Biomedical subjects

Milan Vasák

Publications and source records attributed to Milan Vasák.

6 recordsLinked to original sources

PubMed2003

Redox labile site in a Zn4 cluster of Cu4,Zn4-metallothionein-3.

Human metallothionein-3 (MT-3) is a neuronal inhibitory factor mainly expressed in brain and downregulated in Alzheimer's disease. The neuroinhibitory activity has been established for native Cu(4),Zn(3)-MT-3 and recombinant Zn(7)-MT-3. However, there is only limited knowledge about the structure and properties of the former metalloform. We have now generated native-like MT-3 through direct Cu(I) and Zn(II) incorporation into the recombinant apoprotein. Its characterization revealed monomeric Cu(4),Zn(4)-MT-3 containing metal-thiolate clusters located in two mutually interacting protein domains, a Cu(4) cluster in the beta-domain and a Zn(4) cluster in the alpha-domain. Using the PC12 cell line, the nontoxic nature of the protein was demonstrated. The results of electronic absorption and Cu(I) luminescence at 77 K showed that the Cu(4) cluster possesses an unprecedented stability in air. In contrast, the Zn(4) cluster is air sensitive. Its oxidation results in the release of one Zn(II) and the formation of a Zn(3) cluster, i.e., Cu(4),Zn(3)-MT-3. This process can be prevented or reversed under reducing conditions. The determined apparent stability constant for the Zn(4) cluster of 2.4 x 10(11) M(-1) is similar to that obtained for other zinc-containing MTs. This suggests that a substantially increased nucleophilic reactivity of specific thiolate ligands is responsible for this effect. Thus, the Zn(4) cluster in MT-3 may play a redox-dependent regulatory role.

Amino Acid Sequence↗

PubMed2003

Activity of metal-responsive transcription factor 1 by toxic heavy metals and H2O2 in vitro is modulated by metallothionein.

Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H2O2 additionally requires the presence of zinc-saturated metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.

Animals↗

PubMed2002

Zn(II)-free dimethylargininase-1 (DDAH-1) is inhibited upon specific Cys-S-nitrosylation.

The endogenous nitric oxide synthase inhibitors L-N(omega)-methylarginine and L-N(omega),N(omega)-dimethylarginine are catabolized by the enzyme dimethylargininase. Dimethylargininase-1 from bovine brain contains one tightly bound Zn(II) coordinated by two cysteine sulfur and two lighter ligands. Activity measurements showed that only the apo-enzyme is active and that the holo-enzyme is activated by zinc removal. In this work, the effect of NO on dimethylargininase-1 structure and its activity was investigated using 2-(N,N-dimethylamino)-diazenolate-2-oxide as an NO source. The results showed that whereas the holo-form was resistant to S-nitrosylation, the apo-form could be modified. The results of absorption spectroscopy, mass spectrometry, and fluorometric S-NO quantification revealed that two of five cysteine residues reacted with NO yielding cysteine-S-NO. The modification reaction is specific, because by liquid chromatography/mass spectrometry experiments of digested S-NO-dimethylargininase-1, cysteines 221 and 273 could be identified as cysteine-NO. Because Zn(II) protects the enzyme against nitrosation, it is suggested that both cysteines are involved in metal binding. However, specific cysteine-S-NO formation occurred in the absence of a characteristic sequence motif. Based on a structural model of dimethylargininase-1, the activation of both cysteines may be accomplished by the close proximity of charged residues in the tertiary structure of the enzyme.

Amidohydrolases↗

PubMed2002

A distinct Cu(4)-thiolate cluster of human metallothionein-3 is located in the N-terminal domain.

Metallothionein-3 (MT-3), also known as neuronal growth inhibitory factor, is a metalloprotein expressed almost exclusively in the brain. Isolated MT-3 contains four Cu(I) and three Zn(II) ions organized in homometallic metal-thiolate clusters located in two independent protein domains. In this work a Cu(I) binding to metal-free MT-3 has been studied, aiming at the better understanding of the domain specificity for this metal ion. The cluster formation was followed by electronic absorption, circular dichroism, and by luminescence spectroscopy at room temperature and 77 K. The stepwise incorporation of Cu(I) into recombinant human apo-MT-3 revealed the cooperative formation of two Cu(4)S(9) clusters in succession, formed in both protein domains, i.e. Cu(4)- and Cu(8)-MT-3. Further binding of four Cu(I) caused an expansion of these Cu(I) cores, leading to fully metal-loaded Cu(12)-MT-3 containing Cu(6)S(9) and Cu(6)S(11) clusters in the beta- and alpha-domains of the protein, respectively. The location of the preferentially formed Cu(4) cluster in the protein was established by immunochemistry. Using domain-specific antibodies, in combination with limited tryptic digestion of a partially metal-occupied Cu(4)-MT-3, we could demonstrate that the Cu(4)S(9) cluster is located in the N-terminal beta-domain of the protein that contains a total of nine cysteine ligands.

Antibody Specificity↗

PubMed2002

Recent developments in quantification methods for metallothionein.

The metallothioneins (MT), a family of proteins with relatively low molecular weight (6-7 kDa), are characterised by the intrinsic presence of 20 cysteinyl groups in their structure, which confers unique metal binding properties to the molecule. Since MT are involved in biological roles, quantification of MT remains an important task. To date, a large number of determination methods have been developed. In this paper recent developments, from 1995 to the present, in methodology employed in quantification studies of total MT and MT polymorphism are described. Different fields were taken into consideration, such as (i) separation techniques and hyphenated systems, (ii) electrochemical methods, (iii) immunological methods and (iv) quantification of MT mRNA. The data presented are based on our own and published results. A brief overview of the use of metallothionein as a biomarker is included as a relevant example of the importance of MT quantification. Finally, general problems associated with determination and evaluation of obtained results within the above four topics are mentioned.

Animals↗

PubMed2002

Advances in the structure and chemistry of metallothioneins.

A low molecular weight (6-7 kDa) class of metalloproteins, designated as metallothioneins (MTs), exhibit repeated sequence motifs of either CxC or CxxC through which mono or divalent d(10) metal ions are bound in polymetallic-thiolate clusters. The preservation of metal-thiolate clusters in an increasing number of three-dimensional structures of these proteins signifies the importance of this structural motif. This review focuses on the recent developments regarding the versatile and striking chemical reactivity of MTs as well as on the existence of conformational/configurational dynamics within their structure. Both properties and their interplay are likely to be essential for the still elusive biological function of these proteins.

Amino Acid Motifs↗