硫黄素T对淀粉样β-蛋白质40聚集成核动力学的双重影响

荧光染料硫黄素T常用于淀粉样纤维聚集过程的定性定量检测。虽然有研究表明,某些抑制淀粉样蛋白质聚集的小分子抑制剂会与硫黄素T相互作用,影响其测试结果。但硫黄素T如何影响淀粉样蛋白质的聚集成核动力学尚不清晰。本文以淀粉样β-蛋白质40 (Aβ40)为模型,系统研究了硫黄素T对Aβ40聚集成核的影响。研究发现：硫黄素T能够显著改变Aβ40的聚集成核动力学,且影响程度与硫黄素T的浓度密切相关。即在低浓度硫黄素T存在下,Aβ40成核速率的延迟时间先随着硫黄素T浓度的升高而缩短,后随着硫黄素T浓度的升高延迟时间反而延长。但延伸的速率却随硫黄素T浓度的升高而缓慢增大。另外,硫黄素T基本不会影响Aβ40的二级结构和纤维形态。同时,等温滴定微量热实验结果表明,硫黄素T结合Aβ40之间的主要作用力为疏水相互作用。据此,本研究提出硫黄素T对Aβ40聚集成核动力学的双重影响机理。这些结果有助于进一步了解硫黄素T与淀粉样蛋白质的作用特点,为今后硫黄素T在Aβ40聚集成核动力学实验中的使用提供参考。     

Quantitative Aspects of Electrochemical Detection of Amyloid‐β Aggregation

The tyrosine based electrochemical analysis of synthetic amyloid‐β (Aβ) peptide – an analog of natural peptide implicated in Alzheimer's disease pathogenesis – was applied for a quantitative estimation of peptide aggregation in vitro. The analysis was carried out by square wave voltammetry (SWV) on carbon screen printed electrodes (SPE). The electrooxidation peak current (I_p) for Aβ42 peptide in different aggregation states was directly compared with the size and structure of Aβ42 aggregates occurring in the analyzed sample. Dynamic light scattering (DLS) and thioflavin T (ThT) based fluorescence assay were employed to estimate the size and structure of Aβ42 aggregates. The I_p was found to decrease in a linear fashion when the average diameter of aggregates and the relative ThT fluorescence in Aβ42 solutions exceeded 35 nm and 3, respectively, while being nearly constant below these values. It was suggested that the electrooxidation current is mostly generated by peptide monomers and that a depletion of the monomer pool due to inclusion of Aβ42 molecules in aggregates is responsible for the decrease of electrooxidation current. The direct electrochemistry is emerging as a method complementary to methods based on aggregates’ detection and commonly employed for monitoring Aβ aggregation. The work further enlarges the basis for application of the cost‐effective and rapid electrochemical techniques, such as SWV on carbon SPE, to in vitro studies of Aβ aggregation.     

Application of an Electrochemical Method to Evaluation of Amyloid‐β Aggregation Inhibitors: Testing the RGKLVFFGR‐NH2 Peptide Antiaggregant

The aggregation of amyloid‐β peptide (Aβ) is believed to play a crucial role in the Alzheimer's disease (AD) pathogenesis and is considered as a therapeutic target for treating AD. The Aβ electrooxidation via a Tyr‐10 residue, sensitive to a depletion of a pool of Aβ monomers and oligomers in the course of Aβ aggregation, may be employed for testing natural and synthetic organic compounds (including short peptides) potentially able to inhibit the pathological Aβ aggregation (antiaggregants). In the present work, using the known peptide antiaggregant RGKLVFFGR‐NH₂ (OR2) and its scrambled variant KGLRVGFRF‐NH₂ as a control, we demonstrate that the electrochemical method based on electrooxidation of an Aβ42 Tyr‐10 residue, when combined with methods allowing for the evaluation of the Aβ42 aggregate structure and size, can provide essential information regarding the antiaggregant impact on Aβ42 aggregation. Electrochemical measurements were performed using square wave voltammetry on carbon screen printed electrodes whereas the Aβ42 aggregate structure and size were analyzed by means of the conventional thioflavin T (ThT) based fluorescence assay and dynamic light scattering. While inhibiting Aβ42 fibrillation as manifested by the unchanged level of ThT fluorescence, the OR2 peptide antiaggregant had no effect on the decrease of Aβ42 electrooxidation current in the course of Aβ42 aggregation. These observations suggest that OR2 does not stop the aggregation but redirects it into a pathway where amorphous rather than fibrillar aggregates are formed. Hence, the direct electrochemistry appears to offer a simple and cost‐effective approach for probing potential peptide antiaggregants, which is complementary to methods based on detecting Aβ aggregates.     

Exploring the binding mechanism of thioflavin-T to the β-amyloid peptide by blind docking method

Using blind dock method,we find that thioflavin-T(ThT) can bind to both monomers and fibrils of the full-length β-amyloid peptide(Aβ1-42) and has a higher binding affinity to the fibrils.It is shown that the hydrophobic interaction between the ligand(ThT) and substrate(Aβ1-42) are stronger than hydrogen bonds.Furthermore,ThT tends to be located near the C-terminus of Aβ monomer through hydrophobic and electrostatic interactions,while it tends to contact the residues Met35 and Gly27 of the fibril surface mainly through hydrophobic interaction.Finally,according to the docking results and ThT fluorescence assay,a kinetic equation is proposed to deduce the aggregation rate coefficient of Aβ1-42.     

AFM and fluorescence spectrascopy investigation for disaggregation of existing Aβ fibrils by baicalein

The wavelength for the peak of fluorescence emission of thioflavin T（ThT） was changed from 445 nm to 481 nm when ThT was added in Aβ solution which indicating theβ-sheet structure of Aβ fibril.The significant decrease in the intensity of fluorescence at 481 nm was observed when the baicalein was added in mixed solution of Aβ and ThT,suggesting that the depolymerization of Aβ fibrils happened and there were Aβfibrils left to react with ThT to keep the initial fluorescence intensity.And the existing Aβfibrils are disaggregated by baicalein in a time- and dose-dependent manner.AFM images of the morphologies of the Aβ_1-42 fibrils obviously changed smaller and more dispersive when baicalein added indicating also the depolymerization of Aβ.The results demonstrate a basis for development of a potential herb drug candidate for the treatment of Alzheimer’s disease（AD）.     

Bifunctional compounds for controlling metal-mediated aggregation of the aβ42 peptide

Abnormal interactions of Cu and Zn ions with the amyloid β (Aβ) peptide are proposed to play an important role in the pathogenesis of Alzheimer's disease (AD). Disruption of these metal-peptide interactions using chemical agents holds considerable promise as a therapeutic strategy to combat this incurable disease. Reported herein are two bifunctional compounds (BFCs) L1 and L2 that contain both amyloid-binding and metal-chelating molecular motifs. Both L1 and L2 exhibit high stability constants for Cu(2+) and Zn(2+) and thus are good chelators for these metal ions. In addition, L1 and L2 show strong affinity toward Aβ species. Both compounds are efficient inhibitors of the metal-mediated aggregation of the Aβ(42) peptide and promote disaggregation of amyloid fibrils, as observed by ThT fluorescence, native gel electrophoresis/Western blotting, and transmission electron microscopy (TEM). Interestingly, the formation of soluble Aβ(42) oligomers in the presence of metal ions and BFCs leads to an increased cellular toxicity. These results suggest that for the Aβ(42) peptide-in contrast to the Aβ(40) peptide-the previously employed strategy of inhibiting Aβ aggregation and promoting amyloid fibril dissagregation may not be optimal for the development of potential AD therapeutics, due to formation of neurotoxic soluble Aβ(42) oligomers.     

Study of thioflavin-T immobilized in porous silicon and the effect of different organic vapors on the fluorescence lifetime

Steady-state and time-resolved emission techniques have been employed to study the fluorescence properties of thioflavin-T (ThT) adsorbed on oxidized porous silicon (PSi) surfaces, with an average pore size of ～10 nm. We found that the average fluorescence decay time of ThT, when it is adsorbed on the PSi surface, is rather long, τ(av) = 1.3 ns. We attribute this relatively long emission lifetime to the effect of the immobilization of ThT on the PSi surface, which inhibit the rotation of the aniline with respect to the benzothiazole moieties of ThT. We also measured the fluorescence properties of ThT in PSi samples in equilibrium with vapors of several liquids, such as methanol, acetonitrile, and water. We found that the fluorescence intensity drops by a factor of 10, and the average decay time, measured by a time-correlated single-photon counting technique, decreases by a factor of 3. We explain these results in terms of liquid condensation of the vapors in the PSi pores, which leads to partial dissolution of the ThT molecules in the liquid pools.     

Antibody-free lanthanide-based fluorescent probe for determination of protein tyrosine kinase and phosphatase activities

A single-labeled peptide probe for measuring peptide phosphorylation status was developed by using a phosphate sensitive terbium chelate. The activity of Abl protein tyrosine kinase and T-cell protein Tyrosine phosphatase (TC PTP) was monitored in real time. To study the probe design in detail, variable substrate peptide sequences, where the enzyme target site was located from two to five amino acids apart from the nearest tyrosine residue, were synthesized. The maximum change observed in fluorescence intensity after phosphorylation was up to 320%, when the phosphorylated tyrosine was located two amino acids from the lysine coupled to the phosphate sensitive terbium chelate, demonstrating an excellent performance for a homogeneous assay. Also the longer distance of five amino acids between the phosphorylated tyrosine residue and terbium chelate resulted up to 260% change in fluorescence intensity.

Quenching of fluorescence from pyrene in micellar solutions by cationic quenchers

The fluorescence from the S₁ state of pyrene solubilized within the hydrophobic region of sodium dodecyl sulfate micelles has been studied as a function of concentration of some cationic additives to the aqueous phase. At concentrations of added quencher below ca. 10^?3 mol dm^?3 the fluorescence decay follows a complex rate law; at long time, the decay is exponential with the same time constant as that of pyrene (S₁) without added quencher. The initial slope of the decay curve depends linearly on the concentration of added cation. These results are discussed in terms of the recently proposed model which requires the water-hydrocarbon interface to penetrate the micelle in the region of the probe. An analytical treatment is presented which, on the premises stated, serves to explain the observed kinetic features.     

Real‐time Amyloid Aggregation Monitoring with a Photonic Crystal‐based Approach

We propose the application of a new label‐free optical technique based on photonic nanostructures to real‐time monitor the amyloid‐beta 1‐42 (Aβ(1‐42)) fibrillization, including the early stages of the aggregation process, which are related to the onset of the Alzheimer’s Disease (AD). The aggregation of Aβ peptides into amyloid fibrils has commonly been associated with neuronal death, which culminates in the clinical features of the incurable degenerative AD. Recent studies revealed that cell toxicity is determined by the formation of soluble oligomeric forms of Aβ peptides in the early stages of aggregation. At this phase, classical amyloid detection techniques lack in sensitivity. Upon a chemical passivation of the sensing surface by means of polyethylene glycol, the proposed approach allows an accurate, real‐time monitoring of the refractive index variation of the solution, wherein Aβ(1‐42) peptides are aggregating. This measurement is directly related to the aggregation state of the peptide throughout oligomerization and subsequent fibrillization. Our findings open new perspectives in the understanding of the dynamics of amyloid formation, and validate this approach as a new and powerful method to screen aggregation at early stages.     

Cryptotanshinone against vascular dementia through inhibition of Aβ aggregation and inflammatory responses in cerebrovascular endothelial cells

Abnormal aggregation of amyloid-β (Aβ) peptides and associated inflammation and apoptosis in cerebrovascular endothelial cells are prelude to inhibition of onset of vascular dementia (VaD). Although small molecules have been widely used to mitigate the cell damage induced by aggregated species of Aβ, its molecular mechanism based on anti-amyloid properties and corresponding mitigation of cytotoxicity against cerebrovascular endothelial cells have not been elucidated. Herein, we used cryptotanshinone as the major bioactive compound from the root of Salvia miltiorrhiza Bunge to effectively inhibit Aβ fibrillation and associated cytotoxicity. Thoflavin T (ThT) and 1-Anilino-8-naphthalene sulfonate (ANS) fluorescence, Congo red, and circular dichroism (CD) analyses indicted that cryptotanshinone potentially inhibit Aβ_1-42 aggregation through elongation of nucleation phase, apparent decrease in the slope of the growth phase, and the final fluorescence intensity in a concentration-dependent manner. Also, cell viability, inflammation and capsae-3 assays showed that co-incubation of Aβ_1-42 peptide with cryptotanshinone in the aggregation buffer not only mitigated their cytotoxicity, but also reduced the levels of TNF-α, IL-1β, IL-6 and caspase-3 activity in cerebrovascular endothelial cells induced by Aβ_1-42. This study suggested that cryptotanshinone may show a great promise in the development of small molecule-based platforms for the treatment of VaD.     

Short polyglutamine peptide forms a high-affinity binding site for thioflavin-T at the N-terminus

Thioflavin-T is one of the most important amyloid specific dyes and has been used for more than 50 years; however, the molecular mechanism of staining is still not understood. Chemically synthesized short polyglutamine peptides (Q(n), n = 5-10) were subjected to the thioflavin-T (ThT) staining assay. It was found that the minimum Q(n) peptide that stained positive to ThT was Q(6). Two types of ThT-binding sites, a high-affinity site (k(d1) = 0.1-0.17 μM) and a low-affinity site (k(d2) = 5.7-7.4 μM), were observed in short polyQs (n = 6-9). (13)C{(2)H}REDOR NMR experiments were carried out to extract the local structure of ThT binding sites in Q(8) peptide aggregates by observing the intermolecular dipolar coupling between [3-Me-d(3)]ThT and natural abundance Q(8) or residue-specific [1,2-(13)C(2)] labeled Q(8)s. (13)C{(2)H}REDOR difference spectra of the [3-Me-d(3)]ThT/natural abundance Q(8) (1/9) complex indicated that all of the five carbons of the glutamine residue participated in the formation of ThT-binding sites. (13)C{(2)H}DQF-REDOR experiments of [3-Me-d(3)]ThT/residue-specific [1,2-(13)C(2)] labeled Q(8) (1/50) complexes demonstrated that the N-terminal glutamine residue had direct contact with the ThT molecule at the high-affinity ThT-binding sites.     

基于ThT荧光寿命检测蛋清溶菌酶蛋白寡聚体

采用时间分辨荧光技术, 检测了不同形态蛋白聚集体的荧光染料硫磺素T(ThT)荧光寿命. 利用蛋清溶菌酶体外制备了蛋白聚集体; 采用透射电子显微镜(TEM)及ThT稳态荧光检测了结合蛋白纤维生长的动力学曲线, 确定其形成寡聚体及纤维样聚集体的特征和时间. 通过时间相关单光子计数(TCSPC)技术测定了蛋清溶菌酶单体、 寡聚体和淀粉样纤维的ThT荧光寿命曲线, 并拟合、 计算其荧光寿命. 根据圆二色谱(CD)分析结果推测聚集体的结构不同导致其与ThT的结合状态不同, 从而影响ThT荧光寿命. 结果表明, 通过测定ThT荧光寿命可以区分蛋白单体、 寡聚体和纤维样聚集体, 并监测蛋白寡聚体的形成, 为后续病理蛋白聚集过程中形成寡聚体物质的监测提供了研究基础.     

Spectroscopic investigations of metalloporphyrin-oligopeptide systems: evidence for peptide aggregation

Anionic pentapeptides consisting of a string of four glutamic acid residues terminated by either tyrosine (Glu4Tyr) or tryptophan (Glu4Trp) were synthesized, and their aggregation properties in buffered (pH = 7.0) aqueous solutions were investigated using two different approaches. In the first approach, the effects of the concentration of peptide used as its own probe (intrinsic probe) on its fluorescence emission, circular dichroism, surface tension, and solution pH yielded similar critical peptide concentrations of around 175 microM. This particular concentration was taken as evidence for peptide aggregation. In the second approach, peptide aggregation was investigated using cationic metalloporphyrins, tetrakis(N-methyl-4-pyridyl)porphyrin (Pd(II)TMPyP(4+) and Zn(II)TMPyP(4+)), as extrinsic probes. The effect of peptide concentration on porphyrin ground-state absorption confirmed peptide aggregation, but at a lower critical peptide concentration near 125 microM. This difference was attributed to the possible distortion introduced by the association of one (or more) large metalloporphyrin molecule with the peptide aggregates. Evidence for peptide aggregation was also demonstrated from the effect of peptide concentration on Pd(II)TMPyP(4+) triplet-state decay. The fast component (k(f), associated with electron transfer from the target Tyr and Trp residues to the porphyrin triplet state) was found to be independent of peptide concentration, implying no noticeable effect of peptide aggregation on the electron-transfer event. This was attributed to the fact that species formed by excitation of porphyrin associated with ion-pair complexes or bound to peptide aggregates and the diffusion together of the separate T(1) and peptide entities in the bulk phase are kinetically similar. On the other hand, the slower component (k(s)) of the decay, which is associated with the diffuse formation of an encounter complex between the free peptide and T(1) porphyrin (bulk phase), was peptide-dependent and displayed a critical peptide concentration near 125 microM, above which it became practically independent of peptide concentration. This invariance of k(s) was taken as an indication that the free peptide concentration in the bulk phase remains constant above 125 microM, the concentration at which peptide molecules prefer to associate as aggregates.     

Inhibiting and Reversing Amyloid‐β Peptide (1–40) Fibril Formation with Gramicidin S and Engineered Analogues

In Alzheimer’s disease, amyloid‐β (Aβ) peptides aggregate into extracellular fibrillar deposits. Although these deposits may not be the prime cause of the neurodegeneration that characterizes this disease, inhibition or dissolution of amyloid fibril formation by Aβ peptides is likely to affect its development. ThT fluorescence measurements and AFM images showed that the natural antibiotic gramicidin S significantly inhibited Aβ amyloid formation in vitro and could dissolve amyloids that had formed in the absence of the antibiotic. In silico docking suggested that gramicidin S, a cyclic decapeptide that adopts a β‐sheet conformation, binds to the Aβ peptide hairpin‐stacked fibril through β‐sheet interactions. This may explain why gramicidin S reduces fibril formation. Analogues of gramicidin S were also tested. An analogue with a potency that was four‐times higher than that of the natural product was identified.     

ThX – a next-generation probe for the early detection of amyloid aggregates

Neurodegenerative diseases such as Alzheimer''s and Parkinson''s are associated with protein misfolding and aggregation. Recent studies suggest that the small, rare and heterogeneous oligomeric species, formed early on in the aggregation process, may be a source of cytotoxicity. Thioflavin T (ThT) is currently the gold-standard fluorescent probe for the study of amyloid proteins and aggregation processes. However, the poor photophysical and binding properties of ThT impairs the study of oligomers. To overcome this challenge, we have designed Thioflavin X, (ThX), a next-generation fluorescent probe which displays superior properties; including a 5-fold increase in brightness and 7-fold increase in binding affinity to amyloidogenic proteins. As an extrinsic dye, this can be used to study unique structural amyloid features both in bulk and on a single-aggregate level. Furthermore, ThX can be used as a super-resolution imaging probe in single-molecule localisation microscopy. Finally, the improved optical properties (extinction coefficient, quantum yield and brightness) of ThX can be used to monitor structural differences in oligomeric species, not observed via traditional ThT imaging.

Introducing ThX, a next-generation ThT derivative that allows for the early detection of amyloid aggregates at the bulk and single-aggregate levels.     

On the dynamics of the TPPS4 aggregation in aqueous solutions: successive formation of H and J aggregates

The dynamics of aggregation of meso-tetrakis (p-sulfonatofenyl) porphyrin (TPPS4) in function of its concentration, pH and ionic strength was studied by optical absorption, fluorescence and resonance light scattering (RLS) techniques. In the region of pH, where TPPS4 exists in biprotonated form, the addition of NaCl induces the TPPS4 aggregation due to the formation of the "cloud" of counter ions around the TPPS4 molecule thus reducing electrostatic repulsion between the porphyrin molecules. The formation of this "cloud" shifts the pKa value to acid region (from 5.0 in the absence of salt to 4.5 at [NaCl] = 0.4 M), reduces the TPPS4 absorption in all spectral range and quantum yield and lifetime of fluorescence (from 0.27 to 0.17 and from 4.00+/-0.04 to 3.00+/-0.03 ns in the absence of salt and in the presence of NaCl, respectively). The aggregation process involves two successive stages: initially H aggregates are formed, which in time are transformed in J ones. The existence of these two stages was confirmed by the fluorescence and RLS data. The kinetics of the formation of J aggregates is characterized by the induction time t1 and the average growth time t2, which depend on both TPPS4 and salt concentrations. The induction period t1 appears as a result of initial formation of H aggregates and their successive transformation in J ones. At very high TPPS4 concentrations, the J aggregates are united in more complex structures such as hollow cylinders or helixes.     

Optical trapping for the characterization of amyloid-beta aggregation kinetics

Alzheimer's disease (AD) is marked by the accumulation of neuronal plaques from insoluble amyloid-beta (Aβ) peptides. Growing evidence for the role of Aβ oligomers in neuronal cell cytotoxicity and pathogenesis has prompted the development of novel techniques to better understand the early stages of aggregation. Near infrared (NIR) optical trapping was applied to characterize the early stages of Aβ aggregation in the presence of a β-sheet intercalating dye, Congo Red (CR), as the fluorescent marker. The integration of fluorescence analysis with NIR optical trapping has provided a new outlook into the first two hours of Aβ aggregation.     

On the Protein Fibrillation Pathway: Oligomer Intermediates Detection Using ATR-FTIR Spectroscopy

Oligomeric intermediates on the pathway of amyloid fibrillation are suspected as the main cytotoxins responsible for amyloid-related pathogenicity. As they appear to be a part of the lag phase of amyloid fibrillation when analyzed using standard methods such as Thioflavin T (ThT) fluorescence, a more sensitive method is needed for their detection. Here we apply Fourier transform infrared spectroscopy (FTIR) in attenuated total reflectance (ATR) mode for fast and cheap analysis of destabilized hen-egg-white lysozyme solution and detection of oligomer intermediates of amyloid fibrillation. Standard methods of protein aggregation analysis— Thioflavin T (ThT) fluorescence, atomic force microscopy (AFM), and 8-anilinonaphthalene-1-sulphonic acid (ANS) fluorescence were applied and compared to FTIR spectroscopy data. Results show the great potential of FTIR for both, qualitative and quantitative monitoring of oligomer formation based on the secondary structure changes. While oligomer intermediates do not induce significant changes in ThT fluorescence, their secondary structure changes were very prominent. Normalization of specific Amide I region peak intensities by using Amide II peak intensity as an internal standard provides an opportunity to use FTIR spectroscopy for both qualitative and quantitative analysis of biological samples and detection of potentially toxic oligomers, as well as for screening of efficiency of fibrillation procedures.     

基于芬顿反应和硫磺素T构建免标记荧光传感器用于葡萄糖的检测

基于芬顿反应和硫磺素T(ThT)构建新奇的免标记荧光传感器用于葡萄糖的检测。当无葡萄糖存在时,ThT诱导富G-DNA探针形成G-四链体/ThT复合物,ThT的荧光强度显著增强;当葡萄糖存在时,葡萄糖氧化酶催化葡萄糖产生H2 O2,在Fe^2+催化的芬顿反应作用下,H2 O2转化为羟基自由基(·OH),·OH引发DNA的氧化损伤导致富G-DNA探针裂解为短寡核苷酸片段而丧失形成G-四链体/ThT的能力,ThT的荧光强度显著降低,从而实现对葡萄糖的检测。在优化的检测条件下,G-四链体/ThT荧光强度变化和葡萄糖浓度在0.5~45μmol/L的范围内呈现较好的线性关系(R^2=0.99268),检出限为0.1μmol/L。利用本法对葡萄糖加标的血液样品进行分析,葡萄糖的回收率为90.7%~118.3%,相对标准偏差为1.7%~5.8%,方法可用于血糖检测。