Control of Abnormal Metal-Protein Interactions in Neurodegenerative Disorders by Metallothionein-3

In the brain, zinc and copper homeostasis is regulated by a small metalloprotein, metallothionein-3 (Zn7MT-3), which is down-regulated in neurodegenerative diseases such as Alzheimer (AD), Creutzfeldt-Jacob and Parkinson. These disorders share common pathological hallmarks including misfolding of amyloid-? (A?), prion protein and ?-synuclein, the formation of protein aggregates, abnormal metal-protein interactions and oxidative stress. In AD, Cu(II) and Zn(II) areinvolved in the disease progression by modulating the formation and toxicity of soluble and insoluble oligomers and aggregates of the A? peptide. Whereas the copper-induced A? aggregation is related to the ROS production and neurotoxicity, the zinc-induced A? aggregation is considered neuroprotective. The protective effect of extracellular Zn7MT-3 from A? toxicity in neuronal cell cultures is not understood. We show that Zn7MT-3 not only scavenges freeCu(II) ions, but also removes Cu(II) bound to A?. A metal swap between Zn7MT-3 and soluble and aggregated A?-Cu(II) is the underlying molecular mechanism by which the ROS production and related cellular toxicity is abolished. In this process, copper is reduced by the protein thiolates forming Cu(I)4Zn4MT-3, in which an air stable Cu(I)4-thiolate cluster and two disulfide bonds are present. To examine whether the discovered effect represents ageneral protective role of this protein in other metal-linked neurodegenerative pathologies, similar studies using prion peptides in complex with Cu(II) were conducted. Zn7MT-3 by a similar metal swap reaction removes abnormally bound Cu(II) from the prion protein, impeding the ROS production. This finding signifies a so far unrecognized protective role of this protein in the brain

Chemistry and biology of mammalian metallothioneins

Metallothioneins (MTs) are a class of ubiquitously occurring low molecular mass, cysteine- and metal-rich proteins containing sulfur-based metal clusters formed with Zn(II), Cd(II), and Cu(I) ions. In mammals, four distinct MT isoforms designated MT-1 through MT-4 exist. The first discovered MT-1/MT-2 are widely expressed isoforms, whose biosynthesis is inducible by a wide range of stimuli, including metals, drugs, and inflammatory mediators. In contrast, MT-3 and MT-4 are noninducible proteins, with their expression primarily confined to the central nervous system and certain squamous epithelia, respectively. MT-1 through MT-3 have been reported to be secreted, suggesting that they may play different biological roles in the intracellular and extracellular space. Recent reports established that these isoforms play an important protective role in brain injury and metal-linked neurodegenerative diseases. In the postgenomic era, it is becoming increasingly clear that MTs fulfill multiple functions, including the involvement in zinc and copper homeostasis, protection against heavy metal toxicity, and oxidative damage. All mammalian MTs are monomeric proteins, containing two metal-thiolate clusters. In this review, after a brief summary of the historical milestones of the MT-1/MT-2 research, the recent advances in the structure, chemistry, and biological function of MT-3 and MT-4 are discusse

Reaction of human metallothionein-3 with cisplatin and transplatin

Human metallothioneins, small cysteine- and metal-rich proteins, play an important role in the acquired resistance to platinum-based anticancer drugs. These proteins contain a M(II)4(CysS)11 cluster and a M(II)3(CysS)9 cluster localized in the α-domain and the β-domain, respectively. The noninducible isoform metallothionein-3 (Zn7MT-3) is mainly expressed in the brain, but was found overexpressed in a number of cancer tissues. Since the structural properties of this isoform substantially differ from those of the ubiquitously occurring Zn7MT-1/Zn7MT-2 isoforms, the reactions of cis-diamminedichloridoplatinum(II) (cisplatin) and trans-diamminedichloridoplatinum(II) (transplatin) with human Zn7MT-3 were investigated and the products characterized. A comparison of the reaction kinetics revealed that transplatin reacts with cysteine ligands of Zn7MT-3 faster than cisplatin. In both binding processes, stoichiometric amounts of Zn(II) were released from the protein. Marked differences between the reaction rates of cisplatin and transplatin binding to Zn7MT-3 and the formation of the Pt-S bonds suggest that the binding of both Pt(II) compounds is a complex process, involving at least two subsequent binding steps. The electrospray ionization mass spectrometry characterization of the products showed that whereas all ligands in cisplatin were replaced by cysteine thiolates, transplatin retained its carrier ammine ligands. The 113Cd NMR studies of Pt1 113Cd6MT-3 revealed that cisplatin binds preferentially to the β-domain of the protein. The rates of reaction of cisplatin and transplatin with Zn7MT-3 were much faster than those of cisplatin and transplatin with Zn7MT-2. The biological consequences of a substantially higher reactivity of cisplatin toward Zn7MT-3 than Zn7MT-2 in the acquired resistance to platinum-based drugs are discusse

Influence of NH - Sγ {\text{NH - }}{{\text{S}}^\gamma } bonding interactions on the structure and dynamics of metallothioneins

Mammalian metallothioneins ($${\text{M}}_7^{\text{IIMTs}}$$) show a clustered arrangement of the metal ions and a nonregular protein structure. The solution structures of Cd3-thiolate cluster containing β-domain of mouse β-MT-1 and rat β-MT-2 show high structural similarities, but widely differing structure dynamics. Molecular dynamics simulations revealed a substantially increased number of $${\text{NH - }}{{\text{S}}^\gamma }$$ hydrogen bonds in β-MT-2, features likely responsible for the increased stability of the Cd3-thiolate cluster and the enfolding protein domain. Alterations in the $${\text{NH - }}{{\text{S}}^\gamma }$$ hydrogen-bonding network may provide a rationale for the differences in dynamic properties encountered in the β-domains of MT-1, -2, and -3 isoforms, believed to be essential for their different biological functio

The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets

Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M8 II-MTC (MisZn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn6-MTC and Cd7-MTC species from M8 II-MTC after treatment with Chelex 100. The NMR characterization of Cd7-MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal-Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1-31) reveal a Cd3Cys9 cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32-64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21Cys are involved in the binding of eight to nine Cu(I) ions (Cu8-9-MTC). The subsequent generation of Cu12-MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MT

Implications on zinc binding to S100A2

AbstractHuman S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn2+ is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals

Mammalian metallothionein-3: new functional and structural insights

Metallothionein-3 (MT-3), a member of the mammalian metallothionein (MT) family, is mainly expressed in the central nervous system (CNS). MT-3 possesses a unique neuronal growth inhibitory activity, and the levels of this intra- and extracellularly occurring metalloprotein are markedly diminished in the brain of patients affected by a number of metal-linked neurodegenerative disorders, including Alzheimer's disease (AD). In these pathologies, the redox cycling of copper, accompanied by the production of reactive oxygen species (ROS), plays a key role in the neuronal toxicity. Although MT-3 shares the metal-thiolate clusters with the well-characterized MT-1 and MT-2, it shows distinct biological, structural and chemical properties. Owing to its anti-oxidant properties and modulator function not only for Zn, but also for Cu in the extra- and intracellular space, MT-3, but not MT-1/MT-2, protects neuronal cells from the toxicity of various Cu(II)-bound amyloids. In recent years, the roles of zinc dynamics and MT-3 function in neurodegeneration are slowly emerging. This short review focuses on the recent developments regarding the chemistry and biology of MT-3

Sodium and Potassium Ions in Proteins and Enzyme Catalysis

The group I alkali metal ions Na(+) and K(+) are ubiquitous components of biological fluids that surround biological macromolecules. They play important roles other than being nonspecific ionic buffering agents or mediators of solute exchange and transport. Molecular evolution and regulated high intracellular and extracellular M(+) concentrations led to incorporation of selective Na(+) and K(+) binding sites into enzymes to stabilize catalytic intermediates or to provide optimal positioning of substrates. The mechanism of M(+) activation, as derived from kinetic studies along with structural analysis, has led to the classification of cofactor-like (type I) or allosteric effector (type II) activated enzymes. In the type I mechanism substrate anchoring to the enzyme active site is mediated by M(+), often acting in tandem with a divalent cation like Mg(2+), Mn(2+) or Zn(2+). In the allosteric type II mechanism, M(+) binding enhances enzyme activity through conformational transitions triggered upon binding to a distant site. In this chapter, following the discussion of the coordination chemistry of Na(+) and K(+) ions and the structural features responsible for the metal binding site selectivity in M(+)-activated enzymes, well-defined examples of M(+)-activated enzymes are used to illustrate the structural basis for type I and type II activation by Na(+) and K(+)

Metallothionein structure and reactivity

The structure and chemistry of mammalian MTs with divalent (ZnII, CdII) and monovalent (CuI) metal ions pertinent to their role in biological systems are discussed. In human four MT isoforms designated MT-1 through MT-4 are found. The characteristic feature of these cysteine- and metal-rich proteins is the presence of two metal-thiolate clusters located in independent protein domains. The structure of these clusters is highly dynamic allowing a fast metal exchange and metal transfer to modulate activity and function of zinc-binding proteins. Despite the fact that the protein thiolates are involved in metal binding, they show a high reactivity toward electrophiles and free radicals leading to cysteine oxidation and/or modification, and metal release. The unusual structural properties of MT-3 are responsible for its neuronal growth inhibitory activity, involvement in trafficking of zinc vesicles in the CNS, and the protection against copper-mediated toxicity in Alzheimer's disease. MT-1/-2 also play a role in cellular resistance against a number of metal-based drugs