Activity of Zn and Mg phthalocyanines and porphyrazines in amyloid aggregation of insulin

Formation of the deposits of protein aggregates—amyloid fibrils in an intracellular and intercellular space—is common to a large group of amyloid‐associated disorders. Among the approaches to develop of therapy of such disorders is the use of agents preventing protein fibrillization. Polyaromatic complexes—porphyrins and phthalocyanines—are known as compounds possessing anti‐fibrillogenic activity. Here, we explore the impact of related macrocyclic complexes—phthalocyanines (Pc) and octaphenyl porphyrazines (Pz) of Mg and Zn—on aggregation of amyloidogenic protein insulin. Pz complexes are firstly reported as compounds able to affect protein fibrillization. The effect of Pc and Pz complexes on the kinetics and intensity of insulin aggregation was studied by the fluorescent assay using amyloid sensitive cyanine dye. This has shown the impact of metal ion on the anti‐fibrillogenic properties of macrocyclic complexes—the effect on the fibrillization kinetics of Mg‐containing compounds is much more pronounced comparing to that of Zn analogues. Scanning electron microscopy experiments have demonstrated that filamentous fibrils are the main product of aggregation both for free insulin and in the presence of macrocyclic complexes. However, those fibrils are distinct by their length and proneness to lateral aggregation. The Pc complexes cause the increase in variation of fibrils length 0.9 to 2.7 nm in opposite to 1.4 to 2.0 nm for free insulin, whereas Pz complexes cause certain shortening of the fibrils to 0.8 to 1.6 nm. The averaged size of the fibrils population was estimated by dynamic light scattering; it correlates with the size of single fibrils detected by scanning electron microscopy.     

Probing the mechanism of insulin aggregation with added metalloporphyrins

The mechanism of inhibition of insulin-based amyloid gel formation by metal derivatives of tetrakis(4-sulfonatophenyl)porphyrin has been investigated. Time-course UV/vis measurements in conjunction with atomic force microscopy (AFM) were used to study the correlation between observed kinetics and amyloid structure for various concentration ranges of added metalloporphyrins. Observed structures include fibrils as well as circular, ring-like structures formed as a result of the interaction of insulin with porphyrin. In addition, binding studies demonstrate that the effectiveness of inhibition of the various metalloporphyrins is directly related to the strength of binding to insulin. It is suggested that both the electron distribution in the porphyrin core and the tendency to form porphyrin dimers affect both the structure of amyloid formed and the kinetic profile of the reaction.     

Quercetin inhibits amyloid fibrillation of bovine insulin and destabilizes preformed fibrils

Growing interest and research efforts have recently been focused on elucidating the molecular mechanism of amyloid formation and the screening of effective inhibitors to interrupt amyloid structures. In the present study, the anti-amyloidogenic effects of quercetin were investigated in vitro using bovine insulin as a model protein. The results demonstrated that quercetin dose-dependently inhibited amyloid formation of insulin. Moreover, quercetin destabilized the preformed insulin fibrils and transformed the fibrils into amorphous aggregates. Hemolysis was observed when human erythrocytes were co-incubated with insulin fibrils. Quercetin inhibited fibril-induced hemolysis in a dose-dependent manner. SDS–PAGE showed that insulin fibrils induced the aggregation of cytoskeletal proteins of erythrocyte membranes and that quercetin attenuated this fibril-induced cytoskeletal aggregation. The results of the present work suggest that quercetin may serve as a lead structure for the design of novel anti-amyloidogenic drugs.     

Tuning amyloidogenic conformations through cosolvents and hydrostatic pressure: when the soft matter becomes even softer

Compact packing, burial of hydrophobic side-chains, and low free energy levels of folded conformations contribute to stability of native proteins. Essentially, the same factors are implicated in an even higher stability of mature amyloid fibrils. Although both native insulin and insulin amyloid are resistant to high pressure and influence of cosolvents, intermediate aggregation-prone conformations are susceptible to either condition. Consequently, insulin fibrillation may be tuned under hydrostatic pressure or-- through cosolvents and cosolutes-- by preferential exclusion or binding. Paradoxically, under high pressure, which generally disfavors aggregation of insulin, an alternative "low-volume" aggregation pathway, which leads to unique circular amyloid is permitted. Likewise, cosolvents are capable of preventing, or altering amyloidogenesis of insulin. As a result of cosolvent-induced perturbation, distinct conformational variants of fibrils are formed. Such variants, when used as templates for seeding daughter generations, reproduce initial folding patterns regardless of environmental biases. By the close analogy, this suggests that the "prion strains" phenomenon may mirror a generic, common feature in amyloids. The susceptibility of amyloidogenic conformations to pressure and cosolvents is likely to arise from their "frustration", as unfolding results in less-densely packed side-chains, void volumes, and exposure of hydrophobic groups. The effects of cosolvents and pressure are discussed in the context of studies on other amyloidogenic protein models, amyloid polymorphism, and "strains".     

Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine‐rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His‐tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low‐pH harsh conditions, however, His‐HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid‐hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full‐length His‐HP when incubated with 10–20% 2,2,2‐trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage.     

Zinc determines dynamical properties and aggregation kinetics of human insulin

Protein aggregation is a widespread process leading to deleterious consequences in the organism, with amyloid aggregates being important not only in biology but also for drug design and biomaterial production. Insulin is a protein largely used in diabetes treatment, and its amyloid aggregation is at the basis of the so-called insulin-derived amyloidosis. Here, we uncover the major role of zinc in both insulin dynamics and aggregation kinetics at low pH, in which the formation of different amyloid superstructures (fibrils and spherulites) can be thermally induced. Amyloid aggregation is accompanied by zinc release and the suppression of water-sustained insulin dynamics, as shown by particle-induced x-ray emission and x-ray absorption spectroscopy and by neutron spectroscopy, respectively. Our study shows that zinc binding stabilizes the native form of insulin by facilitating hydration of this hydrophobic protein and suggests that introducing new binding sites for zinc can improve insulin stability and tune its aggregation propensity.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

Osmolyte controlled fibrillation kinetics of insulin: New insight into fibrillation using the preferential exclusion principle

Amyloid proteins are converted from their native‐fold to long β‐sheet‐rich fibrils in a typical sigmoidal time‐dependent protein aggregation curve. This reaction process from monomer or dimer to oligomer to nuclei and then to fibrils is the subject of intense study. The main results of this work are based on the use of a well‐studied model amyloid protein, insulin, which has been used in vitro by others. Nine osmolyte molecules, added during the protein aggregation process for the production of amyloid fibrils, slow‐down or speed up the process depending on the molecular structure of each osmolyte. Of these, all stabilizing osmolytes (sugars) slow down the aggregation process in the following order: tri > di > monosaccharides, whereas destabilizing osmolytes (urea, guanidium hydrochloride) speed up the aggregation process in a predictable way that fits the trend of all osmolytes. With respect to kinetics, we illustrate, by adapting our earlier reaction model to the insulin system, that the intermediates (trimers, tetramers, pentamers, etc.) are at very low concentrations and that nucleation is orders of magnitude slower than fibril growth. The results are then collated into a cogent explanation using the preferential exclusion and accumulation of osmolytes away from and at the protein surface during nucleation, respectively. Both the heat of solution and the neutral molecular surface area of the osmolytes correlate linearly with two fitting parameters of the kinetic rate model, that is, the lag time and the nucleation rate prior to fibril formation. These kinetic and thermodynamic results support the preferential exclusion model and the existence of oligomers including nuclei and larger structures that could induce toxicity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Formation of low-dimensional crystalline nucleus region during insulin amyloidogenesis process

Insulin, as other amyloid proteins, can form amyloid fibrils at certain conditions. The self-assembled aggregation process of insulin can result in a variety of conformations, starting from small oligomers, going through various types of protofibrils, and finishing with bundles of fibrils. One of the most common consensuses among the various self-assembly processes that are suggested in the literature is the formation of an early stage nucleus conformation. Here we present an additional insight for the self-assembly process of insulin. We show that at the early lag phase of the process (prior to fibril formation) the insulin monomers self-assemble into ordered nanostructures. The most notable feature of this early self-assembly process is the formation of nanocrystalline nucleus regions with a strongly bound electron-hole confinement, which also change the secondary structure of the protein. Each step in the self-assembly process is characterized by an optical spectroscopic signature, and possesses a narrow size distribution. By following the spectroscopic signature we can measure the potency of amyloid fibrils inhibitors already at the lag phase. We further demonstrate it by the use of epigallocatechin gallate, a known inhibitor for insulin fibrils. The findings can result in a spectroscopic-based application for the analysis of amyloid fibrils inhibitors.     

Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ～20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.     

Photo-induced inhibition of insulin amyloid fibrillation on online laser measurement

Protein aggregation and amyloid fibrillation can lead to several serious diseases and protein drugs ineffectiveness; thus, the detection and inhibition of these processes have been of great interest. In the present study, the inhibition of insulin amyloid fibrillation by laser irradiation was investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), far-UV circular dichroism (far-UV CD), and thioflavin T (ThT) fluorescence. During heat-induced aggregation, the size distribution of two insulin solutions obtained by online and offline dynamic light scattering were different. The laser-on insulin in the presence of 0.1 M NaCl exhibited fewer fibrils than the laser-off insulin, whereas no insulin fibril under laser irradiation was observed in the absence of 0.1 M NaCl for 45 h incubation. Moreover, our CD results showed that the laser-irradiated insulin solution maintained mainly an α-helical conformation, but the laser-off insulin solution formed bulk fibrils followed by a significant increase in β-sheet content for 106 h incubation. These findings provide an inhibition method for insulin amyloid fibrillation using the laser irradiation and demonstrate that the online long-time laser measurements should be carefully used in the study of amyloid proteins because they may change the original results.     

Rational design of amyloid β peptide–binding proteins: Pseudo‐Aβ β‐sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Aβ

Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid β‐peptide (Aβ) is thought to be a toxic agent by self‐assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo‐Aβ β‐sheet surfaces and evaluated their abilities to bind to Aβ and inhibit Aβ oligomerization. Two GFP variants P13H and AP93Q bound tightly to Aβ, K_d = 260 nM and K_d = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Aβ oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Aβ‐derived sequences was designed and bound more tightly to Aβ (K_d = 100 nM) than those having only two pseudo‐Aβ strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Aβ more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo‐Aβ β‐sheet structures onto a protein surface arranged in the β‐barrel structure is useful to construct molecules capable of binding tightly to Aβ and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Disulfide bridges remain intact while native insulin converts into amyloid fibrils

Amyloid fibrils are β-sheet-rich protein aggregates commonly found in the organs and tissues of patients with various amyloid-associated diseases. Understanding the structural organization of amyloid fibrils can be beneficial for the search of drugs to successfully treat diseases associated with protein misfolding. The structure of insulin fibrils was characterized by deep ultraviolet resonance Raman (DUVRR) and Nuclear Magnetic Resonance (NMR) spectroscopy combined with hydrogen-deuterium exchange. The compositions of the fibril core and unordered parts were determined at single amino acid residue resolution. All three disulfide bonds of native insulin remained intact during the aggregation process, withstanding scrambling. Three out of four tyrosine residues were packed into the fibril core, and another aromatic amino acid, phenylalanine, was located in the unordered parts of insulin fibrils. In addition, using all-atom MD simulations, the disulfide bonds were confirmed to remain intact in the insulin dimer, which mimics the fibrillar form of insulin.     

Novel fluorescent trimethine cyanine dye 7519 for amyloid fibril inhibition assay

Abstract

Fluorescence spectroscopy was used to study the ability of dye 7519 to follow the transition of monomeric insulin into fibrils and applicability of the dye to the insulin aggregation inhibition assay. The commercially available classic amyloid stain, thioflavin T, was used as the reference dye. For selecting potential inhibitors, the QSAR approach was applied. Dye 7519 appeared to be suitable for monitoring insulin aggregation into fibrils in vitro. The properties of the dye allowed us to test it as a potential probe in the screening assay of potential inhibitors of insulin fibrillization. One hundred forty-four flavonoids were tested as potential inhibitors of amyloid fibril formation using the quantitative structure activity relationship approach. Among them, 10 candidates with high indexes of inhibition were selected for tests in vitro using dye 7519 and the reference amyloid dye thioflavine T. Using dye 7519 fluorescence, we found that two compounds had inhibitory effects on insulin amyloid formation. These results agree with inhibition data using the thioflavine T assay. Our studies demonstrated that the fluorescent cyanine dye 7519 is a sensitive probe for quantitative detection of insulin amyloid formation and can be applied to screen agents capable of affecting aggregation of amyloid proteins.     

Amyloid‐like aggregation of designer bolaamphiphilic peptides: Effect of hydrophobic section and hydrophilic heads

Amyloid‐like aggregation of natural proteins or polypeptides is an important process involved in many human diseases as well as some normal biological functions. Plenty of works have been done on this ubiquitous phenomenon, but the molecular mechanism of amyloid‐like aggregation has not been fully understood yet. In this study, we showed that a series of designer bolaamphiphilic peptides could undergo amyloid‐like aggregation even though they didn't possess typical β‐sheet secondary structure. Through systematic amino acid substitution, we found that for the self‐assembling ability, the number and species of amino acid in hydrophobic section could be variable as long as enough hydrophobic interaction is provided, while different polar amino acids as the hydrophilic heads could change the self‐assembling nanostructures with their aggregating behaviors affected by pH value change. Based on these results, novel self‐assembling models and aggregating mechanisms were proposed, which might provide new insight into the molecular basis of amyloid‐like aggregation.     

Microscopic Factors that Control β-Sheet Registry in Amyloid Fibrils Formed by Fragment 11-25 of Amyloid β Peptide: Insights from Computer Simulations

Short fragments of amyloidogenic proteins are widely used as model systems in studies of amyloid formation. Fragment 11-25 of the amyloid β protein involved in Alzheimer's disease (Aβ11-25) was recently shown to form amyloid fibrils composed of anti-parallel β-sheets. Interestingly, fibrils grown under neutral and acidic conditions were seen to possess different registries of their inter-β-strand hydrogen bonds. In an effort to explain the microscopic origin of this pH dependence, we studied Aβ11-25 fibrils using methods of theoretical modeling. Several structural models were built for fibrils at low and neutral pH levels and these were examined in short molecular dynamics simulations in explicit water. The models that displayed the lowest free energy, as estimated using an implicit solvent model, were selected as representative of the true fibrillar structure. It was shown that the registry of these models agrees well with the experimental results. At neutral pH, the main contribution to the free energy difference between the two registries comes from the electrostatic interactions. The charge group of the carboxy terminus makes a large contribution to these interactions and thus appears to have a critical role in determining the registry.     

The impact of binding of macrocyclic metal complexes on amyloid fibrillization of insulin and lysozyme

Amyloid fibrils are insoluble protein aggregates whose accumulation in cells and tissues is connected with a range of pathological diseases. We studied the impact of 2 metal complexes (axially coordinated Hf phthalocyanine and iron (II) clathrochelate) on aggregation of insulin and lysozyme. For both proteins, the host‐guest interaction with these compounds changes the kinetics of fibrillization and affects the morphology of final aggregates. The Hf phthalocyanine is a very efficient inhibitor of insulin fibrillization; in its presence, only very low amounts of fibrils with the diameters of 0.8 to 5 nm and spherical aggregates were found. Effective concentration of fibrillization inhibition (IC₅₀) was estimated to be 0.11 ± 0.04 μM. The clathrochelate induced the formation of thin fibrils with the diameters of 0.8 to 2.5 nm; IC₅₀ was estimated as 20 ± 9 μM. The lysozyme fibrillization remained quite intensive in the presence of the studied compounds; they induced the formation of long filaments (the length up to 2.5 μm, the diameters of 1.5‐3.5 nm). These fibrils noticeably differed from those of free lysozyme short linear species (the diameters of 3‐5 nm, the length up to 0.6 μm). Thinning and elongation of fibrils suggest that the metal complexes bind mainly to the grooves of protofilaments; this hinders the stacking of early aggregates or protofilaments together but does not hinder their growth. The image of the fibril separated into 2 protofilaments allows suggesting that the fibril formation occurs via the growth of the parallel protofilaments with their subsequent twisting in the fibril. The changes of the lysozyme intrinsic fluorescence indicate that both metal complexes interact with the protein during the stage of the fibrillar seeds formation.     

Design and study of lipopeptide inhibitors on preventing aggregation of human islet amyloid polypeptide residues 11‐20

Type 2 diabetes mellitus, a kind of conformational disease, has become an epidemic disease, which seriously endangers the quality of life and health of human beings. The deposition of human islet amyloid polypeptide (hIAPP) has been considered as one of the major pathological features of type 2 diabetes mellitus. As lipopeptides have some hydrophobic groups, which are similar to the reported aggregation inhibitors, and some lipopeptides could prevent cells from depositing of amyloid fibrils, several potential lipopeptide inhibitors have been engineered and synthesized, which have been assessed for their inhibitory effect in preventing amyloid fibrils formation of hIAPP_11‐20 by using the conventional thioflavin‐T fluorescence assay and new technique microscale thermophoresis (MST). The final amyloid fibrils of hIAPP_11‐20 were characterized by transmission electron microscopy. Results suggested that with the increasing length of alkyl chain, the antiaggregation efficiency of lipopeptide inhibitors towards hIAPP_11‐20 increased gradually. Meanwhile, the amount of arginines, which represent the head groups of lipopeptides, may also have some influence. The binding events also showed that the inhibitory efficiency of these lipopeptide inhibitors was enhanced with the increase of affinities between lipopeptides and hIAPP_11‐20, which were obtained from MST. This study demonstrated the efficiency of lipopeptides in inhibiting the aggregation of hIAPP_11‐20 and proved that MST could be regarded as an appropriate and rapid method to screen potential inhibitors of hIAPP_11‐20 or other amyloid proteins. This study also broadens the types of inhibitors on inhibiting amyloid formation of hIAPP.     

Inhibition of insulin fibrillogenesis with targeted peptides

Under conditions of acidic pH and elevated temperature, insulin partially unfolds and aggregates into highly structured amyloid fibrils. Aggregation of insulin leads to loss of activity and can trigger an unwanted immune response. Compounds that prevent protein aggregation have been used to stabilize insulin; these compounds generally suppress aggregation only at relatively high inhibitor concentrations. For example, effective inhibition of aggregation of 0.5 mM insulin required arginine concentrations of > or =100 mM. Here, we investigate a targeted approach toward inhibiting insulin aggregation. VEALYL, corresponding to residues B12-17 of full-length insulin, was identified as a short peptide that interacts with full-length insulin. A hybrid peptide was synthesized that contained this binding domain and hexameric arginine; this peptide significantly reduced the rate of insulin aggregation at near-equimolar concentrations. An effective binding domain and N-terminal placement of the arginine hexamer were necessary for inhibitory activity. The data were analyzed using a simple two-step model of aggregation kinetics. These results are useful not only in identifying an insulin aggregation inhibitor but also in extending a targeted protein strategy for modifying aggregation of amyloidogenic proteins.