patients with pancreatitis. In acute pancreatitis, MT plays an essential role as the antioxidant. In chronic exacerbated pancreatitis, MT and Cu/Zn SOD act jointly with each other; however, in patients with chronic pancreatitis, these antioxidants complement each other [7].

6 MT interacts with endocytic receptors One of the best documented interactions of MT with other proteins is its interaction with endocytic receptors. Numerous studies confirmed MT interaction with low-density lipoprotein receptors (LDLRs) [4, 74, 75]. It has been shown that LDLRs, especially megalin and lipoprotein receptor-related protein 1 (LRP1), are involved in the uptake of MT. Additionally, cubilin – another endocytic receptor, which is structurally different from the LDLR family, but has some common ligands with megalin, is presumed to interact with MT [4]. LDLRs are transmembrane endocytic receptors, which occur on different types of cells [76, 77]. LRP is widely expressed in neurons, hepatocytes and fibroblasts. It is considered as a receptor binding more than 15 structurally and functionally different ligands, which include apolipoprotein E, amyloid precursor protein, α2-macroglobulin and tissue plasminogen activator. Moreover, recent studies have shown that in addition to the function of mediating endocytosis, the receptor is involved in the direct transmission of extracellular signals across the cell membrane [76]. Megalin is expressed in many tissues in the body, including the renal proximal tubules, the nervous system - in the choroid plexus and neurons [74]. This receptor



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binds a variety of ligands with different affinity. It performs the functions of the scavenger receptor, which mediates the uptake of different proteins [78]. Megalin, LRP1B and LRP1 have four ligand binding domains, other members contain only one [79]. Feature of the LDLR is that ligand interaction with these receptors can be antagonized by a receptorassociated protein (RAP), and it is often used in studies concerning the function of LDLR and analysis of their ligands [76]. They are involved in signal transduction pathways, in delivery of ligands, including apolipoprotein, proteases, growth factors, transport proteins, to endosomes or lysosomes [74]. In the study conducted by Klassen et al., a specific receptor capable of binding to MT1/2 and mediating its uptake in the brush border in the kidney, lipoprotein receptor – megalin, was identified [4]. This receptor is also known as LRP2 [4, 74]. It is known that megalin binds proteins such as β2-microglobulin, cytochrome C and retinol binding protein, but also antibiotics such as gentamicin. Cubilin also binds several ligands, including megalin’s ligands [78]. It has been demonstrated that the uptake of β2-microglobulin and MT in rat proximal tubules is not blocked by each other. The observations which indicate that megalin mediates the uptake of β2-microglobulin lead to indirect evidence that this receptor might also bind MT. Klassen et al. [4] showed that megalin can bind and mediates renal uptake of MT complexes with heavy metal. Three evidences were shown to confirm this interaction. First, by SPR, was shown that megalin could bind MT-1/2 depending on MT concentration. What is more, oligomerized MT bound more effectively to megalin than monomer. Using the tetramer as a basis for calculations, one may estimate a 100-fold change in Kdys (7 x 10-7 M). Second, anti-megalin antibody and megalin ligands inhibited the uptake of MT. β2microglobulin, which is a megalin ligand, competed with MT for uptake by megalin. Third, by using receptor antibodies, colocalization of these proteins was demonstrated. Megalin,



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cubilin and MT were colocalized on the cell surface. Additionally, a critical site for binding MT to megalin is conserved hinge or interdomain region of MT, centered on a lysine repeat [4]. While the studies clearly indicate that megalin dominates in the mechanism of MT uptake, the same role of cubilin cannot be excluded. In the studies using SPR, binding of MT with cubilin was not observed. However, the co-localization of MT with both megalin and cubilin, during the early stages of uptake and internalization into endosomes, was demonstrated. It should be mentioned, that megalin is a molecular chaperon for cubilin and facilitates the internalization of its ligand. Thus research should be continued [4, 75]. Whether cubilin participates in the uptake of MT requires further study. The uptake of MT complexes with heavy metals in the kidneys, distorts numerous functions within proximal tubules. Cadmium enters the organism primarily via the alimentary and respiratory tracts [80]. This one, that enters the organism with food, mostly is transported in complexes with MT. In this form, it is delivered to the kidneys. Cd-MT complex is so small (approx. 7 kDa), that can be freely filtered by the glomeruli, then passes through the renal tubules, and is absorbed by the S1 segment of the proximal tubule [4, 75]. It was found that megalin mediates internalization of Cd-MT. It was suggested that after the uptake of CdMT complex by endocytosis, Cd2+ is released and is transported outside the endo/lysosomes, where free Cd2+ triggers apoptosis. RAP, ligand for megalin was used and as a result of competition binding, the uptake of Cd-MT and cell death, which could be due to internalization of the complex, were reduced. With the use of anti-megalin antibody, the amount of free megalin was reduced, so internalization of the Cd-MT complex in proximal tubules was lowered, resulting in lower toxicity and increased cell viability [75]. In conclusion, megalin enhances internalization of Cd-MT complex. As the result, harmful Cd is released. It can be concluded that there is a mechanism which leads to the reduction of toxicity caused by endocytosis of Cd-MT. The addition of another megalin ligand can



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compete with Cd-MT for binding to it, thereby limiting megalin availability on the cell surface [75]. MT has well documented protective effect in the mammalian brain, especially after physical damage and ischemia or the occurrence of neurodegenerative diseases. In recent years, it has become obvious that MT is present in the CNS and it interacts with LDLRs, which are located on the cell surface – especially with LRP1 and megalin. This interaction activates intracellular pathways, which are in accordance with the observed effects of MT activity in CNS, including influence on neurons, glial cells and cells of the immune system [74]. In the nervous system, MT was identified in the brain, spinal cord, and in the entire peripheral nervous system, and is represented by three isoforms (MT-1, MT-2, MT-3) [74, 81]. It is possible that extracellular MT may be delivered from the blood to the CNS and it is be able to cross the blood-brain barrier. There is no published report on this topic, however, the ability of MT-1/2 to cross the blood-brain barrier must remain possible due to the presence of the transport mechanism based on megalin receptors in the choroid plexus. This receptor is expressed in the choroid plexus, and is responsible for the bidirectional transport of different substances, through the blood-brain barrier, such as the transport of amyloid β from the cerebrospinal fluid into the bloodstream. It is therefore possible the uptake of MT1/2 by megalin. There is also the possibility, that MT-1/2 getting into CNS by the damaged blood-brain barrier, which may be associated with neurodegenerative diseases, whether caused by physical injury [74]. Alternatively, the extracellular MT may be produced by cells of CNS (in CNS, astrocytes are main cells producing MT-1/2) [82]. In contrast to these two isoforms, MT-3 is expressed predominantly in the hippocampus and cerebral cortex and in less quantities is produced by astrocytes [74, 83]. What emerges is that MT has the potential



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to contribute to a variety of processes, including neuroprotection, regeneration, and even cognitive functions [84]. Exogenously applicated MT-1/2 favors the development of neurite growth and regeneration of cultured neurons. MT-3 has a similar role as an anti-apoptotic agent and protects against toxic molecules in the intracellular environment [74]. Although its structure is highly homologous to the MT-1/2, it has different characteristics, which give it the ability to inhibit neurite growth under certain conditions, hence the MT-3 is referred as the growth inhibitory neurons called growth inhibitory factor [85]. It is interesting that, in a medium which does not contain the brain extract, MT-3 acts as a neuroprotective agent [83]. Studies have shown that MT-1/2 are internalized by megalin and LRP1 in vitro [86]. However, there is no information whether MT-3 binds to these receptors [74]. By binding to megalin, MT-1/2 may promote survival of neurons. It may be due to the activation of signaling molecules, extracellular signal-regulated kinase signal-regulated kinase, phosphoinositide 3 kinase, Akt kinase and protein binding of cAMP response element and cAMP-response element binding protein. LRP1 promotes MT-1/2 uptake to neurons and causes an increase of its survival involving the same signaling molecules [86]. Cd-MT uptake in the kidneys by megalin resulted in nephrotoxicity, suggesting that the internalized Cd is released from the MT. If such a situation occurs in neurons, it is possible that the internalized MT-1/2 is forwarded to other cellular compartments within neurons, such as nucleus. There, MT may protect DNA from oxidative damage or play a role in the regulation of gene expression by controlling the availability of Zn to transcription factors [86]. For neuroprotective action, MT may regulate the survival and regeneration of retinal ganglion cell axons. This effect is obtained by its interaction with megalin, which is known to participate in the binding and uptake of MT [87]. MT-1/2 is also expressed in the retina and retinal pigment epithelium. It is believed that MT may contribute to the prolonged survival of photoreceptors in the course of retinal



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degeneration. Studies have confirmed the presence of megalin in the retina, both in the inner, and in the outer layer [88]. It has been shown that MT stimulates neurite growth of retinal ganglion cells involving megalin. It is suggested that the neurite growth is triggered by activation of signal transduction pathways by NPxY motive of megalin cytoplasmic tail. Perhaps megalin is MT receptor in retinal ganglion cells. Potentially, MT may be a factor involving neuroregenerative action of megalin [87].

7 Concluding remarks Functions that can be assigned for MT interaction with other proteins are: (1) the maintenance of homeostasis, storage and transfer of essential metals such as Zn and Cu to metalloproteins, including transcription factors and enzymes, (2) the protection against the toxic metals, by binding to heavy metals (detoxification), which prevents damage to cell structures. The reduction of toxicity can be carried out by endocytosis of Cd-MT dependent on megalin, (3) scavenging - ROS and RNS, which are generated in normal metabolism, or oxidative stress induced by toxic metals, inflammatory processes or physical damage (inflammatory, antioxidant, anti-apoptotic functions) [9]. Redox cycle of MT and its interaction with GSH/GSSG regulates Zn release from MT and its availability for other proteins. (4) the repair of cells, regeneration after partial removal of the organ and chemical damage, neurite outgrowth regulated by interaction with LDL receptors [74]. MT interactions with other proteins, presented in this work, were summarized in Figure 4. MT interacts with ferritin, transcription factors, enzymes and membrane receptors The formation and disintegration of MT complexes can be crucial in the transfer and release of metal ions necessary in the formation of metalloenzymes and transcription factors. The



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results contribute to the understanding the mechanism of recognition molecules forming functional complexes with MT. Some proteins interacting with MT have already been identified, however their significance is still not really known. In this regard, there follows specific questions, for which to find answers has fundamental importance cognitive and practical. This would create the basis for the new area of research concerning on the availability of metals regulating, among others, gene expression. Interactions can both activate and deactivate the proteins and regulate processes in which it participates.

The authors have declared no conflict of interest



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References: [1] Sutherland, D. E. K., Stillman, M. J., The „magic numbers” of metallothionein. Metallomics 2011, 3, 444-463. [2] Margoshes, M., Vallee, B. L., A cadmium protein from equine kidney cortex. J. Am. Chem. Soc. 1957, 79, 4813-4814. [3] Bizoń, A., Milnerowicz-Nabzdyk, E., Zalewska, M., Zimmer, M., Milnerowicz, H. Changes in pro/antioxidant balance in smoking and non-smoking pregnant women with intrauterine growth restriction. Reprod Toxicol. 2011, 32, 360-367. [4] Klassen, R. B,. Crenshaw, K., Kozyraki, R., Verroust, P. J. et al., Megalin mediates renal uptake of heavy metal metallothionein complexes. Am. J. Physiol. Renal Physiol. 2004, 287, 393-403. [5] Manso, Y., Adlard, P. A., Carrasco, J., Vašák, M., Hidalgo, J., Metallothionein and brain inflammation. J. Biol. Inorg. Chem. 2011, 16, 1103-1113. [6]

Milnerowicz,

H.,

Chmarek,

M.,

Rabczyński,

J.,

Milnerowicz,

S.

et

al.,

Immunohistochemical localization of metallothioneins in chronic pancreatitis. Pancreas 2004, 29, 28-32. [7] Milnerowicz, H., Jabłonowska, M., Bizoń, A., Change of zinc, copper, and metallothioneins concentrations and the copper-zinc superoxide dismutase activity in patients with pancreatitis. Pancreas 2009, 38, 681-688. [8] Milnerowicz, H., Influence of tobacco smoking on metallothioneins isoforms contents in human placenta, amniotic fluid and milk. Int J Occup Med Environ Health 1997, 10, 395403.



Page 33

Proteomics

[9] Sabolic, I., Breljak, D., Skarica, M., Herak-Kramberger C. M., Role of metallothionein in cadmium traffic and toxicity in kidneys and other mammalian organs. Biometals 2010, 23, 897-926. [10] Śliwińska-Mossoń, M., Milnerowicz, H., Jabłonowska, M., Milnerowicz, S., Nabzdyk, S., Rabczyński, J., The effect of smoking on expression of IL-6 and antioxidants in pancreatic fluids and tissues in patients with chronic pancreatitis. Pancreatology 2012, 12, 295-304. [11] Takahashi, S., Molecular functions of metallothionein and its role in the hematological malignancies. J. Hematol. Oncol. 2012, 5, 1-8. [12] Asmussen, J. W., Sperling, M. L., Penkowa, M., Intraneuronal signaling pathways of metallothionein. J. Neurosci. Res. 2009, 87, 2926-2936. [13] Sutherland, D. E. K., Willans, M. J., Stillman, M. J., Single domain metallothioneins: Supermetalation of human MT 1a. J. Am. Chem. Soc. 2012, 134, 3290-3299. [14] Raudenska, M., Gumulec, J., Podlaha, O., Sztalmachova, M., et al., Metallothionein polymorphisms in pathological processes. Metallomics 2014, 6, 55-68. [15] Hijova, E., Metallothioneins and zinc: their functions and interactions. Bratisl. Lek. Listy. 2004, 105, 230-234. [16] Ruttkay-Nedecky, B., Nejdl, L., Gumulec, J., Zitka O. et al., The role of metallothionein in oxidative stress. Int. J. Mol. Sci. 2013, 14, 6044-6066. [17] Milnerowicz, H., Chmarek, M., Influence of smoking on metallothioneins level and other proteins binding essential metals in human milk. Acta Paediatr. 2005, 94, 402-406. [18] Milnerowicz, H., Bizoń, A., Determination of metallothioneins in biological fluids using enzyme-linked immunoassay with commercial antibody. Acta Biochim Pol. 2010, 57, 99104.



[19]

Śliwińska-Mossoń,

Page 34

M.,

Milnerowicz, H.,

Proteomics

Rabczyński,

J., Milnerowicz,

S.,

Immunohistochemical localization of metallothioneins and p53 protein in pancreatic serous cystadenomas. Arch Immunol Ther Exp. 2009, 57, 295-301. [20] Maret, W., Larsen, K. S., Vallee, B. L., Coordination dynamics of biological zinc "clusters" in metallothioneins and in the DNA-binding domain of the transcription factor Gal4. Proc. Natl. Acad. Sci. USA 1997, 94, 2233-2237. [21] Davis, S. R., Cousins, R. J., Metallothionein expression in animals: a physiological perspective on function. J. Nutr. 2000, 130, 1085-1088. [22] Sutherland, D. E., Stillman, M. J., Challenging conventional wisdom: single domain metallothioneins. Metallomics 2014, DOI: 10.1039/C3MT00216K. [23] Klassen, C. D., Liu, J., Diwan, B. A., Metallothionein protection of cadmium toxicity. Toxicol. Appl. Pharmacol. 2009, 238, 215-220. [24] Colvin, R.A., Holmes, W.R., Fontaine, C.P., Maret, W., Cytosolic zinc buffering and muffling: their role in intracellular zinc homeostasis. Metallomics 2010, 2, 306-317. [25] Krizkova, S., Ryvolova, M., Hrabeta, J., Adam, V., et al., Metallothioneins and zinc in cancer diagnosis and therapy. Drug Metab. Rev. 2012, 44, 287-301. [26] Babula, P., Masarik, M., Adam, V., Eckschlager, T., et al., Mammalian metallothioneins: properties and functions. Metallomics 2012, 4, 739-50. [27] Orihuela, R., Fernández, B., Palacios, O., Valero, E. et al., Ferritin and metallothionein: dangerous liaisons. Chem. Commun. 2011, 47, 12155-12157. [28] De Sole, P., Rossi, C., Chiarpotto, M., Ciasca, G. et al., Possible relationship between Al/ferritin complex and Alzheimer's disease. Clin. Biochem. 2013, 46, 89-93. [29] Harrison, P. M., Arosio, P., The ferritins molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta 1996, 1275, 161-203.



Page 35

Proteomics

[30] Joshi, J. G., Sczekan, S. R., Fleming, J. T., Ferritin − a general metal detoxicant. Biol. Trace Elem. Res. 1989, 21, 105-110. [31] Sakamoto, T., Ogasawara, Y., Ishii, K., Takahashi, H., Tanabe, S., Accumulation of aluminum in ferritin isolated from rat brain. Neurosci. Lett. 2004, 366, 264-267. [32] Goyer, RA., Toxic and essential metal interactions. Annu Rev Nutr. 1997, 17, 37-50. [33] Hidalgo, J., Garvey, J. S., Armario, A., On the metallothionein, glutathione and cysteine relationship in rat liver. J Pharmacol Exp Ther. 1990, 255, 554-64. [34] Wu, G., Fang ,Y. Z., Yang, S., Lupton, J. R., Turner, N. D., Glutathione metabolism and its implications for health. J. Nutr. 2004, 134, 489-492. [35] Kang, Y. J., Metallothionein redox cycle and function. Exp. Biol. Med. 2006, 231, 14591467. [36] Chen, Y., Maret, W., Catalytic selenols couple the redox cycles of metallothionein and glutatione. Eur. J. Biochem. 2001, 268, 3346-3353. [37] Peroza, E.A., dos Santos Cabral, A., Wan, X., Freisinger, E., Metal ion release from metallothioneins: proteolysis as an alternative to oxidation. Metallomics 2013, 5, 12041214. [38] Maret, W., Fluorescent probes for the structure and function of metallothionein. J. Chromatogr. B 2009, 877, 3378-3383. [39] Pedersen, M. Ø., Larsen, A., Stoltenberg, M., Penkowa, M., The role of metallothionein in oncogenesis and cancer prognosis. Prog. Histochem. Cytochem. 2009a, 44, 29-64. [40] Jiang, L. J., Maret, W., Vallee B. L., The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase. Proc. Natl. Acad. Sci. USA 1998, 95, 3483-3488. [41] Ngu, T. T., Dryden, M. D., Stillman, M. J., Arsenic transfer between metallothionein proteins at physiological pH. Biochem Biophys Res Commun. 2010, 8, 69-74.



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[42] Maynard, A. T., Covell, D. G., Reactivity of zinc finger cores: analysis of protein packing and electrostatic screening. J. Am. Chem. Soc. 2001, 123, 1047-1058. [43] Zeng, J., Heuchel, R., Schaffner, W., Kägi, J. H., Thionein (apometallothionein) can modulate DNA binding and transcription activation by zinc finger containing factor Sp1. FEBS Lett. 1991a, 279, 310-312. [44] Ostrakhovitch, E. A., Olsson, P. E., Jiang, S., Cherian, M. G., Interaction of metallothionein with tumor suppressor p53 protein. FEBS Lett. 2006, 580, 1235-1238. [45] Okorokov, A. L., Sherman, M. B., Plisson, C., Grinkevich V. et al., The structure of p53 tumour suppressor protein reveals the basis for its functional plasticity. EMBO J. 2006, 25, 5191-5200. [46] Xia, N., Liu, L., Yi, X., Wang, J., Studies of interaction of tumor suppressor p53 with apo-MT using surface plasmon resonance. Anal. Bioanal. Chem. 2009, 395, 2569-2575. [47] Ryan, K. M., Phillips, A. C., Vousden K. H., Regulation and function of the p53 tumor suppressor protein. Curr. Opin. Cell Biol. 2001, 13, 332-337. [48] Ostrakhovitch, E. A., Olsson, P. E., von Hofsten, J., Cherian, M. G., P53 mediated regulation of metallothionein transcription in breast cancer cells. J. Cell Biochem. 2007, 102, 1571-1583. [49] Cayrol, C., Knibiehler, M., Ducommun, B., p21 binding to PCNA causes G1 and G2 cell cycle arrest in p53-deficient cells. Oncogene 1998, 16, 311-320. [50] Méplan, C., Richard, M. J., Hainaut, P., Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of p53 after exposure to metal chelators in vitro and in intact cells. Oncogene 2000, 19, 5227-5236. [51] Butcher, H. L., Kennette, W. A., Collins, O., Zalups, R. K., Koropatnick, J., Metallothionein mediates the level and activity of nuclear factor κB in murine fibroblasts. J. Pharmacol. Exp. Ther. 2004, 310, 589-598.



Page 37

Proteomics

[52] Abdel-Mageed, A. B., Agrawal, K. C., Activation of nuclear factor κB: potential role in metallothionein-mediated mitogenic response. Cancer Res. 1998, 58, 2335-2338. [53] Vallee, B. L., Coleman, J. E., Auld, D. S., Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains. Proc. Natl. Acad. Sci. USA 1991, 88, 999-1003. [54] Kothinti, R., Blodgett, A., Tabatabai, N. M., Petering, D. H., Zinc finger transcription factor Zn(3)-SP1 reactions with Cd(2+). Chem. Res. Toxicol. 2010, 23, 405-412. [55] Posewitz, M.C.; Wilcox, D.E., Properties of the SP1 zinc-finger-3 peptide-coordination chemistry, redox reactions, and metal-binding competition with metallothioneins. Chem. Res. Toxicol. 1995, 8, 1020–1028. [56] Zeng, J., Vallee, B. L., Kägi, J. H., Zinc transfer from transcription factor IIIA fingers to thionein clusters. Proc. Natl. Acad. Sci. USA 1991b, 15, 9984-9988. [57] Petering, D. H., Huang, M., Moteki, S., and Shaw, C. F. 3rd., Cadmium and lead interactions with transcription factor IIIA from Xenopus laevis: a model for zinc finger protein reactions with toxic metal ions and metallothionein. Mar. Environ. Res. 2000, 50, 89-92. [58] Huang, M., Shaw, C. F. 3rd., Petering, D. H., Interprotein metal exchange between transcription factor IIIa and apo-metallothionein. J. Inorg. Biochem. 2004, 98, 639 – 648. [59] Cano-Gauci, D. F., Sarkar, B., Reversible zinc exchange between metallothionein and the estrogen receptor zinc finger. FEBS Lett. 1996, 386, 1-4. [60] Roesijadi, G., Bogumil, R., Vasák, M., Kägi ,J. H., Modulation of DNA binding of a tramtrack zinc finger peptide by the metallothionein-thionein conjugate pair. J. Biol. Chem. 1998, 273, 17425-17432. [61] Malgieri, G., Zaccaro, L., Leone, M., Bucci, E., et al., Zinc to cadmium replacement in the A. thaliana SUPERMAN Cys2His2 zinc finger induces structural rearrangements of typical DNA base determinant positions. Biopolymers 2011, 95, 801-810.



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Proteomics

[62] Kuwahara, J., and Coleman, J. E. Role of the zinc(II) ions in the structure of the 3-finger DNA-binding domain of the Sp1 transcription factor. Biochemistry 1990, 29, 8627-8631. [63] Goering, P. L., Fowler, B. A., Regulatory roles of high-affinity metal-binding proteins in mediating lead effects on delta-aminolevulinic acid dehydratase. Ann. N. Y. Acad. Sci. 1987, 514, 235-247. [64] Zitka, O., Krizkova, S., Huska, D., Adam, V. et al., Chip gel electrophoresis as a tool for study of matrix metalloproteinase 9 interaction with metallothionein. Electrophoresis 2011, 32, 857-860. [65] Liu, S. X., Fabisiak, J. P., Tyurin, V. A., Borisenko, G. G. et al., Reconstitution of aposuperoxide dismutase by nitric oxide-induced copper transfer from metallothioneins. Chem Res Toxicol. 2000, 13, 922-931. [66] Feng, W., Cai, J., Pierce, W. M., Franklin, R. B. et al., Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts. Biochem. Biophys. Res. Commun. 2005, 8, 853-858. [67] Richards, M.P., Recent developments in trace element metabolism and function: role of metallothionein in copper and zinc metabolism. J. Nutr. 1989, 119, 1062-1070. [68] Nagase, H., Visse, R., Murphy, G., Structure and function of matrix metalloproteinases and TIMPs. Cardiovascular Research 2006, 69, 562-573. [69] Tomokuni, K., Interaction of zinc and other metal on the activity of erythrocyte deltaaminolevulinic acid dehydratase in vitro. J. Toxicol. Sci. 1979, 4, 11-17. [70] Miyayama, T., Ishizuka, Y., Iijima, T., Hiraoka, D., Ogra, Y., Roles of copper chaperone for superoxide dismutase 1 and metallothionein in copper homeostasis. Metallomics 2011, 3, 693-701.



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Proteomics

[71] Park, L., Min, D., Kim, H., Park, J. et al., The combination of metallothionein and superoxide dismutase protects pancreatic β cells from oxidative damage. Diabetes Metab Res Rev. 2011, 27, 802-808. [72] Suzuki, K. T., Kuroda, T., Transfer of copper and zinc from ionic and metallothioneinbound forms to Cu, Zn--superoxide dismutase. Res. Commun. Mol. Pathol. Pharmacol. 1995, 87, 287–296. [73] Koh, M., Kim, H. J., The effects of metallothionein on the activity of enzymes involved in removal of reactive oxygen species. Bull. Korean Chem. Soc. 2001, 22, 362-366. [74] West, A. K., Leung, J. Y. K., Chung, R. S., Neuroprotection and regeneration by extracellular metallothionein via lipoprotein-receptor-related proteins. J. Biol. Inorg. Chem. 2011, 16, 1115-1121. [75] Wolff, N. A., Abouhamed, M., Verroust, P. J., Thévenod, F., Megalin-dependent internalization of cadmium-metallothionein and cytotoxicity in cultured renal proximal tubule cells. J. Pharmacol. Exp. Ther. 2006, 318, 782-791. [76] Li, Y., Cam, J., Bu, G., Low-density lipoprotein receptor family. Mol. Neurobiol. 2001, 23, 53-67. [77] Herz, J., Chen, Y., Masiulis, I., Zhou L., Expanding functions of lipoprotein receptors. J. Lipid Res. 2009, 50, 287-292. [78] Christensen, E. I., Birn, H., Megalin and cubilin: multifunctional endocytic receptors. Nat. Rev. Mol. Cell. Biol. 2002, 3, 256-266. [79] Herz, J., Strickland, D. K., LRP: a multifunctional scavenger and signaling receptor. Clin. Invest. 2001, 108, 779-784. [80] Amin, A.S and Gouda A.A., Utility of solid phase spectrophotometry for the modified determination of trace amounts of cadmium in food samples. Food Chemistry, 2012, 132, 518-524.



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Proteomics

[81] Hidalgo, J., García, A., Oliva, A. M., Giralt, M. et al., Effect of zinc, copper and glucocorticoids on metallothionein levels of cultured neurons and astrocytes from rat brain. Chem Biol Interact. 1994, 93, 197-219. [82] Manso, Y., Carrasco, J., Comes, G., Adlard, P. A. et al., Characterization of the role of the antioxidant proteins metallothioneins 1 and 2 in an animal model of Alzheimer's disease. Cell. Mol. Life Sci. 2012a, 69, 3665-3681. [83] Manso, Y., Carrasco, J., Comes, G., Meloni, G. et al., Characterization of the role of metallothionein-3 in an animal model of Alzheimer's disease. Cell Mol. Life Sci. 2012b, 69, 3683-3700. [84] West, A. K., Hidalgo, J., Eddins, D., Levin, E. D, Aschner, M., Metallothionein in the central nervous system: Roles in protection, regeneration and cognition. Neurotoxicology 2008, 29, 489-503. [85] Uchida, Y., Takio, K., Titani, K., Ihara, Y., Tomonaga, M., The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein. Neuron 1991, 7, 337-347. [86] Pedersen, M. Ø., Jensen, R., Pedersen, D. S., Skjolding A. D. et al., Metallothionein-I+II in neuroprotection. Biofactors 2009b, 35, 315-325. [87] Fitzgerald, M., Nairn, P., Bartlett, C. A., Chung, R. S. et al., Metallothionein-IIA promotes neurite growth via the megalin receptor. Exp. Brain Res. 2007, 183, 171-180. [88] Wunderlich, K. A., Leveillard, T., Penkowa, M., Zrenner, E., Perez, M. T., Altered expression of metallothionein-I and -II and their receptor megalin in inherited photoreceptor degeneration. Invest. Ophthalmol. Vis. Sci. 2010, 51, 4809-4820. [89] Maret, W., Redox biochemistry of mammalian metallothioneins. J. Biol. Inorg. Chem. 2011, 16, 1079-1086.



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Figure 1. Comparison of human MTs isoforms sequences. Sequence comparisons were made by ClustulX program. The sequences were derived from the database SWISS PROT (http://www.expasy.org/sprot/). '*' Indicates positions which have the same residues.



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Figure 2. Model of possible structure for the supermetalated Cd8-βα-rhMT1a [13].



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Figure 3. Forms of MT present in cells and tissues [89].


Proteomics


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Figure 4. MT interaction with other proteins. MT interacts with ferritin, transcription factors, enzymes, and membrane receptors. In many of these interactions, there is a zinc ions exchange that can be modulated by GSH/GSSG ratio. ALAD, δ-aminolevulinic dehydratase; DBH, dopamine β-monooxygenase; ER, estrogen receptor; LRP1, low density lipoprotein receptor protein; MMP-9, matrix metalloproteinase-9; NF-κB, nuclear factor-κB; Sp1, specificity protein 1; TFIIIA, transcription factor IIIA; TTK, tramtrack.


Study of metallothionein-quantum dots interactions.

The Role of Electrostatic Interactions in Folding of β-Proteins.

The role of packing interactions in stabilizing folded proteins.

Cardiolipin Interactions with Proteins.

The role of surface proteins in cell proliferation as studied with thrombin and other proteases.

Interactions of delta9-tetrahydrocannabinol with other drugs.

Role of anisotropic interactions for proteins and patchy nanoparticles.

Metal redistribution in largemouth bass (Micropterus salmoides) in response to restrainment stress and dietary cadmium: role of metallothionein and other metal-binding proteins.

The interactions of cis-diamminedichloroplatinum with metallothionein and glutathione in rat liver and kidney.

The role of interactions between the cholinergic system and other neuromodulatory systems in learning and memory.

Interactions of indomethacin with lysosomes and proteins.

Role of APP Interactions with Heterotrimeric G Proteins: Physiological Functions and Pathological Consequences.

Anesthesiologist's interactions with other medical departments outside the operating room.

Role of Backbone Dipole Interactions in the Formation of Secondary and Supersecondary Structures of Proteins.

Heparan sulfate and heparin interactions with proteins.

The role of metal ions in proteins and other biological molecules.

Predicting the other in cooperative interactions.

Chemical interactions of cardiolipin with daunorubicin and other intercalating agents.

Interactions of Methylotrophs with Plants and Other Heterotrophic Bacteria.

Pharmacologic implications of heparin interactions with other drugs.

enzymes and other food components, and their effects on food quality.

Diamide: mechanisms of action and interactions with other sensitizers.

Platelets and Their Interactions with Other Immune Cells.

Conjugates of Small Molecule Drugs with Antibodies and Other Proteins.