Concentration-Dependent Effect of Nickel Ions on Amyloid Fibril Formation Kinetics of Hen Egg White Lysozyme:a Raman Spectroscopy Study

Nickel,an important transi-tion metal element,is one of the trace elements for hu-man body and has a crucial impact on life and health.Some evidences show the excess exposure to metal ions might be associated with neurological diseases.Herein,we applied Raman spectroscopy to study the Ni(II)ion effect on kinetics of amyloid fibrillation of hen egg white lysozyme(HEWL)in thermal and acidic conditions.Using the well-known Raman indicators for protein tertiary and secondary structures,we monitored and analyzed the concentration effect of Ni(II)ions on the unfolding of tertiary structures and the transformation of sec-ondary structures.The experimental evidence validates the accelerator role of the metal ion in the kinetics.Notably,the additional analysis of the amide I band profile,combined with thioflavin-T fluorescence assays,clearly indicates the inhibitory effect of Ni(II)ions on the formation of amyloid fibrils with organizedβ-sheets structures.Instead,a more significant promotion influence is affirmed on the assembly into other aggregates with disordered struc-tures.The present results provide rich information about the specific metal-mediated protein fibrillation.

Nanomechanical Properties of Amyloid Fibrils Formed in a Water Nanofilm on Mica Surface

The assessment of nanomechanical properties of a single amyloid fibril in a confined space provides important information for understanding the role of fibrils in a cell microenvironment. In this study, the structure and nanomechanical properties of different fibrils formed in water nanofilms on mica surface are carefully investigated by using the new atomic force microscopy imaging mode-peak force quantitative nanomechanics （PF-QNM）. We find that two types of fibrils with different morphologies are formed in water nanofilm on mica. The compression elasticities of these two types of fibrils are 3.9±0.9 and 2.5±0.6 GPa, respectively. The remarkable difference is possibly due to the structural discrepancy in two types of fibrils.

The LBFGS quasi-Newtonian method for molecular modeling prion AGAAAAGA amyloid fibrils

Experimental X-ray crystallography, NMR (Nuclear Magnetic Resonance) spectroscopy, dual polarization interferometry, etc. are indeed very powerful tools to determine the 3-Dimensional structure of a protein (including the membrane protein);theoretical mathematical and physical computational approaches can also allow us to obtain a description of the protein 3D structure at a submicroscopic level for some unstable, noncrystalline and insoluble proteins. X-ray crystallography finds the X-ray final structure of a protein, which usually need refinements using theoretical protocols in order to produce a better structure. This means theoretical methods are also important in determinations of protein structures. Optimization is always needed in the computer-aided drug design, structure-based drug design, molecular dynamics, and quantum and molecular mechanics. This paper introduces some optimization algorithms used in these research fields and presents a new theoretical computational method—an improved LBFGS Quasi-Newtonian mathematical optimization method—to produce 3D structures of prion AGAAAAGA amyloid fibrils (which are unstable, noncrystalline and insoluble), from the potential energy minimization point of view. Because the NMR or X-ray structure of the hydrophobic region AGAAAAGA of prion proteins has not yet been determined, the model constructed by this paper can be used as a reference for experimental studies on this region, and may be useful in furthering the goals of medicinal chemistry in this field.

Amyloid fibril-supported Pd nanoparticles as electrocatalyst for hydrogen peroxide reduction

Palladium nanoparticles(Pd NPs) were fabricated by using insulin amyloid fibrils(INSAFs) as biotemplates.Atomic force microscopy measurements showed that ultrasmall Pd NPs were well adsorbed and dispersed on surfaces of INSAFs. X-ray photoelectron spectroscopy confirmed the partial reduction of Pd ion into metallic Pd(0) probably due to the presence of Cys groups on surface of the insulin fibrils. The electrochemical performance of Pd/INSAFs to reduction of H_2O_2 was further evaluated by cyclic voltammetry. The remarkably high electrocatalytic activity, low detection limitation and excellent stability make the Pd/INSAFs a promising bio-nanoelectrocatalyst.

Suppression of Sup35 amyloid fibril formation by group II chaperonin from <i>Thermoplasma acidophilum</i>

The Group II chaperonin from Thermoplasma acidophilum was added to the in vitro amyloid fibrillation reaction of yeast Sup35NM protein to assess its effects. By measuring the formation of Sup35NM fibrils in real time using the fluorescent dye Thioflavin T, we found that the addition of T. acidophilum-cpn α16, α1, and β1 proteins suppressed fibril formation. Addition of a 0.1 molar-equivalent T. acidophilum-cpn α16 relative to Sup35NM prolonged the initial lag-time of fibril formation and decreased the rate of fibril extension. Addition of 1 or 3 molar-equivalents of T. acidophilum-cpn monomers also produced a similar effect. Delayed addition of these chaperonins after the initial lag phase did not suppress fibril formation. Interestingly, these effects were also observed upon adding only the apical domain segments of α and β-subunits, and we also found that deletion of the helical protrusion in the apical domain of these segments led to an abolishment of the suppression effects. A synthetic peptide whose sequence corresponded to the helical protrusion also displayed a suppression effect, which indicated that archaeal group II chaperonin binds to Sup35NM through the helical protrusion of the apical domain. These findings suggest that group II chaperonin might be actively involved in suppressing amyloid fibril formation, in addition to acting as a protein folding assistant.

Synchrotron-Infrared Microscopy Analysis of Amyloid Fibrils Irradiated by Mid-Infrared Free-Electron Laser

Amyloid fibrils are widely recognized as a cause of serious amyloidosis such as Alzheimer’s disease. Although dissociation of amyloid fibril aggregates is expected to lead to a decrease in the toxicity of the fibrils in cells, the fibril structure is robust under physiological conditions. We have irradiated amyloid fibrils with a free-electron laser (FEL) tuned to mid-infrared frequencies to induce dissociation of the aggregates into monomer forms. We have previously succeeded in dissociating fibril structures of a short peptide of the thyroid hormone by tuning the oscillation frequency to the amide I band, but the detailed structural changes of the peptide have not yet been determined at a high spatial resolution. Synchrotron-radiation infrared microscopy (SR-IRM) is a powerful tool for in situ analysis of minute structural changes of various materials, and in this study, the feasibility of SR-IRM for analyzing the microscopic conformational changes of amyloid fibrils after FEL irradiation was investigated. Reflection spectra of the amyloid fibril surface showed that the amide I peaks shifted to higher wave numbers after the FEL irradiation, indicating that the initial β-sheet-rich structure transformed into a mixture of non-ordered and turn-like peptide conformations. This result demonstrates that conformational changes of the fibril structure after the FEL irradiation can be observed at a high spatial resolution using SR-IRM analysis and the FEL irradiation system can be useful for dissociation of amyloid aggregates.

Use of a Mid-Infrared Free-Electron Laser (MIR-FEL) for Dissociation of the Amyloid Fibril Aggregates of a Peptide

Amyloid fibrils are deposited in various tissues in the body, and are linked to the putative causes of serious diseases such as amyloidosis. Although the conditions of the disease would be expected to improve if the fibril structure could be destroyed, the aggregated structure is stable under physiological conditions. Recently, we found that the amyloid fibrils of lysozyme could be refolded into their active form by using a mid-infrared free-electron laser (MIR-FEL) tuned to the amide I band (corresponding to the C=O stretch vibration), with the MIR-FEL having specific oscillation characteristics of a picosecond pulse structure, a tunable wavelength within mid-infrared frequencies, and high photon density. In the study, we tested the usability of the FEL for dissociation of aggregates of pathological amyloid fibrils by using a short peptide of human thyroid hormone. The fibrils (after being placed on a glass slide) were irradiated using the FEL tuned to the amide I band (1644 cm?1), and those in situ were analyzed by Congo-Red assay, scanning-electron microscopy, and transmission-electron microscopy. All of the results obtained using these microscopic analyses indicated that the amyloid fibril formation was considerably decreased by FEL irradiation. Moreover, upon irradiation, a strong fibril peak at the amide I band in the infrared spectrum was transformed into a broad peak. These results imply that the β-sheet-rich structure of the amyloid fibrils changed into non-ordered or unspecified structures after the FEL irradiation. This FEL irradiation system, combined with various analytical methods, shows promise for the dissociation of amyloid aggregates.

Apolipoproteins and amyloid fibril formation in atherosclerosis

Amyloid fibrils arise from the aggregation of misfolded proteins into highly-ordered structures.The accumulation of these fibrils along with some non-fibrillar constituents within amyloid plaques is associated with the pathogenesis of several human degenerative diseases.A number of plasma apolipoproteins,including apolipoprotein(apo)A-I,apoA-II,apoC-II and apoE are implicated in amyloid formation or influence amyloid formation by other proteins.We review present knowledge of amyloid formation by apolipoproteins in disease,with particular focus on atherosclerosis.Further insights into the molecular mechanisms underlying their amyloidogenic propensity are obtained from in vitro studies which describe factors affecting apolipoprotein amyloid fibril formation and interactions.Additionally,we outline the evidence that amyloid fibril formation by apolipoproteins might play a role in the development and progression of atherosclerosis,and highlight possible molecular mechanisms that could contribute to the pathogenesis of this disease.

Effect of metallic nanoparticles on amyloid fibrils and their influence to neural cell toxicity

The modification of amyloid fibrils cytotoxicity through exogenous nanomaterials is crucial to understand the processes controlling the role of protein aggregation in the related diseases.The influence of nanoparticles on amyloid stability yields great interest due to the small size and high surface area-to-volume ratio of nanoparticles.Various physico-chemical parameters play a role in the interaction of proteins and nanoparticles in solution,thus influencing the disaggregation of preformed fibrils.We have examined the influence of two kinds of metallic nanoparticles on lysozyme amyloid fibrils using a multi-technique approach and focalized their impact on cytotoxicity on human neuroblastoma cells(SH-SY5Y).In particular,fluorescence,infrared and circular dichroism spectroscopies,optical and atomic force microscopy experiments have been carried out;the results are analyzed to rationalize the effects of these complexes on neural cell viability.It is remarkable,that the fibrils in the presence of AuNPs,unlike fibrils alone or with AgNPs,do not generate a significant cytotoxic effect even at high concentration and an amyloid degradation effect is visible.

Polarity-activated super-resolution imaging probe for the formation and morphology of amyloid fibrils

The formation of amyloid plaques usually occurs in the early-stage of Alzheimer’s disease(AD).Stimulated emission depletion(STED)imaging provided a powerful tool for visualizing amyloid structures on the nanometer scale.However,many commercial probes adopted in detecting amyloid fibrils are inapplicable to STED imaging,owing to their unmatched absorption and emission wavelengths,small Stokes'shift,easy photo-bleaching,etc.Herein,we demonstrated a polarity-activated STED probe based on an intramolecular charge transfer donor(D)-7c-acceptor(A)compound.The electron-rich carbazole group and the electron-poor pyridinium bromide group,linked by 7i-conjugated thiophen-bridge,ensure strong near infrared(NIR)emission with a Stokes'shift larger than 200 nm.The tiny change in polarity before and after binding with amyloid plaques leads to a transition from weakly emission charge-transfer(CT)state(Φ<0.04)to highly emissive locally-excited(LE)state(Φ=0.57),giving rise to a fluorescence Turn-On probe.Together with large Stokes'shift,good photostability and high depletion efficiency,the super-resolution imaging of the formation and morphology of amyloid fibrils in vitro based on this probe was realized with a lateral spatial resolution better than 33 nm at an extremely low depletion power.Moreover,the ex-vivo super-resolution imaging of(E)-1-butyl-4(2-(5-(9-ethyl-9Hcarbazol-3-yl)thiophen-2-yl)vinyl)pyridinium bromide(CTPB)probe in Aβ plaques in the brain slices of a Tg mouse was demonstrated.This research provides a demonstration of the super resolution imaging probe of amyloid fibrils based on polarity-response mechanism,providing a new approach to the development of future amyloid probes.

Nanomechanics of individual amyloid fibrils using atomic force microscopy

Compression elasticity of glucagon amyloid fibrils in the transverse direction was investigated by a nanoindentation approach based on atomic force microscopy (AFM).With force-volume mapping, we obtained the correlations between radially applied force and compression of amyloid fibrils, from which the radial compressive elasticity can be deduced.The estimated elastic modulus at three typical locations of fibrils varied from (0.72±0.80) GPa to (1.26±0.62) GPa under small external forces, imply-ing the structural heterogeneity of different fibrils.

姜黄素-氧化石墨烯纳米复合材料抑制淀粉样蛋白聚集

到目前为止,已经发现20多种人类退行性疾病与淀粉样纤维有紧密的关系,包括帕金森综合症、阿尔茨海默病、全身性非神经性淀粉样变性等。利用氧化石墨烯(GO)大比表面积和近红外光响应的优点,制备了姜黄素-氧化石墨烯(Cur-GO)纳米复合材料。Cur-GO的Cur负载量为213 mg/g,可在近红外光下实现控释。硫黄素T(ThT)分析表明,Cur-GO以剂量依赖的方式抑制淀粉样蛋白原纤维的聚集(抑制效率为85%)。设计制备的Cur-GO纳米复合材料可为淀粉样变疾病的治疗和控制提供新的药物材料和研究基础。

绿豆球蛋白淀粉样纤维的形成、结构表征及乳化特性

基于不同酸热处理时间探讨绿豆球蛋白淀粉样纤维(mung bean globulin amyloid fibrils,MBGFs)形成过程中的结构演变规律,同时探究各阶段形成的MBGFs乳化特性。结果表明:加热0~12 h过程中,绿豆球蛋白(mung bean globulin,MBG)的亚基逐渐降解,水解成以小分子多肽为主的纤维结构单元;并产生大量β-折叠结构,相对含量从0 h的(18.28±0.75)%提升到12 h的(53.61±1.15)%,与硫磺素T结合后荧光强度增强;MBGFs形态逐渐变得细长、柔韧,在此过程中发生了定向的纤维化聚集;在加热16~24 h过程中,成熟的MBGFs结构逐渐发生解离,纤维结构特征被破坏;与MBG相比,MBGFs乳液的乳化活性指数、乳化稳定性指数、蛋白吸附率和界面蛋白含量均有明显提高,其中酸热处理4 h形成的MBGFs乳化效果最优;4 h MBGFs乳液油滴体积较小、分布均匀有序,表观黏度最高,具有弹性凝胶结构。综上,不同酸热处理时间对MBGFs结构和乳化特性具有显著影响,MBGFs与MBG相比具有更优异的乳化性能。本研究为明晰MBGFs形成规律提供理论支撑,为高效食品级乳化剂的研发提供思路。

胰岛素淀粉样纤维的近红外聚集诱导发光荧光探针

为了快速检测淀粉样纤维以及深入研究淀粉样纤维化过程的动力学和结构特性的机理,实现对阿尔茨海默病(AD)等的早期监测,本文设计了一种两亲性的聚集诱导发光(AIE)荧光探针(E)-1-丁基-4-(4-(二苯胺基)苯乙烯基)吡啶-1-溴化鎓(BSTI)。荧光探针BSTI表现出高的结合亲和力、高的开关比、高的光稳定性和良好的双光子激发荧光特性,并兼具适当的相对分子质量以及可穿越血脑屏障的潜能。当其与牛胰岛素淀粉样原纤维结合后,表现出强烈的近红外(NIR)荧光发射,开关比>100。此外,基于BSTI的淀粉样原纤维的双光子荧光成像也表现出较高的信噪比。BSTI有望成为检测体内淀粉样原纤维的理想探针,并应用于AD的早期监测。

酸性调控对大豆分离蛋白纤维功能性质的影响

近年来,蛋白淀粉样纤维因其独特的材料学特性,在结构化和功能化食品领域引起广泛的关注。然而人们对蛋白纤维基材料的功能特性及相关调控机理还不甚了解。本文研究了不同酸诱导剂对大豆分离蛋白(SPI)自组装纤维的结构、乳化特性、凝胶特性及成膜性质的影响。结果表明,经盐酸或醋酸诱导均能形成纤维化蛋白,蛋白质的二级结构则由α-螺旋转变为β-折叠结构。相较于盐酸诱导的短而硬的蛋白纤维(H-SPI),醋酸诱导的蛋白纤维(C-SPI)形状更加柔顺且规则。除此之外,C-SPI拥有更大的粒径、更高的凝胶硬度、更优的乳化稳定性和成膜性。因此,通过选择适宜的酸诱导剂种类能够改善蛋白纤维的理化和功能性质。

Evaluation of the inhibitory effect of docosahexaenoic acid and arachidonic acid on the initial stage of amyloid β1-42 polymerization by fluorescence correlation spectroscopy

Amyloid β(Aβ)1-42 fibrillation is a crucial step in the development of pathological hallmarks, such as neuritic plaques and neurofibrillary tangles, of Alzheimer’s disease (AD). In this study, we evaluated the effects of free docosahexaenoic acid (DHA), an essential brain polyunsaturated fatty acid (PUFA), on the inhibition of Aβ1-42 fibrillation by fluorescence correlation spectroscopy (FCS), a technique capable of detecting molecular movements and interactions in solution. We also examined whether free arachidonic acid (AA), eicosapentaenoic acid (EPA), and metabolites of DHA, including neuroprotectin D1 (NPD1, 10S, 17S-dihydroxy-DHA), resolvin D1 (RvD1, 7S, 8R, 17S-trihydroxy-DHA), and didocosahexaenoyl glycerol (diDHA), affect Aβ1-42 polymerization. The results of the FCS study reveal that DHA and AA significantly reduced the diffusion time of TAMRA (5-carboxytetramethylrhoda-mine)-Aβ1-42 by 28% and 31%, respectively, while EPA, NPD1, RvD1, and diDHA had no effects on diffusion time. These results indicate that DHA and AA inhibited Aβ1-42 polymerization and that their inhibitory effects occurred at the initial stage of Aβ1-42 polymerization. This study will advance the research on PUFAs in preventing AD progression.

Hofmeister序列阴阳离子对溶菌酶淀粉样纤维的影响

应用原子力显微镜(AFM)技术和傅里叶变换红外光谱(FTIR)技术对Hofmeister阴阳离子序列中9种阴离子(SO_(4)^(2-)>F^(-)>COO^(-)>Cl^(-)>Br^(-)>NO_(3)^(-)>I^(-)>ClO_(4)^(-)>SCN^(-))和9种阳离子(NH_(4)^(+)>K^(+)>Na^(+)>Cs^(+)>Li^(+)>Rb^(+)>Mg^(2+)>Ca^(2+)>Ba^(2+))对溶菌酶淀粉样纤维化的影响进行了研究.通过AFM技术研究,发现Hofmeister序列中不同位置的阴离子对溶菌酶淀粉样纤维的微观形貌存在不同的影响,其中受SO_(4)^(2-)、F^(-)、COO^(-)这3种亲水阴离子影响生成的纤维呈现纤维分枝和增厚的现象,而受Cl^(-)、Br^(-)、NO_(3)^(-)、I^(-)、ClO_(4)^(-)、SCN^(-)这6种疏水阴离子影响的纤维则为常见的细长纤维.本研究将出现这种现象的原因归因于这2类阴离子对溶菌酶淀粉样纤维化二次成核过程的影响差异.与阴离子效应相反,研究中发现9种Hofmeister序列阳离子对溶菌酶淀粉样纤维的形貌影响差异不大.利用红外光谱分峰拟合技术,探究了不同阴阳离子影响下生成的淀粉样纤维中各种二级结构含量由多到少的排序与Hofmeister序列的相关性.希望本工作中的这些新发现能为人们更加深入地理解淀粉样纤维化的影响因素提供有益参考.

米谷蛋白淀粉样纤维聚集体自组装过程中蛋白质结构演变、功能性质及体外消化性研究

蛋白质自组装形成淀粉样纤维聚集体,是改善和拓宽食品蛋白质功能性质的重要手段。本文研究了pH 2.0和85℃加热不同时间形成的米谷蛋白淀粉样纤维聚集体(RAFA)自组装过程中蛋白质结构演变、黏度和热力学等功能性质,以及RAFA在模拟胃液和小肠液中的消化行为。结果显示,在pH 2.0、85℃条件下,天然米谷蛋白高分子量亚基逐渐水解成12 ku以下的小分子肽。随着加热时间的延长,RAFA的β-折叠构含量从0 h的(22.76±0.49)%增至峰值6 h时的(32.11±0.52)%,表明RAFA的形成是蛋白质先水解后多肽重组聚集的过程。另外,RAFA表现出较高的热稳定性,其放热峰的Tmax值从0 h的(70.34±0.51)℃增至峰值4 h时的(174.55±0.34)℃。体外消化的透射电镜结果显示,热处理6 h和10 h的纤维消化趋势一致,胃蛋白酶消化60 min后,一部分具有分枝状的长纤维被酶解成短小纤维,同时观察到较大的聚集体颗粒。胰酶作用后纤维结构减少,大的聚集体颗粒被酶解为小聚集体颗粒,即RAFA表现出胃蛋白酶抗性。本研究结果为利用蛋白质纤维聚集体构建新型食品提供了理论参考。

Self-Assembled Protein Hybrid Nanofibrils for Photosynthetic Hydrogen Evolution

In artificial photosynthesis systems,synthetic diiron complexes are popular[FeFe]-hydrogenase mimics,which are attractive for the fabrication of photocatalyst-protein hybrid structures to amplify hydrogen(H2)generation capability.However,constructing a highly bionic and efficient catalytic hybrid system is a major challenge.Notably,we designed an ideal hybrid nanofibrils system that incorporates the crucial components:(1)a[FeFe]-H2ase mimic,which has a three-arm architecture(named triFeFe)for more interaction sites and higher catalytic activity and(2)uniform hybrid nanofibrils as the biological environment in which cysteine-catalyst coordination and the hydrogen-bonding network play a vital role in both catalyst binding and hydrogen evolution reaction activity.The assembled hybrid nanofibrils achieve efficient H2 generation with a turnover number of 2.3×103,outperforming previously reported diiron catalyst-protein hybrid systems.Additionally,the hybrid nanofibrils work with photosynthetic thylakoids to produce H2,without extra photosensitizers or electron shuttle proteins,which advances the bioengineering of living systems for solar-driven biofuel production.

全长朊粒蛋白淀粉样纤维引发细胞毒性的机制研究

目的 朊病毒病(prion disease)是一类由朊粒蛋白(PrP)发生错误折叠、聚集形成致病性的PrPSc导致的具有高致死率的神经退行性疾病。本文在细胞和动物水平开展了PrP纤维诱导内源PrP聚集和毒性机制的研究。方法 通过超速离心结合蛋白质免疫印迹实验检测PrP聚集;通过氧化压力实验,使用Annexin V-FITC/PI双染检测细胞凋亡;运用细胞超薄切片技术检测细胞线粒体形态;在动物水平,分离新生小鼠的前额叶,进行横断切片培养,在脑片上接种PrP纤维。结果 PrP纤维种子可以诱导内源PrP聚集,PrP纤维可以诱导细胞内氧化压力升高和细胞凋亡,PrP纤维可以引起线粒体损伤,PrP纤维可以诱导小鼠前额叶内源PrP聚集。结论 本文在细胞和动物水平证实体外组装的PrP淀粉样纤维具有细胞毒性和潜在的感染性。