Interactions of an Artificial Zinc Finger Protein with Cd(II) and Hg(II): Competition and Metal and DNA Binding

Cys2His2 zinc finger proteins are important for living organisms, as they—among other functions—specifically recognise DNA when Zn(II) is coordinated to the proteins, stabilising their ββα secondary structure. Therefore, competition with other metal ions may alter their original function. Toxic metal ions such as Cd(II) or Hg(II) might be especially dangerous because of their similar chemical properties to Zn(II). Most competition studies carried out so far have involved small zinc finger peptides. Therefore, we have investigated the interactions of toxic metal ions with a zinc finger proteins consisting of three finger units and the consequences on the DNA binding properties of the protein. Binding of one Cd(II) per finger subunit of the protein was shown by circular dichroism spectroscopy, fluorimetry and electrospray ionisation mass spectrometry. Cd(II) stabilised a similar secondary structure to that of the Zn(II)-bound protein but with a slightly lower affinity. In contrast, Hg(II) could displace Zn(II) quantitatively (logβ′ ≥ 16.7), demolishing the secondary structure, and further Hg(II) binding was also observed. Based on electrophoretic gel mobility shift assays, the Cd(II)-bound zinc finger protein could recognise the specific DNA target sequence similarly to the Zn(II)-loaded form but with a ~0.6 log units lower stability constant, while Hg(II) could destroy DNA binding completely

Módszerfejlesztés fehérje-glikoziláció analízisére = Method development for protein glycosylation analysis

Az extracelluláris glikoziláció tanulmányozása némiképp elhanyagolt kutatási terület. Ennek az egyik oka az elképesztő heterogenitás: egy adott pozíció hol módosított, hol nem, és számtalan különböző cukor-szerkezetet viselhet, így a glikopeptidek többnyire szubsztöchiometrikus mennyiségben fordulnak elő. Ráadásul a poszt-transzlációs módosítások vizsgálatára általában használatos tömegspektrometria is nehezebben boldogul a glikopeptidekkel. Mi szérum-fehérjék Ser és Thr oldalláncát módosító gyakori és egyszerű cukrok vizsgálatára fókuszáltunk. Marhaszérummal dolgoztunk. Egy dúsítási eljárást dolgoztunk ki egy cukorkötő-fehérje (lektin) segítségével. A glikopeptid-elegyet egy új MS/MS technika: elektron-transzfer disszociáció (ETD) segítségével analizáltuk. Ennek sikeréhez az adatbázis-lekereső szoftvert is optimalizálni kellett az ETD adatokhoz. Kutatásunk során kb. 40 új glikozilációs helyet azonosítottunk. Ennyit eddig senkinek nem sikerült egyetlen kísérlet-sorozatból. Sejten belül is előfordul O-glikoziláció, egyetlen GlcNAc kerül a Ser/Thr oldalláncokra, regulációs és jelátviteli szerepe van. Bár biológiai szempontból nagyon eltér az extracelluláris rokonságtól, hasonló analitikai kihívást jelent. Erre a módosításra is kidolgoztunk egy dúsítási eljárást. | Studying extracellular glycosylation is a somewhat neglected research area. Partly because the incredible heterogeneity glycoproteins feature both in site occupancy and in the number of different sugar structures modifying the same site. Thus, glycopeptides almost always are present in substoichiometric quantities. In addition, these modifications are a bit difficult to tackle with mass spectrometry that is generally used for the analysis of post-translational modifications. We focused on some simple and frequently occurring sugars modifying the side-chains of Ser and Thr residues of serum proteins. We worked with bovine serum. We developed an enrichment method using a carbohydrate-binding protein (lectin). We characterized the glycopeptide mixtures utilizing a novel MS/MS technique, electron-transfer dissociation (ETD). For this purpose the softwer used for database searching also had to be optimized. We identified ~40 novel glycosylation sites, more than anybody ever assigned in a single study. O-glycosylation occurs within the cell too: a single GlcNAc is deposited on Ser/Thr side chains. It fulfills a regulatory, signaling function. Though biologically very distant from its extracellular relatives, it represents a similar analytical challenge. We developed an enrichment method for this modification too

Hydrolytic Mechanism of a Metalloenzyme Is Modified by the Nature of the Coordinated Metal Ion

The nuclease domain of colicin E7 cleaves double-strand DNA non-specifically. Zn2+ ion was shown to be coordinated by the purified NColE7 as its native metal ion. Here, we study the structural and catalytic aspects of the interaction with Ni2+, Cu2+ and Cd2+ non-endogenous metal ions and the consequences of their competition with Zn2+ ions, using circular dichroism spectroscopy and intact protein mass spectrometry. An R447G mutant exerting decreased activity allowed for the detection of nuclease action against pUC119 plasmid DNA via agarose gel electrophoresis in the presence of comparable metal ion concentrations. It was shown that all of the added metal ions could bind to the apoprotein, resulting in a minor secondary structure change, but drastically shifting the charge distribution of the protein. Zn2+ ions could not be replaced by Ni2+, Cu2+ and Cd2+. The nuclease activity of the Ni2+-bound enzyme was extremely high in comparison with the other metal-bound forms, and could not be inhibited by the excess of Ni2+ ions. At the same time, this activity was significantly decreased in the presence of equivalent Zn2+, independent of the order of addition of each component of the mixture. We concluded that the Ni2+ ions promoted the DNA cleavage of the enzyme through a more efficient mechanism than the native Zn2+ ions, as they directly generate the nucleophilic OH− ion

Neuropeptidek radioaktív jelölése = Radioactive labelling of neuropeptides

Új jelölési módszereket fejlesztettünk ki neuropeptidek tríciummal való jelölésére. A módszerekhez új aminosavak és új neuropeptid prekurzorokra volt szükségünk. Aliciklikus béta-aminosavakat és ezek telítetlen analógjait szintetizáltunk vagy vásároltunk, racém vagy optikailag aktív formában. Az új aminosavakat felhasználtuk prekurzor peptidek szintéziséhez. A diasztereomer peptidek elválasztása radioaktív vagy nem radioaktív formában HPLC-vel történt. A radiojelölés általában trícium gázzal kettős kötés telítésével vagy dehalogénezéssel történt Pd katalizátorok jelenlétében. Az új módszerek között van a diszulfid kötést tartalmazó peptidek előállítása, valamint alaninban és valinban történő jelölés. Az aliciklikus béta-aminosavak radioaktív jelölése az első alkalommal valósult meg a jelöléstechnikában így ez a módszerünk úttörőnek tekinthető. Az így nyert radioaktív peptidek sokkal stabilabbak mint a természetes peptidek a biológiai rendszerekben. A 13 új radioaktív peptidünk (opioid peptidek, Substance P (1-7), Arg-vazopresszin) közül 3 peptidünk (Try-3H-ACPC-Phe-Phe-NH2, Dmt-3H-Pro-Phe-Phe-NH2, 3HDmt-DArg-Phe-Lys-NH2) kereskedelmi termék lett, ezen túlmenően radioaktív peptidjeink nemzetközi kutatási együttműködéseket és nemzetközi pályázatot is eredményezett. | Novel tritium labeling methods were developed for labeling of neuropeptides. New unnatural amino acids and neuropeptide precursors were needed for the labeling methods. Alicyclic-beta-amino acids and their analogues with unsaturated bond were synthesized or bought in raceme or optical pure forms. The new amino acids were used to the synthesis of the precursor peptides. The radio labeling occurred with tritium gas using catalytic saturation or dehalogenation methods using Pd catalyst. The diastereomer peptides in radioactive or no radioactive form were separated by HPLC. Among the new methods was the preparation of peptide containing disulphide bridge and radioactive labeling of peptides where the labels were in the alanine or valine residue. Pioneer method was developed when the label was introduced into the alicyclic beta-amino acids. These peptides were more stable than natural peptides in biological systems. Our 3 radioactive peptides (Dmt-DArg-Phe-Lys-NH2, Dmt-Pro-Phe-Phe-NH2, Tyr-Acpc-Phe-Phe-NH2) among 13 new peptides (opioid peptides, Substance P (1-7, 8Arg-vasopressin) became commercial product . Our radioactive peptides resulted in international research cooperation and international project

Oxygen-Dependent Changes in the N-Glycome of Murine Pulmonary Endothelial Cells

Supplemental oxygen is frequently used together with mechanical ventilation to achieve sufficient blood oxygenation. Despite the undoubted benefits, it is vigorously debated whether too much oxygen can also have unpredicted side-effects. Uncertainty is also due to the fact that the molecular mechanisms are still insufficiently understood. The lung endothelium is covered with an exceptionally broad glycocalyx, carrying N- and O-glycans, proteoglycans, glycolipids and glycosaminoglycans. Glycan structures are not genetically determined but depend on the metabolic state and the expression level and activity of biosynthetic and glycan remodeling enzymes, which can be influenced by oxygen and the redox status of the cell. Altered glycan structures can affect cell interactions and signaling. In this study, we investigated the effect of different oxygen conditions on aspects of the glycobiology of the pulmonary endothelium with an emphasis on N-glycans and terminal sialylation using an in vitro cell culture system. We combined a proteomic approach with N-glycan structure analysis by LC-MS, qRT-PCR, sialic acid analysis and lectin binding to show that constant and intermittent hyperoxia induced time dependent changes in global and surface glycosylation. An siRNA approach identified St6gal1 as being primarily responsible for the early transient increase of alpha 2-6 sialylated structures in response to hyperoxia

Interrogating the Dimerization Interface of the Prion Protein Via Site-Specific Mutations to p-Benzoyl-L-Phenylalanine

Transmissible spongiform encephalopathies are centered on the conformational transition of the prion protein from a mainly helical, monomeric structure to a beta-sheet rich ordered aggregate. Experiments indicate that the main infectious and toxic species in this process are however shorter oligomers, formation of which from the monomers is yet enigmatic. Here, we created 25 variants of the mouse prion protein site-specifically containing one genetically-incorporated para-benzoyl-phenylalanine (pBpa), a cross-linkable non-natural amino acid, in order to interrogate the interface of a prion protein-dimer, which might lie on the pathway of oligomerization. Our results reveal that the N-terminal part of the prion protein, especially regions around position 127 and 107, is integral part of the dimer interface. These together with additional pBpa-containing variants of mPrP might also facilitate to gain more structural insights into oligomeric and fibrillar prion protein species including the pathological variants. (C) 2018 Elsevier Ltd. All rights reserved

Maternal alterations in the proteome of the medial prefrontal cortex in rat

Proteomic differences between rat dams and control mothers deprived of their pups immediately after delivery were investigated in the medial prefrontal cortex (mPFC).A 2-D DIGE minimal dye technique combined with LC-MS/MS identified 32 different proteins that showed significant changes in expression in the mPFC, of which, 25 were upregulated and 7 were downregulated in dams. The identity of one significantly increased protein, the small heat-shock protein alpha-crystallin B chain (Cryab), was confirmed via Western blot analysis. Alpha-crystallin B chain was distributed in scattered cells in the mPFC, as demonstrated by immunohistochemistry. Furthermore, it was found to be localized in parvalbumin-containing neurons using double labeling. The elevation of its mRNA level in rat dams was also demonstrated via RT-PCR.The functional classification of the altered proteins was conducted using the UniProt and Gene Ontology protein databases. The identified proteins predominantly participate in synaptic transport and plasticity, neuron development, oxidative stress and apoptosis, and cytoskeleton organization. A common regulator and target analysis of these proteins determined using the Elsevier Pathway Studio Platform suggests that protein level changes associated with pup nursing are driven by growth factors and cytokines, while the MAP kinase pathway was identified as a common target. A high proportion of the proteins that were found to be altered in the mPFC are associated with depression. Biological significance: The behavior and emotional state of females change robustly when they become mothers. The brain, which governs these changes, may also undergo molecular alterations in mothers. As no proteomics approaches have been applied regarding maternal changes in the brain, we addressed this issue in the mPFC as this brain area is the uppermost cortical center of maternal control and the associated mood changes. The high number of protein-level alterations found between mothers taking care of their litter and those without pups indicates that pup nursing is associated with cortical protein-level changes. Alterations in proteins participating in synaptic transport, plasticity and neuron development suggest neuroplastic changes in the maternal brain. In turn, the relatively high number of altered proteins in the mPFC associated with depression suggests that the physiological effects of the protein-level alterations in the maternal mPFC could promote the incidence of postpartum depression. Cryab, a protein confirmed to be increased during maternal behaviors, was selectively found in parvalbumin cells, which, as fast-spiking interneurons, are associated with depression. The function of Cryab should be further investigated to establish whether it can be used to identify drug targets for future drug development. © 2016 Elsevier B.V