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Tobacco smoking is deleterious to the lungs because it exposes them to many toxic substances. These include transition metal ions, such as cadmium. However, there is a lack of information about the influence of endogenous metal-binding peptides, such as His-Leu (HL), on the lung distribution of transition metals in smokers. To address this, we administered HL subcutaneously to rats exposed to tobacco smoke for six weeks, then we measured the concentrations of transition metal ions in the lungs. We found that exposure to tobacco smoke elevates the concentrations of Cd(II) and Cu(II). Administration of the HL peptide, whose elevation is a consequence of angiotensin receptor blocker anti-hypertension therapy, increases the concentration of Fe in the lungs of rats exposed to smoke. These findings suggest that smoking is a risk factor for patients receiving angiotensin receptor blockers to treat hypertension.

Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu2+ as part of their normal functions. GHK is found at its highest concentration in the albumin-rich fraction of plasma, leading to speculation that HSA and GHK form a ternary Cu2+ complex. Although preliminary evidence was presented 40 years ago, the structure and stability of such a complex have remained elusive. Here, we show that two ternary Cu(GHK)NImHSA complexes are formed between GHK and the imino nitrogen (NIm) of His side chains of HSA. We identified His3 as one site of ternary complex formation (conditional binding constant cKCu(GHK)NImHis3Cu(GHK) = 2900 M-1 at pH 7.4), with the second site (cKCu(GHK)NImHisXCu(GHK) = 1700 M-1) likely being supplied by either His128 or His510. Together with the established role of HSA as a molecular shuttle in the blood, these complexes may aid the transport of the exchangeable Cu2+ pool and the functional form of GHK.

Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV–vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter., Gly-His-Lys (GHK) is a human bioactive tripeptide thought to be activated by Cu(II) binding, but little is known about the molecular aspects of its action. UV−vis, circular dichroism (CD), EPR, and NMR spectroscopies, and cyclic voltammetry were used to examine the reduction of Cu(II)GHK with glutathione (GSH), the most abundant biological thiol. A semistable GSH-Cu(II)GHK reaction intermediate was discovered, with properties suitable for delivering Cu(I) to biological transport proteins.

ATCUN (amino terminal Cu(II) and Ni(II) binding) motifs chelate Cu(II) ions strongly. However, the impact of the phosphorylation of neighboring residues on such complexation has not been elucidated. The copper(II) dissociation constants of original and phosphorylated peptides from human histatin-1 and human serum albumin were compared using spectroscopic methods. Phosphorylation markedly weakened Cu(II) binding. Thus, these results indicate that phosphorylation may be a vital mechanism governing metal ion binding., The phosphorylation of serine residues within or nearby an ATCUN motif markedly weakens Cu(II) binding. It indicates that phosphorylation may be a vital mechanism of metal ion binding regulation.

Amyloid-β (Aβ) peptides are involved in Alzheimer's disease (AD) development. The interactions of these peptides with copper and zinc ions also seem to be crucial for this pathology. Although Cu(II) and Zn(II) ions binding by Aβ peptides has been scrupulously investigated, surprisingly, this phenomenon has not been so thoroughly elucidated for N-truncated Aβ

NiO nanoparticles and non-stoichiometric black NiO were shown to be effective sources of Ni2+ ions causing sequence-selective peptide bond hydrolysis. NiO nanoparticles were as effective in this reaction as their molar equivalent of soluble Ni(ii) salt. These findings highlight the efficacy of delivery of toxic Ni2+ by these environmentally available particles.

Human serum albumin (HSA) and the growth factor glycyl-l-histidyl-l-lysine (GHK) bind Cu

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu2+ -binding ligand in blood and cerebrospinal fluid. Using a combination of electronic absorption, circular dichroism and room-temperature electron paramagnetic resonance spectroscopies, together with potentiometric titrations, we competed the tripeptide GGH against HSA to reveal a conditional binding constant of log c K Cu Cu ( HSA ) =13.02±0.05 at pH 7.4. This rigorously determined value of the Cu2+ affinity has important implications for understanding the extracellular distribution of copper.

Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1 , His2 , and His3 ) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2 , thus, non-saturating the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) reduction to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those containing His1 , His2 , or His3 . Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the analysis. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the respective sequences with the probability of interaction with divalent copper.

Currently available chemotherapeutic treatments for blood cancers (leukemia) usually have strong side effects. More selective, efficient and less toxic anticancer agents are needed. We synthesized seven, new, optically pure (12aS)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12-(2H,11H)-dione derivatives and examined their cytotoxicity towards eight cancer cell lines, including urinary bladder (TCC-SUP, UM-UC-3, KU-19-9), colon (LoVo), and breast (MCF-7, MDA-MB-231) cancer representatives, as well as two leukemic cell lines (MV-4-11, CCRF-CEM) and normal murine fibroblasts (Balb/3T3) as a reference cell line. Three of the seven newly-obtained compounds ((12aS)-8-bromo-2-(3-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione, (12aS)-8,9-dimethoxy-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione and (12aS)-8-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione, showed enhanced activity and selectivity toward the leukemic MV-4-11 cell line when compared to our previously reported compounds, with IC50 values in the range of 2.9-5.6 µM. Additionally, (12aS)-9-nitro-2-(4-phenylbenzoyl)-1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12(2H,11H)-dione exhibited a strong cytotoxic effect against the leukemic CCRF-CEM (IC50 = 6.1 µM) and MV-4-11 (IC50 = 11.0 µM) cell lines, a moderate cytotoxic effect toward other tumor lines (IC50 = 31.8-55.0 µM) and very weak cytotoxic effect toward the Balb/3T3 reference cell line. Selected compounds were further evaluated for their potential to induce apoptotic cell death in MV-4-11 cells by measuring caspase-3 activity. We also obtained the crystal structure of three products and investigated the effect of 22 derivatives of 1,3,4,12a-tetrahydropyrazino[2,1-c][1,4]benzodiazepine-6,12-(2H,11H)-dione on the activity of the cancer-associated enzyme autotaxin. All compounds proved to be weak inhibitors of autotaxin, although some (R) and (S) enantiomers had Ki values of 10-19 µM. The obtained results showed that the tested compounds exhibited a selective anti-leukemic effect, which appeared not to be related directly to autotaxin. Molecular targets responsible for this effect remain to be identified. The newly obtained compounds can be used in the search for new, selective anticancer therapies.

Sporadic Alzheimer's disease (AD) is associated with an inefficient clearance of the β-amyloid (Aβ) peptide from the central nervous system. The protein levels and activity of the Zn2+-dependent endopeptidase neprilysin (NEP) inversely correlate with brain Aβ levels during aging and in AD. The present study considered the ability of Cu2+ ions to inhibit human recombinant NEP and the role for NEP in generating N-truncated Aβ fragments with high-affinity Cu2+ binding motifs that can prevent this inhibition. Divalent copper noncompetitively inhibited NEP ( Ki = 1.0 μM), while proteolysis of Aβ yielded the soluble, Aβ4-9 fragment that can bind Cu2+ with femtomolar affinity at pH 7.4. This provides Aβ4-9 with the potential to act as a Cu2+ carrier and to mediate its own production by preventing NEP inhibition. Enzyme inhibition at high Zn2+ concentrations ( Ki = 20 μM) further suggests a mechanism for modulating NEP activity, Aβ4-9 production, and Cu2+ homeostasis.

Three crystal structures are presented of nematode thymidylate synthases (TS), including Caenorhabditis elegans (Ce) enzyme without ligands and its ternary complex with dUMP and Raltitrexed, and binary complex of Trichinella spiralis (Ts) enzyme with dUMP. In search of differences potentially relevant for the development of species-specific inhibitors of the nematode enzyme, a comparison was made of the present Ce and Ts enzyme structures, as well as binary complex of Ce enzyme with dUMP, with the corresponding mammalian (human, mouse and rat) enzyme crystal structures. To complement the comparison, tCONCOORD computations were performed to evaluate dynamic behaviors of mammalian and nematode TS structures. Finally, comparative molecular docking combined with molecular dynamics and free energy of binding calculations were carried out to search for ligands showing selective affinity to T. spiralis TS. Despite an overall strong similarity in structure and dynamics of nematode vs mammalian TSs, a pool of ligands demonstrating predictively a strong and selective binding to TsTS has been delimited. These compounds, the E63 family, locate in the dimerization interface of TsTS where they exert species-specific interactions with certain non-conserved residues, including hydrogen bonds with Thr174 and hydrophobic contacts with Phe192, Cys191 and Tyr152. The E63 family of ligands opens the possibility of future development of selective inhibitors of TsTS and effective agents against trichinellosis.

Nickel is harmful to humans, being both carcinogenic and allergenic. However, the mechanisms of this toxicity are still unresolved. We propose that Ni(II) ions disintegrate proteins by hydrolysis of peptide bonds preceding the Ser/Thr-Xaa-His sequences. Such sequences occur in nuclear localization signals (NLSs) of human phospholipid scramblase 1, Sam68-like mammalian protein 2, and CLK3 kinase. We performed spectroscopic experiments showing that model nonapeptides derived from these NLSs bind Ni(II) at physiological pH. We also proved that these sequences are prone to Ni(II) hydrolysis. Thus, the aforementioned NLSs may be targets for nickel toxicity. This implies that Ni(II) ions disrupt the transport of some proteins from cytoplasm to cell nucleus.

The apparent affinity of human serum albumin (HSA) for divalent copper has long been the subject of great interest, due to its presumed role as the major Cu

We describe the adsorption behaviour and rheological properties of a calf skin type I collagen, and of its hydrolysates obtained using a Clostridium histolyticum collagenase (CHC) under moderate conditions (pH 7, 37°C). The effect of CHC concentration (2×10(-9)-2×10(-6)M) and incubation time (35-85min) was studied and optimised to achieve the highest decrease of surface tension and the highest dilational surface viscoelasticity of the adsorbed layers. SDS-PAGE electrophoresis and reverse-phase high performance liquid chromatography (RP-HPLC) were used to characterise the hydrolysis products. The results show that even simple modifications (heat treatment, pH change, partial hydrolysis) of collagen enhances its surface properties, especially in terms of surface dilational elasticity modulus. The use of low enzyme concentration (CHC-to-collagen molar ratio of 4×10(-3)) and short incubation time (

The A&beta, 4&minus, 42 peptide is a major beta-amyloid species in the human brain, forming toxic aggregates related to Alzheimer&rsquo, s Disease. It also strongly chelates Cu(II) at the N-terminal Phe-Arg-His ATCUN motif, as demonstrated in A&beta, 16 and A&beta, 9 model peptides. The resulting complex resists ROS generation and exchange processes and may help protect synapses from copper-related oxidative damage. Structural characterization of Cu(II)A&beta, x complexes by NMR would help elucidate their biological function, but is precluded by Cu(II) paramagneticism. Instead we used an isostructural diamagnetic Pd(II)-A&beta, 16 complex as a model. To avoid a kinetic trapping of Pd(II) in an inappropriate transient structure, we designed an appropriate pH-dependent synthetic procedure for ATCUN Pd(II)A&beta, 16, controlled by CD, fluorescence and ESI-MS. Its assignments and structure at pH 6.5 were obtained by TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-15N HSQC NMR experiments, for natural abundance 13C and 15N isotopes, aided by corresponding experiments for Pd(II)-Phe-Arg-His. The square-planar Pd(II)-ATCUN coordination was confirmed, with the rest of the peptide mostly unstructured. The diffusion rates of A&beta, 16, Pd(II)-A&beta, 16 and their mixture determined using PGSE-NMR experiment suggested that the Pd(II) complex forms a supramolecular assembly with the apopeptide. These results confirm that Pd(II) substitution enables NMR studies of structural aspects of Cu(II)-A&beta, complexes.

The tripeptide NH2&ndash, Gly&ndash, His&ndash, Lys&ndash, COOH (GHK), cis-urocanic acid (cis-UCA) and Cu(II) ions are physiological constituents of the human body and they co-occur (e.g., in the skin and the plasma). While GHK is known as Cu(II)-binding molecule, we found that urocanic acid also coordinates Cu(II) ions. Furthermore, both ligands create ternary Cu(II) complex being probably physiologically functional species. Regarding the natural concentrations of the studied molecules in some human tissues, together with the affinities reported here, we conclude that the ternary complex [GHK][Cu(II)][cis-urocanic acid] may be partly responsible for biological effects of GHK and urocanic acid described in the literature.

Nickel is harmful for humans, but molecular mechanisms of its toxicity are far from being fully elucidated. One of such mechanisms may be associated with the Ni(II)-dependent peptide bond hydrolysis, which occurs before Ser/Thr in Ser/Thr-Xaa-His sequences. Human annexins A1, A2, and A8, proteins modulating the immune system, contain several such sequences. To test if these proteins are potential molecular targets for nickel toxicity we characterized the binding of Ni(II) ions and hydrolysis of peptides Ac-KALTGHLEE-am (A1-1), Ac-TKYSKHDMN-am (A1-2), and Ac-GVGTRHKAL-am (A1-3), from annexin A1, Ac-KMSTVHEIL-am (A2-1) and Ac-SALSGHLET-am (A2-2), from annexin A2, and Ac-VKSSSHFNP-am (A8-1), from annexin A8, using UV-vis and circular dichroism (CD) spectroscopies, potentiometry, isothermal titration calorimetry, high-performance liquid chromatography (HPLC), and electrospray ionization mass spectrometry (ESI-MS). We found that at physiological conditions (pH 7.4 and 37 °C) peptides A1-2, A1-3, A8-1, and to some extent A2-2 bind Ni(II) ions sufficiently strongly in 4N complexes and are hydrolyzed at sufficiently high rates to justify the notion that these annexins can undergo nickel hydrolysis in vivo. These results are discussed in the context of specific biochemical interactions of respective proteins. Our results also expand the knowledge about Ni(II) binding to histidine peptides by determination of thermodynamic parameters of this process and spectroscopic characterization of 3N complexes. Altogether, our results indicate that human annexins A1, A2, and A8 are potential molecular targets for nickel toxicity and help design appropriate cellular studies.

In view of previous crystallographic studies, N4-hydroxy-dCMP, a slow-binding thymidylate synthase inhibitor apparently caused “uncoupling” of the two thymidylate synthase-catalyzed reactions, including the N5,10-methylenetetrahydrofolate one-carbon group transfer and reduction, suggesting the enzyme's capacity to use tetrahydrofolate as a cofactor reducing the pyrimidine ring C(5) in the absence of the 5-methylene group. Testing the latter interpretation, a possibility was examined of a TS-catalyzed covalent self-modification/self-inactivation with certain pyrimidine deoxynucleotides, including 5-fluoro-dUMP and N4-hydroxy-dCMP, that would be promoted by tetrahydrofolate and accompanied with its parallel oxidation to dihydrofolate. Electrophoretic analysis showed mouse recombinant TS protein to form, in the presence of tetrahydrofolate, a covalently bound, electrophoretically separable 5-fluoro-dUMP-thymidylate synthase complex, similar to that produced in the presence of N5,10-methylenetetrahydrofolate. Further studies of the mouse enzyme binding with 5-fluoro-dUMP/N4-hydroxy-dCMP by TCA precipitation of the complex on filter paper showed it to be tetrahydrofolate-promoted, as well as to depend on both time in the range of minutes and the enzyme molecular activity, indicating thymidylate synthase-catalyzed reaction to be responsible for it. Furthermore, the tetrahydrofolate- and time-dependent, covalent binding by thymidylate synthase of each 5-fluoro-dUMP and N4-hydroxy-dCMP was shown to be accompanied by the enzyme inactivation, as well as spectrophotometrically confirmed dihydrofolate production, the latter demonstrated to depend on the reaction time, thymidylate synthase activity and temperature of the incubation mixture, further documenting its catalytic character.

Crystal structures were solved of the binary complexes Trichinella spiralis and Caenorhabditis elegans thymidylate synthases with deoxyuridine monophosphate (dUMP), with crystals obtained by the vapor diffusion method in hanging drops. For the T. spiralis thymidylate synthase-dUMP complex, the diffraction data were collected at the BESSY Synchrotron to 1.9 Å resolution. The crystal belongs to the space group P1 with two dimers in the asymmetric unit (ASU). For the C. elegans TS-dUMP complex crystal, the diffraction data were collected at the BESSY Synchrotron to 2.48 Å resolution, and the crystal belongs to the space group P 32 2 1, with two monomers (one dimer) in the ASU. Structural comparisons were made of both structures and each of them with the corresponding mouse thymidylate synthase complex.

Endogenous thymidylate synthases, isolated from tissues or cultured cells of the same specific origin, have been reported to show differing slow-binding inhibition patterns. These were reflected by biphasic or linear dependence of the inactivation rate on time and accompanied by differing inhibition parameters. Considering its importance for chemotherapeutic drug resistance, the possible effect of thymidylate synthase inhibition by post-translational modification was tested, e.g. phosphorylation, by comparing sensitivities to inhibition by two slow-binding inhibitors, 5-fluoro-dUMP and N(4)-hydroxy-dCMP, of two fractions of purified recombinant mouse enzyme preparations, phosphorylated and non-phosphorylated, separated by metal oxide/hydroxide affinity chromatography on Al(OH)3 beads. The modification, found to concern histidine residues and influence kinetic properties by lowering Vmax, altered both the pattern of dependence of the inactivation rate on time from linear to biphasic, as well as slow-binding inhibition parameters, with each inhibitor studied. Being present on only one subunit of at least a great majority of phosphorylated enzyme molecules, it probably introduced dimer asymmetry, causing the altered time dependence of the inactivation rate pattern (biphasic with the phosphorylated enzyme) and resulting in asymmetric binding of each inhibitor studied. The latter is reflected by the ternary complexes, stable under denaturing conditions, formed by only the non-phosphorylated subunit of the phosphorylated enzyme with each of the two inhibitors and N(5,10)-methylenetetrahydrofolate. Inhibition of the phosphorylated enzyme by N(4)-hydroxy-dCMP was found to be strongly dependent on [Mg(2+)], cations demonstrated previously to also influence the activity of endogenous mouse TS isolated from tumour cells.

International audience; Phosphorylation of proteins is an essential signaling mechanism in eukaryotic and prokaryotic cells. Although N-phosphorylation of basic amino acid is known for its importance in biological systems, it is still poorly explored in terms of products and mechanisms. In this study, two mass spectrometry fragmentation methods, electron-capture dissociation (ECD) and collision-induced dissociation (CID), were tested as tools for analysis of N-phosphorylation of three model peptides, RKRSRAE, RKRARKE, and PLSRTLSVAAKK. The peptides were phosphorylated by the reaction with monopotassium phosphoramidate. The results were confirmed by the 1H NMR and 31P NMR studies. The ECD method was found useful for the localization of phosphorylation sites in unstable lysine-phosphorylated peptides. Its main advantage is a significant reduction of the neutral losses related to phosphoramidate moiety. Moreover, the results indicate that the ECD mass spectrometry may be useful for analysis of regioselectivity of the N-phosphorylation reaction. Stabilities of the obtained lysine-phosphorylated peptides under various conditions were also tested.

In search of an activity-preserving protein thiophosphorylation method, with thymidylate synthase recombinant protein used as a substrate, potassium thiophosphoramidate and diammonium thiophosphoramidate salts in Tris- and ammonium carbonate based buffer solutions were employed, proving to serve as a non-destructive environment. Using potassium phosphoramidate or diammonium thiophosphoramidate, a series of phosphorylated and thiophosphorylated amino acid derivatives was prepared, helping, together with computational (using density functional theory, DFT) estimation of (31)P NMR chemical shifts, to assign thiophosphorylated protein NMR resonances and prove the presence of thiophosphorylated lysine, serine and histidine moieties. Methods useful for prediction of (31)P NMR chemical shifts of thiophosphorylated amino acid moieties, and thiophosphates in general, are also presented. The preliminary results obtained from trypsin digestion of enzyme shows peak at m/z 1825.805 which is in perfect agreement with the simulated isotopic pattern distributions for monothiophosphate of TVQQQVHLNQDEYK where thiophosphate moiety is attached to histidine (His(26)) or lysine (Lys(33)) side-chain.

Thymidylate synthase (TS) may undergo phosphorylation endogenously in mammalian cells, and as a recombinant protein expressed in bacterial cells, as indicated by the reaction of purified enzyme protein with Pro-Q® Diamond Phosphoprotein Gel Stain (PGS). With recombinant human, mouse, rat, Trichinella spiralis and Caenorhabditis elegans TSs, expressed in Escherichia coli, the phosphorylated, compared to non-phosphorylated recombinant enzyme forms, showed a decrease in Vmax(app), bound their cognate mRNA (only rat enzyme studied), and repressed translation of their own and several heterologous mRNAs (human, rat and mouse enzymes studied). However, attempts to determine the modification site(s), whether endogenously expressed in mammalian cells, or recombinant proteins, did not lead to unequivocal results. Comparative ESI-MS/analysis of IEF fractions of TS preparations from parental and FdUrd-resistant mouse leukemia L1210 cells, differing in sensitivity to inactivation by FdUMP, demonstrated phosphorylation of Ser(10) and Ser(16) in the resistant enzyme only, although PGS staining pointed to the modification of both L1210 TS proteins. The TS proteins phosphorylated in bacterial cells were shown by (31)P NMR to be modified only on histidine residues, like potassium phosphoramidate (KPA)-phosphorylated TS proteins. NanoLC-MS/MS, enabling the use of CID and ETD peptide fragmentation methods, identified several phosphohistidine residues, but certain phosphoserine and phosphothreonine residues were also implicated. Molecular dynamics studies, based on the mouse TS crystal structure, allowed one to assess potential of several phosphorylated histidine residues to affect catalytic activity, the effect being phosphorylation site dependent.

Accumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system.

Human alpha-1 antitrypsin (AAT) is an abundant serum protein present at a concentration of 1.0-1.5 g L(-1). AAT deficiency is a genetic disease that manifests with emphysema and liver cirrhosis due to the accumulation of a misfolded AAT mutant in hepatocytes. Lung AAT amount is inversely correlated with chronic obstructive pulmonary disease (COPD), a serious and often deadly condition, with increasing frequency in the aging population. Exposure to cigarette smoke and products of fossil fuel combustion aggravates AAT deficiency and COPD according to mechanisms that are not fully understood. Taking into account that these fumes contain particles that can release nickel to human airways and skin, we decided to investigate interactions of AAT with Ni(ii) ions within the paradigm of Ni(ii)-dependent peptide bond hydrolysis. We studied AAT protein derived from human blood using HPLC, SDS-PAGE, and mass spectrometry. These studies were aided by spectroscopic experiments on model peptides. As a result, we identified three hydrolysis sites in AAT. Two of them are present in the N-terminal part of the molecule next to each other (before Thr-13 and Ser-14 residues) and effectively form one N-terminal cleavage site. The single C-terminal cleavage site is located before Ser-285. The N-terminal hydrolysis was more efficient than the C-terminal one, but both abolished the ability of AAT to inhibit trypsin in an additive manner. Nickel ions bound to hydrolysis products demonstrated an ability to generate ROS. These results implicate Ni(ii) exposure as a contributing factor in AAT-related pathologies.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a key eukaryotic enzyme,catalyzing the NAD+ dependent poly(ADP-ribosyl)ation of protein substrates, crucial for major DNA repair pathways, and involved in other fundamental cellular processes, such as transcription, cell cycle control, and apoptosis. Its ability to bind DNA depends on two CCHC zinc finger domains, in short, PARPzf1 and PARPzf2. Using spectroscopic methods and competitive titrations with Zn(II), Co(II), and Ni(II) ions, we determined conditional dissociation constants for Zn(II) complexes of PARPzf1 and PARPzf2 at pH 7.4 (HEPESbuffer) as 26 ± 4 nM and 4 ± 1 pM, respectively. The former value indicates an extremely low affinity of PARPzf1 toward metal ions, meaning that under cellular conditions PARP1zf might be largely present in a “metal-free” state. This finding provides a clue to the high susceptibility of PARP-1 to oxidative stress but also raises questions regarding the activation of PARPzf1 under cellular conditions. We also determined conditional dissociation constants for Ni(II) complexes of PARPzf1 and PARPzf2 under the same conditions as 0.78 ± 0.04 μM and 0.26 ± 0.05 nM, respectively.

cis-Urocanic acid, a derivative of histidine, is one of the essential components of human skin. We found that it can bind nickel(II) ions in a pH-dependent manner, with the dissociation constant in the low millimolar range, as revealed by potentiometry, and confirmed by isothermal titration calorimetry and UV-vis spectroscopy. The binding occurs within the physiological skin pH range. Considering the fact that cis-urocanic acid is present in the human skin in concentrations as high as millimolar, this molecule may be a physiologically important player in nickel trafficking in the human organism.

2-Amino-2-hydroxymethyl-propane-1,3-diol, or tris(hydroxymethyl)aminomethane (Tris), is probably the most common biochemical buffer used alone or in combination with other buffers because it is stable, unreactive, and compatible with most proteins and other biomolecules. Being nontoxic, it has even found applications in medicine. Tris is known, however, to coordinate transition metal ions, Cu(II) among them. Although often ignored, this feature affects interactions of Cu(II) ions with biomolecules, as Tris is usually used in high molar excess. Therefore, it is important to have precise knowledge on the stoichiometry, stability, and reactivity of cupric Tris complexes. The literature data are incoherent in this respect. We reinvestigated the complex formation in the Tris-Cu(II) system by potentiometry, UV-vis, ESI-MS, and EPR at a broad range of concentrations and ratios. We found, contrary to several previous papers, that the maximum stoichiometry of Tris to Cu(II) is 2 and at neutral pH, dimeric complexes are formed. The apparent affinity of Tris buffer for Cu(II), determined by the competitivity index (CI) approach [Krezel, A.; Wojcik, J.; Maciejczyk, M.; Bal, W. Chem. Commun. 2003, 6, 704-705] at pH 7.4 varies between 2 × 10(6) and 4 × 10(4) M(-1), depending on the Tris and Cu(II) concentrations and molar ratio.

Highly purified preparations of thymidylate synthase, isolated from calf thymus, and L1210 parental and FdUrd-resistant cells, were found to be nitrated, as indicated by a specific reaction with anti-nitro-tyrosine antibodies, suggesting this modification to appear endogenously in normal and tumor tissues. Each human, mouse and Ceanorhabditis elegans recombinant TS preparation, incubated in vitro in the presence of NaHCO(3), NaNO(2) and H(2)O(2) at pH 7.5, underwent tyrosine nitration, leading to a V(max)(app) 2-fold lower following nitration of 1 (with human or C. elegans TS) or 2 (with mouse TS) tyrosine residues per monomer. Enzyme interactions with dUMP, meTHF or 5-fluoro-dUMP were not distinctly influenced. Nitration under the same conditions of model tripeptides of a general formula H(2)N-Gly-X-Gly-COOH (X = Phe, Tyr, Trp, Lys, Arg, His, Ser, Thr, Cys, Gly), monitored by NMR spectroscopy, showed formation of nitro-species only for H-Gly-Tyr-Gly-OH and H-Gly-Phe-Gly-OH peptides, the chemical shifts for nitrated H-Gly-Tyr-Gly-OH peptide being in a very good agreement with the strongest peak found in (15)N-(1)H HMBC spectrum of nitrated protein. MS analysis of nitrated human and C. elegans proteins revealed several thymidylate synthase-derived peptides containing nitro-tyrosine (at positions 33, 65, 135, 213, 230, 258 and 301 in the human enzyme) and oxidized cysteine (human protein Cys(210), with catalytically critical Cys(195) remaining apparently unmodified) residues.

Regulation by phosphorylation is a well-established mechanism for controlling biological activity of proteins. Recently, phosphorylation of serine 124 in human thymidylate synthase (hTS) has been shown to lower the catalytic activity of the enzyme. To clarify a possible mechanism of the observed influence, molecular dynamics (MD), essential dynamics (ED) and MM-GBSA studies were undertaken. Structures derived from the MD trajectories reveal incorrect binding alignment between the pyrimidine ring of the substrate, dUMP, and the pterine ring of the cofactor analogue, THF, in the active site of the phosphorylated enzyme. The ED analysis indicates changes in the behavior of collective motions in the phosphorylated enzyme, suggesting that the formation of the closed ternary complex is hindered. Computed free energies, in agreement with structural analysis, predict that the binding of dUMP and THF to hTS is favored in the native compared to phosphorylated state of the enzyme. The paper describes at the structural level how phosphorylation at the distant site influences the ligand binding. We propose that the ‘phosphorylation effect’ is transmitted from the outside loop of Ser 124 into the active site via a subtle mechanism initiated by the long-range electrostatic repulsion between the phosphate groups of dUMP and Ser124. The mechanism can be described in terms of the interplay between the two groups of amino acids: the link (residues 125–134) and the patch (residues 189–192), resulting in the change of orientation of the pyrimidine ring of dUMP, which, in turn, prevents the correct alignment between the latter ring and the pterin ring of THF.

Crystal structures of mouse thymidylate synthase (mTS) in complexes with (1) sulfate anion, (2) 2′-deoxyuridine 5′-monophosphate (dUMP) and (3) 5-fluoro-dUMP (FdUMP) and N 5,10-methylenetetrahydrofolate (meTHF) have been determined and deposited in Protein Data Bank under the accession codes 3IHI, 4E5O and 5FCT, respectively. The structures show a strong overall similarity to the corresponding structures of rat and human thymidylate synthases (rTS and hTS, respectively). Unlike with hTS, whose unliganded and liganded forms assume different conformations (“inactive” and “active,” respectively) in the loop 181–197, in each of the three mTS structures, the loop 175–191, homologous to hTS loop 181–197, populates the active conformer, with catalytic Cys 189 buried in the active site and directed toward C(6) of the pyrimidine ring of dUMP/FdUMP, pointing to protein’s inability to adopt the inactive conformation. The binary structures of either dUMP- or sulfate-bound mTS, showing the enzyme with open active site and extended C-terminus, differ from the structure of the mTS–5-FdUMP–meTHF ternary complex, with the active site closed and C-terminus folded inward, thus covering the active site cleft. Another difference pertains to the conformation of the Arg44 side chain in the active site-flanking loop 41–47, forming strong hydrogen bonds with the dUMP/FdUMP phosphate moiety in each of the two liganded mTS structures, but turning away from the active site entrance and loosing the possibility of H-bonding with sulfate in the sulfate-bound mTS structure.

Reversible phosphorylation is the most widespread posttranslational protein modification, playing regulatory role in almost every aspect of cell life. The majority of protein phosphorylation research has been focused on serine, threonine and tyrosine that form acid-stable phosphomonoesters. However, protein histidine, arginine and lysine residues also may undergo phosphorylation to yield acid-labile phosphoramidates, most often remaining undetected in conventional studies of protein phosphorylation. It has become increasingly evident that acid-labile protein phosphorylations play important roles in signal transduction and other regulatory processes. Beside acting as high-energy intermediates in the transfer of the phosphoryl group from donor to acceptor molecules, phosphohistidines have been found so far in histone H4, heterotrimeric G proteins, ion channel KCa3.1, annexin 1, P-selectin and myelin basic protein, as well as in recombinant thymidylate synthase expressed in bacterial cells. Phosphoarginines occur in histone H3, myelin basic protein and capsidic protein VP12 of granulosis virus, whereas phospholysine in histone H1. This overview of the current knowledge on phosphorylation of protein basic amino-acid residues takes into consideration its proved or possible roles in cell functioning. Specific requirements of studies on acid-labile protein phosphorylation are also indicated.