Shubo Han

Effect of 4-Hydroxy-2-nonenal Modification on α-Synuclein Aggregation

Several observations have implicated oxidative stress and aggregation of the presynaptic protein α-synuclein in the pathogenesis of Parkinson disease. α-Synuclein has been shown to have affinity for unsaturated fatty acids and membranes enriched in polyunsaturated fatty acids, which are especially sensitive to oxidation under conditions of oxidative stress. One of the most important products of lipid oxidation is 4-hydroxy-2-nonenal (HNE), which has been implicated in the pathogenesis of Parkinson disease. Consequently, we investigated the effects of the interaction of HNE with α-synuclein. Incubation of HNE with α-synuclein at pH 7.4 and 37 °C resulted in covalent modification of the protein, with up to six HNE molecules incorporated as Michael addition products. Fourier transform infrared and CD spectra indicated that HNE modification of α-synuclein resulted in a major conformational change involving increased β-sheet. HNE modification of α-synuclein led to inhibition of fibrillation in an HNE concentration-dependent manner. This inhibition of fibrillation was shown to be due to the formation of soluble oligomers based on size exclusion high pressure liquid chromatography and atomic force microscope data. Small angle x-ray scattering analysis indicated that the HNE-induced oligomers were compact and tightly packed. Treatment with guanidinium chloride demonstrated that the HNE-induced oligomers were very stable with an extremely slow rate of dissociation. Addition of 5 μm HNE-modified oligomers to primary mesencephalic cultures caused marked neurotoxicity because the integrity of dopaminergic and GABAergic neurons was reduced by 95 and 85%, respectively. Our observations indicate that HNE modification of α-synuclein prevents fibrillation but may result in toxic oligomers, which could therefore contribute to the demise of neurons subjected to oxidative damage.

A methylene blue-mediated enzyme electrode for the determination of trace mercury(II), mercury(I), methylmercury, and mercury–glutathione complex

A methylene blue-mediated enzyme biosensor has been developed for the detection of inhibitors including mercury(II), mercury(I), methylmercury, and mercury-glutathione complex. The inhibition to horseradish peroxidase was apparently reversible and noncompetitive in the presence of HgCl2 in less than 8 s and irreversibly inactivated when incubated with different concentrations of HgCl2 for 1-8 min. The binding site of horseradish peroxidase with HgCl2 probably was a cysteine residue SH. Mercury compounds can be assayed amperometrically with the detection limits 0.1 ng ml(-1) Hg for HgCl2 and methylmercury, 0.2 ng ml(-1) Hg for Hg2(NO3)2 and 1.7 ng ml(-1) Hg for mercury glutathione complex. Inactivation of the immobilized horseradish peroxidase was displayed in the AFM images of the enzyme membranes.

Formation of disulfide bond in p53 correlates with inhibition of DNA binding and tetramerization.

The p53 tumor suppressor protein is susceptible to oxidation, which prevents it from binding to its DNA response element. The goal of the current research was to determine the nature of the cysteine residue thiol oxidation that prevents p53 from binding its DNA target and its effect on p53 structure. Recombinant p53, purified in the presence of the reducing agent dithiothreitol (DTT), contains five free thiol groups on the surface of the protein. In the absence of DTT, p53 contains only four thiol groups, indicating that an average of one surface thiol group is readily susceptible to oxidation. Sulfite-mediated disulfide bond cleavage followed by reaction with 2-nitro-5-thiosulfobenzoate showed that oxidized p53 contains a single disulfide bond per monomer. By atomic force microscopy, we determined that reduced p53 binds to a double-stranded DNA containing the p53 promoter element of the MDM2 gene. The DNA-bound reduced p53 has an average cross-sectional diameter of 8.61 nm and a height of 4.12 nm. The amount of oxidized p53 that bound to the promoter element was ninefold lower, and it has an 18% larger average cross-sectional diameter. Electromobility shift assays showed that binding of oxidized p53 to DNA was enhanced upon addition of DTT, indicating that oxidation is reversible. The possibility that oxidized p53 contained significant amounts of sulfenic (-SOH), sulfinic (-SO2H), or sulfonic acid (-SO3H) was ruled out. Gel filtration chromatography indicated that oxidation increases the percentage of p53 monomers and high-molecular-weight oligomers (>1,000 kDa) relative to tetrameric p53. Protein modeling studies suggest that a mixed disulfide glutathione adduct on Cys182 could account for the observed stoichiometry of oxidized thiols and structural changes. The glutathione adduct may prevent proper helix-helix interaction within the DNA binding domain and contribute to tetramer dissociation.

Supramolecular inclusion complex formation and application of β-cyclodextrin with heteroanthracene ring cationic dyes

b-Cyclodextrin forms 1:1 inclusion complexes with methylene blue, azure A, toluidine blue, resorcinol blue, neutral red, safranine T, indigo carmine and acridine orange in aqueous media. The formation constants are determined by differential pulse polarography and spectrophotometry. The supramolecular interaction in the inclusion complexes can be employed to immobilize dyes on an electrode. This gives high sensitivity and stable electrochemical behavior for H2O2 detection at the mmol l ˇ1 level by means of the supramolecular interaction between b-CD and dye molecules. # 1999 Elsevier Science B.V.

β-Cyclodextrin polymer as the immobilization matrix for peroxidase and mediator in the fabrication of a sensor for hydrogen peroxide

Abstract A novel immobilization approach based on the supramolecular function between β-cyclodextrin polymer (β-CDP) and cationic dyes, and the condensation polymerization among β-CDP, glutaric dialdehyde and horseradish peroxidase (HRP) in the fabrication of a hydrogen peroxide sensor is described. IR and UV–vis spectroscopy were employed to characterize the structure of the composite membrane modified on the surface of the electrode. AFM was used to visualize the morphology of the composite membrane. The characteristics of supramolecular inclusion compounds between β-CDP and cationic dyes were studied. The fabricated H2O2 sensor responded rapidly to H2O2 in the linear range from 1.0 to 1.1 mmol l−1 with a detection limit of 0.5 mol l−1.

Oxidized quercetin inhibits α-synuclein fibrillization

BACKGROUND α-Synucein is a small (14 kDa), abundant, intrinsically disordered presynaptic protein, whose aggregation is believed to be a critical step in Parkinsons disease (PD). Oxidative stress is reported to be a risk factor for dopamine cell degeneration in PD. Flavonoids are suggested to be important antioxidant against oxidative stress. Flavonoids were reported to inhibit fibrillization and disaggregate the preformed fibrils of α-synucein, but the molecular mechanism was still not clear. METHODS Quercetin, a well-recognized flavonoid antioxidant, was tested for its inhibition of α-synucein aggregation by thioflavin T assay, light scattering measurement, size-exclusion high performance liquid chromatography, atomic force microscopy, etc. RESULTS The pre-incubated quercetin exhibited a noticeably stronger inhibition behavior to the fibril formation than that of the freshly prepared. The inhibition is significant in the presence of ortho- and para-benzenediol isomers and inconsiderable in the presence of meta-isomer. The oxidized quercetin species (i.e., chalcantrione, benzyfuranone, quercetinchinone, and other derivatives) cause stronger inhibition than quercetin does because of the elevated polarity and hydrophilicity. Presence of quercetin disaggregates α-synucein fibrils, rather than oligomers and amorphous aggregations. CONCLUSIONS Instead of the antioxidant activity, the 1:1 covalent binding of quercetin with α-synucein, and the increased hydophilicity of the covalently modified α-synucein oligomers or monomers, account for the inhibition of α-synucein fibrillation. GENERAL SIGNIFICANCE Clarification of the molecular mechanism of the inhibition and disaggregation may help to screen safer and more effective flavonoid therapeutic in combating PD.

Characterization of the Non-Fibrillar α-Synuclein Oligomers

Under certain in vitro conditions, α-Synuclein is an abundant 14 kDa presynaptic intrinsically disordered protein, involved in the pathogenesis of Parkinsons disease (PD) forms amyloid fibrils which resemble those found in Lewy bodies of PD patients. However, a substantial fraction of α-synuclein molecules (10-20 %) does not form fibrils during fibrillation and exists in a form of soluble oligomers. In this study, we examined these soluble oligomers by a variety of biophysical techniques including atomic force microscopy (AFM), circular dichroism, Fourier-transform infrared spectroscopy and thioflavin T fluorescence. We observed that the fibrillation kinetics is affected by the variation in salt and protein concentrations. Although both high salt and high protein concentrations noticeably accelerated α-synuclein fibrillation, the amount of non-fibrillar oligomers is independent of the salt content. The oligomers formed at low salt concentration adopt more β-sheet structure and are smaller in size than those formed at high salt concentration. AFM analysis shows that the low salt oligomers represent a mixture of small oligomers and some amorphous aggregates, whereas oligomers formed at high salt concentrations are noticeably larger, more homogenous, and are mostly spherical in shape. All the late stage non-fibrillar oligomers do not form fibrils even when seeded with pre-formed fibrils, are characterized by negligible rates of dissociation, likely due to their intertwined structure, and are able to disrupt the integrity of the biological membrane. These findings suggest that these soluble oligomers are important players in the multi-pathway aggregation of α-synuclein and should be taken into account in studies on the molecular mechanisms of this protein fibrillation.

A modified nanosphere lithography for the fabrication of aminosilane/polystyrene nanoring arrays and the subsequent attachment of gold or DNA-capped gold nanoparticles

A modified nanosphere lithographic method for producing arrays of silanized structures at silicon surfaces is described. Polystyrene (PS) particles (600 nm or 1000 nm in diameter) were self-assembled onto a silicon substrate to form a hexagonal close-packed pattern. The resultant patterned surface was then exposed to a solution of 3-aminopropyltriethoxysilane (APTS), which deposited gradually in the interstitial voids of the PS particle array. When such a surface was sonicated in toluene to dislodge the PS particles, a mesoporous network containing truncated PS nanorings/shells was produced. Gold nanoparticles or DNA-capped gold nanoparticles, which are both negatively charged, can be electrostatically attached onto the PS/APTS nanoring array. Atomic force microscopy (AFM) was used to image the surface pattern and structure after each step of the procedure, while X-ray photoelectron spectra (XPS) and UV-visible spectrometry were used to determine the composition of the surface patterns. The mechanisms for forming the PS/APTS nanostructures are discussed. These structures could potentially be used as biosensors, heterogeneous catalysts, and functionalized nano-devices.

A three-dimensional heterogeneous DNA sensing surface formed by attaching oligodeoxynucleotide-capped gold nanoparticles onto a gold-coated quartz crystal

Exposing oligodeoxynucleotide (ODN)-capped Au nanoparticles to a quartz crystal under shear oscillation resulted in the formation of a uniform monolayer containing these nanoparticles or multilayers with islands of the ODN-capped nanoparticles, which, in turn, improved the extent of DNA hybridization.

DJ-1, a Parkinson's disease related protein, aggregates under denaturing conditions and co-aggregates with α-synuclein through hydrophobic interaction

BACKGROUND DJ-1, a small ubiquitously expressed protein implicated in several pathways associated with Parkinsons disease pathogenesis, has been found to interact with α-synuclein and modulate its aggregation, yet the exact mechanisms remain unclear. METHODS The stability and aggregation properties of wild-type DJ-1 under denaturing conditions, such as low pH, high temperature, presence of denaturants were investigated. The interaction between DJ-1 and α-synuclein was tested by SDS-PAGE gel and native gel electrophoresis and by size-exclusion HPLC. Fibrillization was monitored by thioflavin T fluorescence assays and amorphous aggregation was followed by light scattering measurements. The morphology of aggregated species was observed by transmission electron microscopy and atomic force microscopy. Protein secondary structures were characterized by far-UV circular dichroism. RESULTS DJ-1 fibrillization was first observed at low pH or by adding denaturants. Amorphous aggregates formed at neutral pH, and the aggregation was dramatically accelerated by elevated temperature and the presence of α-synuclein. Aggregation of DJ-1 were enhanced by heating and perturbed by the co-occurrence of α-synuclein but strong interactions between the two proteins were not found. CONCLUSIONS Varying environmental factors led to different aggregation pathways of DJ-1 although a simulated physiological condition would not lead to fibrillization. DJ-1 co-aggregating with α-synuclein may result from weak hydrophobic interaction and DJ-1 exhibited chaperon-like activity in the initial time of α-synuclein aggregation at high temperature. GENERAL SIGNIFICANCE This research on DJ-1 presented its aggregation behavior under denaturing conditions and interaction mechanism with α-synuclein that may help to decipher its potential neuroprotective or neurotoxic role in Parkinsons disease.