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

Helpdesk for Computer Network Maintenance Support

Práce se zabývá vytvořením a implementací systému helpdesk s diagnostikou (zpětnou odezvou) pro firmu The Best Network Solution, zabývající se IT outsourcingem, sloužící ke správě jejich počítačových sítí, zařízení a podporu koncových zákazníků.The thesis is dealing with creating and implementation of helpdesk system with diagnostics (feedback) for The Best Network Solution, the IT outsourcing company, serving for maintenance theirs computer networks, equipment and end customers support.

3D Scene Composition with Open-Source Tools

Práce se zabývá vytvořením komplexní 3D scény pomocí open-source nástrojů. Je zde popsán postup vytváření 3D modelů, úprav scény a následný převod do renderovacího programu PovRay, kde je za pomoci úpravy kódu a přidáním vlastností vytvořen výsledný obraz. Cílem je vyrenderovaná scéna metodou Ray-tracing.The work deals with the creation of complex 3D scenes using open-source tools. It describes how to create 3D models, adjustments to the scene and subsequent transfer to renderer program PovRay, where with the help of regulation code and adding features created by the resulting image. The aim is to render scene with Ray-tracing method.

Financial Analysis of the Company Šroubárna Turnov, a.s.

Tématem této bakalářské práce je finanční analýza společnosti Šroubárna Turnov, a. s., která je provedena za období v letech 2011 až 2015. Práce je rozdělena na dvě části. V teoretické části jsou vysvětleny metody finanční analýzy. V praktické části je představena společnost, na které jsou aplikovány metody finanční analýzy uvedené v teoretické části. Dále je podnik porovnán s odvětvím. V závěru jsou shrnuty výsledky finanční analýzy.The theme of this bachelor thesis is the financial analysis of the company Šroubárna Turnov, a. s., which is made in period from 2011 to 2015. The thesis consists of two parts. Explanation of methods of financial analysis is in the theoretical part. The practical part consists of description of the company, application of methods of financial analysis and comparison with industry. Results of the financial analysis are summarized in the end of the thesis

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