Search

Alzheimer's disease (AD) is one of the most common of the multifactorial diseases and is characterized by a range of abnormal molecular processes, such as the accumulation of extracellular plaques containing the amyloid-β (Aβ) peptides and dyshomeostasis of copper in the brain. In this study, we have investigated the effect of CuII on the aggregation of Aβ1-40 and Aβ4-40 , representing the two most prevalent families of Aβ peptides, that is, the full length and N-truncated peptides. Both families are similarly abundant in healthy and AD brains. For either of the studied peptides, substoichiometric CuII concentrations accelerated aggregation, whereas superstoichiometric CuII inhibited fibril formation, likely by stabilizing the oligomers. The addition of either Aβ4-40 or substoichiometric CuII affected the aggregation profile of Aβ1-40 , by yielding shorter and thicker fibrils; amorphous aggregates were formed in the presence of a molar excess of CuII . The similarity of these two effects can be attributed to the increase in the positive charge on the Aβ N terminus, caused both by CuII complexation and N truncation at position 4. Our findings provide a better understanding of the biological Aβ aggregation process as these two Aβ species and CuII coexist and interact under physiological conditions.

Covalent protein kinase inhibitors exploit currently noncatalytic cysteines in the adenosine 5′-triphosphate (ATP)-binding site via electrophiles directly appended to a reversible-inhibitor scaffold. Here, we delineate a path to target solvent-exposed cysteines at a distance >10 Å from an ATP-site-directed core module and produce potent covalent phosphoinositide 3-kinase α (PI3Kα) inhibitors. First, reactive warheads are used to reach out to Cys862 on PI3Kα, and second, enones are replaced with druglike warheads while linkers are optimized. The systematic investigation of intrinsic warhead reactivity (kchem), rate of covalent bond formation and proximity (kinactand reaction space volume Vr), and integration of structure data, kinetic and structural modeling, led to the guided identification of high-quality, covalent chemical probes. A novel stochastic approach provided direct access to the calculation of overall reaction rates as a function of kchem, kinact, Ki, and Vr, which was validated with compounds with varied linker lengths. X-ray crystallography, protein mass spectrometry (MS), and NanoBRET assays confirmed covalent bond formation of the acrylamide warhead and Cys862. In rat liver microsomes, compounds 19 and 22 outperformed the rapidly metabolized CNX-1351, the only known PI3Kα irreversible inhibitor. Washout experiments in cancer cell lines with mutated, constitutively activated PI3Kα showed a long-lasting inhibition of PI3Kα. In SKOV3 cells, compounds 19 and 22 revealed PI3Kβ-dependent signaling, which was sensitive to TGX221. Compounds 19 and 22 thus qualify as specific chemical probes to explore PI3Kα-selective signaling branches. The proposed approach is generally suited to develop covalent tools targeting distal, unexplored Cys residues in biologically active enzymes., Journal of the American Chemical Society, 144 (14), ISSN:0002-7863, ISSN:1520-5126

The stochastic nature of chemical reaction and impact of the stochasticity on their evolution is soundly documented. Both theoretical predictions and emerging experimental evidence indicate the influence of stochastic effects on the equilibrium state of association reaction. In this work simple mathematical formulas are introduced to estimate these effects. First, the dependence of the ratio of observed reactants (apparent association constant, equivalent of macroscopic association constant in stochastic analysis) on the volume and the number of molecules of reagents is discussed and the limiting factors of this effect are shown. Next, the apparent association constant is approximated for nanoscale systems by closed-form formulas derived for this purpose. Finally, an estimation for the macroscopic constant value from the apparent one is provided and validated on the published experimental data. This work was inspired by chemical reactions occurring in biological compartments, but the results can be used for all systems belonging to the stochastic regime of chemical reactions.

Alzheimer's disease (AD) is one of the most common of the multifactorial diseases and is characterized by a range of abnormal molecular processes, such as the accumulation of extracellular plaques containing the amyloid-β (Aβ) peptides and dyshomeostasis of copper in the brain. In this study, we have investigated the effect of Cu

The intracellular exchangeable Zn(II) is usually measured with synthetic fluorescent zinc sensors. 4',5'-Bis[bis(2-pyridylmethyl)aminomethyl]-2',7'-dichlorofluorescein (Zinpyr-1) is a sensor containing the fluorescein platform and a duplicated chelating unit. Its advantages include brightness and a relatively high affinity for Zn(II)

Tetrathiolate zinc fingers are potential targets of oxidative assault under cellular stress conditions. We used the synthetic 37-residue peptide representing the tetrathiolate zinc finger domain of the DNA repair protein XPA, acetyl-DYVICEECGKEFMSYLMNHFDLPTCDNCRDADDKHK-amide (XPAzf) as a working model to study the reaction of its Zn(II) complex (ZnXPAzf) with hydrogen peroxide and S-nitrosoglutathione (GSNO), as oxidative and nitrosative stress agents, respectively. We also used the Cd(II) substituted XPAzf (CdXPAzf) to assess the situation of cadmium assault, which is accompanied by oxidative stress. Using electrospray mass spectrometry (ESI-MS), HPLC, and UV-vis and circular dichroism spectroscopies we demonstrated that even very low levels of H2O2 and GSNO invariably cause irreversible thiol oxidation and concomitant Zn(II) release from ZnXPAzf. In contrast, CdXPAzf was more resistant to oxidation, demonstrating the absence of synergy between cadmium and oxidative stresses. Our results indicate that GSNO cannot act as a reversible modifier of XPA, and rather has a deleterious effect on DNA repair.

Knowledge of the nature of exchangeable (labile) intracellular Zn(II) is increasingly important for biomedical research. The detection and quantitative determination of Zn(II) ions is usually performed by using Zn(II)-specific fluorescent sensors, among which 2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) has been used most widely. Selectivity of this sensor for Zn(II) over other divalent cations was demonstrated, but possible interference in its performance by other compounds has not been investigated. Many potential low molecular weight ligands for Zn(II) ions (LMWLs) are abundant in the cell. In this study we demonstrate that FluoZin-3 is susceptible to competition for Zn(II) from LMWLs and also forms strong ternary complexes with some of them. We determined the set of conditional stability constants

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.

Seleno-substituted model peptides of copper metallochaperone proteins were analyzed for the metal affinity and in vitro anti-oxidative reactivity. An acyclic MTCXXC (X is any amino acid) reference peptide previously analyzed as a potent inhibitor of ROS production underwent substitution of the cysteine residues with selenocysteine to give two singly substituted derivatives C3U and C6U and the doubly substituted analogue C3U/C6U. Presumably due to the softer nature of Se vs. S, all selenocysteine containing peptides demonstrated high affinity to Cu(I), higher than that of the reference peptide, and in the same order of magnitude as that measured for the native protein, Atox1. A stronger impact of residue 3 confirmed previous findings on its more dominant role in metal coordination. In vitro studies on the HT-29 human colon cancer cell line, MEF mice embryonic fibroblasts, and MEF with the knocked-out Atox1 gene (Atox1 −/−) consistently identified C3U/C6U as the most potent inhibitor of ROS cellular production based on the 2′,7′-dichlorodihydrofluorescin diacetate (H2DCF-DA) assay, also in comparison with known drugs employed in the clinic for Wilson's disease. The selenocysteine containing peptides are thus promising drug candidates for chelation therapy of Wilson's disease and related conditions relevant to excessive copper levels.

2-[2-[2-[2-[bis(carboxylatomethyl)amino]-5-methoxyphenoxy]ethoxy]-4-(2,7-difluoro-3-oxido-6-oxo-4a,9a-dihydroxanthen-9-yl)anilino]acetate (FluoZin-3) is used very broadly in life sciences as intra- and extracellular Zn(II) sensor selective for Zn(II) over Co(II), Ca(II) and Mg(II) ions at their physiological concentrations. It has been used for determination of relative and absolute levels of exchangeable Zn(II) in cells and extracellular fluids. Despite its popularity, the knowledge of its acid/base and Zn(II) coordination abilities and of its spectroscopic properties remained very limited. Also the published conditional dissociation constant ((C)Kd) values at pH7.4 are slightly discrepant, (15nM or 8.9nM). In this work we determined the (C)Kd for Zn(II) complexation by FluoZin-3 at pH7.4 with nitrilotriacetic acid (NTA) as competitor using two independent methods: fluorimetry and UV-Vis spectroscopy. For the first time, we investigated FluoZin-3 alone and complexed with Zn(II) in the wide range of pH, determining the total of eight pKa values from fluorescence spectra and from various regions of UV-Vis spectra. The validated values of (C)Kd (9.1±0.4nM; -log (C)Kd=8.04) and of the absolute (pH-independent) stability constant log βZnL (8.16±0.05) were provided by fluorescence spectroscopy experiments performed at 1μM concentrations. Our experiments demonstrated that both of aminocarboxylate moieties of FluoZin-3 bind the Zn(II) ion synergistically.

The effect of position II in the binding sequence of copper metallochaperones, which varies between Thr and His, was investigated through structural analysis and affinity and oxidation kinetic studies of model peptides. A first Cys-Cu(I)-Cys model obtained for the His peptide at acidic and neutral pH, correlated with higher affinity and more rapid oxidation of its complex; in contrast, the Thr peptide with the Cys-Cu(I)-Met coordination under neutral conditions demonstrated weaker and pH dependent binding. Studies with human antioxidant protein 1 (Atox1) and three of its mutants where S residues were replaced with Ala suggested that (a) the binding affinity is influenced more by the binding sequence than by the protein fold (b) pH may play a role in binding reactivity, and (c) mutating the Met impacted the affinity and oxidation rate more drastically than did mutating one of the Cys, supporting its important role in protein function. Position II thus plays a dominant role in metal binding and transport.

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.