Artur Krężel

Synaptic release of zinc from brain slices : Factors governing release, imaging, and accurate calculation of concentration

Cerebrocortical neurons that store and release zinc synaptically are widely recognized as critical in maintenance of cortical excitability and in certain forms of brain injury and disease. Through the last 20 years, this synaptic release has been observed directly or indirectly and reported in more than a score of publications from over a dozen laboratories in eight countries. However, the concentration of zinc released synaptically has not been established with final certainty. In the present work we have considered six aspects of the methods for studying release that can affect the magnitude of zinc release, the imaging of the release, and the calculated concentration of released zinc. We present original data on four of the issues and review published data on two others. We show that common errors can cause up to a 3000-fold underestimation of the concentration of released zinc. The results should help bring consistency to the study of synaptic release of zinc.

Different redox states of metallothionein/thionein in biological tissue.

Mammalian metallothioneins are redox-active metalloproteins. In the case of zinc metallothioneins, the redox activity resides in the cysteine sulfur ligands of zinc. Oxidation releases zinc, whereas reduction re-generates zinc-binding capacity. Attempts to demonstrate the presence of the apoprotein (thionein) and the oxidized protein (thionin) in tissues posed tremendous analytical challenges. One emerging strategy is differential chemical modification of cysteine residues in the protein. Chemical modification distinguishes three states of the cysteine ligands (reduced, oxidized and metal-bound) based on (i) quenched reactivity of the thiolates when bound to metal ions and restoration of thiol reactivity in the presence of metal-ion-chelating agents, and (ii) modification of free thiols with alkylating agents and subsequent reduction of disulfides to yield reactive thiols. Under normal physiological conditions, metallothionein exists in three states in rat liver and in cell lines. Ras-mediated oncogenic transformation of normal HOSE (human ovarian surface epithelial) cells induces oxidative stress and increases the amount of thionin and the availability of cellular zinc. These experiments support the notion that metallothionein is a dynamic protein in terms of its redox state and metal content and functions at a juncture of redox and zinc metabolism. Thus redox control of zinc availability from this protein establishes multiple methods of zinc-dependent cellular regulation, while the presence of both oxidized and reduced states of the apoprotein suggest that they serve as a redox couple, the generation of which is controlled by metal ion release from metallothionein.

The biological inorganic chemistry of zinc ions

The solution and complexation chemistry of zinc ions is the basis for zinc biology. In living organisms, zinc is redox-inert and has only one valence state: Zn(II). Its coordination environment in proteins is limited by oxygen, nitrogen, and sulfur donors from the side chains of a few amino acids. In an estimated 10% of all human proteins, zinc has a catalytic or structural function and remains bound during the lifetime of the protein. However, in other proteins zinc ions bind reversibly with dissociation and association rates commensurate with the requirements in regulation, transport, transfer, sensing, signalling, and storage. In contrast to the extensive knowledge about zinc proteins, the coordination chemistry of the “mobile” zinc ions in these processes, i.e. when not bound to proteins, is virtually unexplored and the mechanisms of ligand exchange are poorly understood. Knowledge of the biological inorganic chemistry of zinc ions is essential for understanding its cellular biology and for designing complexes that deliver zinc to proteins and chelating agents that remove zinc from proteins, for detecting zinc ion species by qualitative and quantitative analysis, and for proper planning and execution of experiments involving zinc ions and nanoparticles such as zinc oxide (ZnO). In most investigations, reference is made to zinc or Zn2+ without full appreciation of how biological zinc ions are buffered and how the d-block cation Zn2+ differs from s-block cations such as Ca2+ with regard to significantly higher affinity for ligands, preference for the donor atoms of ligands, and coordination dynamics. Zinc needs to be tightly controlled. The interaction with low molecular weight ligands such as water and inorganic and organic anions is highly relevant to its biology but in contrast to its coordination in proteins has not been discussed in the biochemical literature. From the discussion in this article, it is becoming evident that zinc ion speciation is important in zinc biochemistry and for biological recognition as a variety of low molecular weight zinc complexes have already been implicated in biological processes, e.g. with ATP, glutathione, citrate, ethylenediaminedisuccinic acid, nicotianamine, or bacillithiol.

Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering. Combinatorial library determination of optimal sequences.

Previously we demonstrated for several examples that peptides having a general internal sequence R(N)-Yaa-Ser/Thr-Xaa-His-Zaa-R(C) (Yaa = Glu or Ala, Xaa = Ala or His, Zaa = Lys, R(N) and R(C) = any N- and C-terminal amino acid sequence) were hydrolyzed specifically at the Yaa-Ser/Thr peptide bond in the presence of Ni(II) ions at alkaline pH (Krezel, A., Mylonas, M., Kopera, E. and Bal, E. Acta Biochim. Polon. 2006, 53, 721-727 and references therein). Hereby we report the synthesis of a combinatorial library of CH(3)CO-Gly-Ala-(Ser/Thr)-Xaa-His-Zaa-Lys-Phe-Leu-NH(2) peptides, where Xaa residues included 17 common alpha-amino acids (except Asp, Glu, and Cys) and Zaa residues included 19 common alpha-amino acids (except Cys). The Ni(II)-dependent hydrolysis at 37 and 45 degrees C of batches of combinatorial peptide mixtures randomized at Zaa was monitored by MALDI-TOF mass spectrometry. The correctness of library-based predictions was confirmed by accurate measurements of hydrolysis rates of seven selected peptides using HPLC. The hydrolysis was strictly limited to the Ala-Ser/Thr bond in all library and individual peptide experiments. The effects of individual residues on hydrolysis rates were quantified and correlated with physical properties of their side chains according to a model of independent contributions of Xaa and Zaa residues. The principal component analysis calculations demonstrated partial molar side chain volume and the free energy of amino acid vaporization for both Xaa and Zaa residues and the amine pK(a) for Zaa residues to be the most significant empirical parameters influencing the hydrolysis rate. Therefore, efficient hydrolysis required bulky and hydrophobic residues at both variable positions Xaa and Zaa, which contributed independently to the hydrolysis rate. This relationship between the peptide sequence and the hydrolysis rate provides a basis for further research, aimed at the elucidation of the reaction mechanism and biotechnological applications of Ni(II)-dependent peptide bond hydrolysis.

Sequence-specific Ni(II)-dependent peptide bond hydrolysis for protein engineering: reaction conditions and molecular mechanism.

Recently we screened a combinatorial library of R(1)-(Ser/Thr)-Xaa-His-Zaa-R(2) peptides (Xaa = 17 common alpha-amino acids, except Asp, Glu, and Cys; Zaa =19 common alpha-amino acids, except Cys; R(1) = CH(3)CO-Gly-Ala, R(2) = Lys-Phe-Leu-NH(2)) and established criteria for selecting Ser/Thr, Xaa, and Zaa substitutions optimal for specific R(1)-Ser/Thr peptide bond hydrolysis in the presence of Ni(II) ions (Krezel, A.; Kopera, E.; Protas, A. M.; Poznanski, J.; Wysłouch-Cieszynska, A.; Bal, W. J. Am. Chem. Soc. 2010, 132, 3355-3366). The screening results were confirmed by kinetic studies of hydrolysis of seven peptides: R(1)-Ser-Arg-His-Trp-R(2), R(1)-Ser-Lys-His-Trp-R(2), R(1)-Ser-Ala-His-Trp-R(2), R(1)-Ser-Arg-His-Ala-R(2), R(1)-Ser-Gly-His-Ala-R(2), R(1)-Thr-Arg-His-Trp-R(2), and R(1)-Thr-His-His-Trp-R(2). In this paper, we used the same seven peptides to investigate the molecular mechanism of the hydrolysis reaction. We studied temperature dependence of the reaction rate at temperatures between 24 and 75 degrees C, measured stability constants of Ni(II) complexes with hydrolysis substrates and products, and studied the course of R(1)-Ser-Arg-His-Trp-R(2) peptide hydrolysis under a broad range of conditions. We established that the specific square planar complex containing the Ni(II) ion bonded to the His imidazole nitrogen and three preceding peptide bond nitrogens (4N complex) is required for the reaction to occur. The reaction mechanism includes the N-O acyl shift, yielding an intermediate ester of R(1) with the Ser/Thr hydroxyl group. This ester hydrolyzes spontaneously, yielding final products. The Ni(II) ion activates the R(1)-Ser peptide bond by destabilizing it directly through peptide nitrogen coordination and, indirectly, by imposing a strain in the peptide chain.

Potentiometric and spectroscopic studies of the interaction of Cu(II) ions with the hexapeptides AcThrAlaSerHisHisLysNH2, AcThrGluAlaHisHisLysNH2, AcThrGluSerAlaHisLysNH2 and AcThrGluSerHisAlaLysNH2, models of C-terminal tail of histone H2A

The hexapeptides AcThrAlaSerHisHisLysNH2, AcThrGluAlaHisHisLysNH2, AcThrGluSerAlaHisLysNH2 and AcThrGluSerHisAlaLysNH2 which represent modifications of the 120 � /125 sequence of histone H2A were synthesized and their interactions with Cu(II) ions were studied with potentiometric and spectroscopic (UV � /Vis, EPR and CD spectroscopy) studies. All peptides coordinate Cu(II) efficiently. At physiological pH, AcThrAlaSerHisHisLysNH2 forms dimeric species while the rest of the peptides form monomers. The dimer is formed when Cu(II) ions coordinate equatorially through the imidazole nitrogen of the His-4 residue and the amide nitrogens of the Ser-3 and His-4 residues, plus the imidazole nitrogen of the His-5 residue of a second peptide molecule after deprotonation. At higher pH peptides AcThrAlaSerHisHisLysNH2 and AcThrGluAlaHisHisLysNH2 are using the second histidine residue for coordination at the apical position while Cu(II) ions coordinate equatorially with the imidazole nitrogen of His-5 or His-4 and three amido nitrogens with the peptides AcThrGluSerAlaHisLysNH2 and AcThrGluSerHisAlaLysNH2. # 2002 Elsevier Science B.V. All rights reserved.

Structure–function relationships in glutathione and its analogues

The results are presented of measurements of protonation constants (potentiometry and NMR), UV spectroscopic properties and redox potentials of GSH and its five analogues, which are modified at the C-terminal glycine residue (gammaGlu-Cys-X, X = Gly, Gly-NH2, Gly-OEt, Ala, Glu, Ser). Strong linear correlations were found between various properties of the thiol and other functions of these peptides. These results allow discussion of the relationships between the structures and properties in glutathione and its analogues, and provide a novel chemical background for the issue of control of GSH reactivity.

Studies of zinc(II) and nickel(II) complexes of GSH, GSSG and their analogs shed more light on their biological relevance.

Glutathione, Υ-Glu-Cys-Gly, is one of the most abundant small molecules in biosphere. Its main form is the reduced monomer (GSH), serving to detoxicate xenobiotics and heavy metals, reduce protein thiols, maintain cellular membranes and deactivate free radicals. Its oxidized dimer (GSSG) controls metal content of metallothionein. The results presented provided a quantitative and structural description of Zn(II)- glutathione complexes, including a novel ternary Zn(II)-GSH-His complex. A solution structure for this complex was obtained using 2D-NMR. The Complexes studied may contribute to both zinc and glutathione physiology. In the case of Ni(ll) complexes an interesting dependence of coordination modes on the ratios of reactants was found. At high GSH excess a Ni(GSH)2 complex is formed, with Ni(ll) bonded through S and N and/or O donor atoms. This complex may exist as a high- or low-spin species. Another goal of the studies presented was to describe the catalytic properties of Ni(II) ions towards GSH oxidation, which appeared to be an important step in nickel carcinogenesis. The pH dependence of oxidation rates allowed to determine the Ni(GSH)2 complex as the most active among the toxicologically relevant species. Protonation and oxidation of metal-free GSH and its analogues were also studied in detail. The monoprotonated form HL2- of GSH is the one most susceptible to oxidation, due to a salt bridge between S- and NH3+ groups, which activates the thiol.

The binding of Ni(II) ions to terminally blocked hexapeptides derived from the metal binding -ESHH- motif of histone H2A

The coordination properties of Ni(II) ions towards the terminally blocked (CH3CONH- and -CONH2) hexapeptides -TESHHK-, -TASHHK-, -TEAHHK-, -TESAHK- and -TESHAK- were studied by using potentiometric and spectroscopic techniques (UV/Vis, CD, NMR). The peptides were chosen in such a way as to compare the effect of Glu, Ser and His residues on the stability, the coordination and hydrolytic abilities of the complexes formed. All peptides bind to Ni(II) ions initially through one or two imidazole nitrogens in weakly acidic and neutral solutions forming slightly distorted octahedral complexes. At higher pH values, a series of square-planar complexes are formed, where Ni(II) ions bind simultaneously through an imidazole and three amide nitrogens in an equatorial plane. This proposed conformation includes the participation of only one imidazole nitrogen, in the case of all peptides, in the coordination sphere of Ni(II) ions. In basic solutions, the peptides -TASHHK- and -TESAHK- were hydrolyzed in a Ni(II)-assisted fashion. No hydrolytic processes were noticed in peptides -TEAHHK- and -TESHAK- where the Ser or His-5 residues are replaced with the Ala residue. The Ni(II)-assisted hydrolysis of the analogues of -TESHHK- may provide an insight into the novel mechanism of genotoxicity, combining the damage to the nucleosome with the generation of further toxic Ni(II) species.

Exploration of Biarsenical Chemistry—Challenges in Protein Research

The fluorescent modification of proteins (with genetically encoded low‐molecular‐mass fluorophores, affinity probes, or other chemically active species) is extraordinarily useful for monitoring and controlling protein functions in vitro, as well as in cell cultures and tissues. The large sizes of some fluorescent tags, such as fluorescent proteins, often perturb normal activity and localization of the protein of interest, as well as other effects. Of the many fluorescent‐labeling strategies applied to in vitro and in vivo studies, one is very promising. This requires a very short (6‐ to12‐residue), appropriately spaced, tetracysteine sequence (CCXXCC); this is either placed at a protein terminus, within flexible loops, or incorporated into secondary structure elements. Proteins that contain the tetracysteine motif become highly fluorescent upon labeling with a nonluminescent biarsenical probe, and form very stable covalent complexes. We focus on the development, growth, and multiple applications of this protein research methodology, both in vitro and in vivo. Its application is not limited to intact‐cell protein visualization; it has tremendous potential in other protein research disciplines, such as protein purification and activity control, electron microscopy imaging of cells or tissue, protein–protein interaction studies, protein stability, and aggregation studies.