Identification of minimal peptide sequences in the (8-20) domain of human islet amyloid polypeptide involved in fibrillogenesis

We have examined a series of overlapping peptide fragments from the 8-20 region of human islet amyloid polypeptide (IAPP) with the objective of defining the smallest fibril-forming domain. Peptide fragments corresponding to LANFLV (residues 12-17) and FLVHSS (residues 15-20) were strong enhancers of beta-sheet transition and fibril formation. Negative stain electron microscopy illustrated the ability of these peptide fragments to form fibrils independently when incubated alone in solution. Circular dichroism analysis revealed that when full-length human IAPP was incubated in the presence of these two fragments, fibrillogenesis was accelerated. While the two fragments, LANFLV and FLVHSS, were able to enhance the recruitment of additional IAPP molecules during fibril formation, the "seeding" activity of these peptides had no effect on altering IAPP-induced cytotoxcity as determined by cell culture studies. Therefore, this study has identified two internal IAPP peptide fragments within the 8-20 domain that may have a role in enhancing the folding and aggregation of human IAPP. These fragments are the smallest sequences identified, within the 8-20 region of hIAPP, that can independently form fibrils, and that can interact with IAPP to assemble into fibrils with characteristics similar as those formed by human IAPP alone.     

Non-electrostatic binding and self-association of amyloid beta-peptide on the surface of tightly packed phosphatidylcholine membranes

Self-association of amyloid β-peptide (Aβ) is considered to be an initial step in the development of Alzheimer’s disease and is known to be promoted by negatively charged lipid membranes. We have examined the possibility of non-electrostatic Aβ-membrane interaction by using neutral phosphatidylcholine lipids. Fluorescence and circular dichroism spectra have clearly shown that Aβ binds to the phosphatidylcholine membrane in the lamellar gel phase but not in the ripple gel or liquid crystalline phase, indicating the importance of the tight lipid packing characteristic of the lamellar gel phase. The Aβ-membrane binding occurs at both low and high salt concentrations, ensuring the non-electrostatic nature of the interaction. The membrane-bound Aβ molecule takes a monomeric α-helical or self-associated β-sheet structure depending on the temperature, peptide/lipid ratio, and salt concentration. The flat surface of tightly packed phosphatidylcholine membranes appears to serve as a platform for non-electrostatic binding and self-association of Aβ.     

Contribution of simple saccharides to the stabilization of amyloid structure

The use of osmolytes or chaperones to stabilize proteins/peptides that misfold in neurodegenerative diseases is an attractive concept for drug development. We have investigated the role of a series of small carbohydrates for protection of the natively structured Alzheimer's amyloid-beta peptides (Abeta). Using circular dichroism spectroscopy to follow the beta-structural transitions and electron microscopy to examine tertiary structural characteristics, we demonstrate that the hydrogen bonding capacity of the carbohydrate determines the inhibition or promotion of fibrillogenesis. Three sugar molecules that vary only in their distribution of potential H-bonding partners promote various structural changes in Abeta. Two of these sugar molecules are excluded from Abeta during aggregation and promote mature fibre growth, while the other binds Abeta promoting nucleation and the accumulation of protofibrils. Our studies suggest that utilization of a combinatorial strategy to alter H-bonding capacity across a simple carbohydrate molecule may represent a novel drug design strategy.     

Shadoo binds lipid membranes and undergoes aggregation and fibrillization

Lipid membrane can enhance prion protein (PrP) pathological fibrillogenesis. A neuronal paralog of PrP, named Shadoo (Sho), is localized to similar membrane environment as PrP and can also convert to amyloid-like fibrilles. To gain insight into the role of Sho in prion diseases, we studied Sho interactions with cellular membrane models. Sho was found to bind anionic lipid vesicles. Spectroscopic and microscopic data showed that membrane-associated Sho slowly converted into amyloid fibers. Furthermore, binding of Sho to anionic liposomes has a disruptive effect on the integrity of the lipid bilayer leading to the formation of supramolecular lipid–protein complexes. In consequence, the role of Sho in prion diseases might depend on the oligomerization state of Sho but also the nature of these lipoprotein assembles.     

Influence of hydrophobic Teflon particles on the structure of amyloid beta-peptide

The amyloid beta-protein (Abeta) constitutes the major peptide component of the amyloid plaque deposits of Alzheimer's disease in humans. The Abeta changes from a nonpathogenic to a pathogenic conformation resulting in self-aggregation and deposition of the peptide. It has been established that denaturing factors (such as the interaction with membranes) are involved in the structural transition. This work is aimed at determining the effect of hydrophobic Teflon on the conformation of the Abeta (1-40). Prior to adsorption, the secondary structure and self-aggregation state of the Abeta in solution were established as a function of pH. Three different species coexist: unordered monomers/dimers, small oligomers in mainly a regular beta-sheet structure, and bigger aggregates having a twisted beta-sheet conformation. Transferring the Abeta from the solution to the Teflon surface strongly promotes alpha-helix formation. Furthermore, increasing the degree of coverage of the Teflon by the Alphabeta protein leads to a conformational change toward a more enriched beta-sheet structure.     

Effect of curcumin on the amyloid fibrillogenesis of hen egg-white lysozyme

At least twenty human proteins can fold abnormally to form pathological deposits that are associated with several degenerative diseases. Despite extensive investigation on amyloid fibrillogenesis, its detailed molecular mechanisms remain unknown. This study is aimed at exploring the inhibitory activity of curcumin against the fibrillation of hen lysozyme. We found that the formation of amyloid fibrils at pH 2.0 in vitro was inhibited by curcumin in a dose-dependent manner. Moreover, quenching analysis confirmed the existence of an interaction between curcumin and lysozyme, and Van't Hoff analysis indicated that the curcumin–lysozyme interaction is predominantly governed by Van Der Waals force or hydrogen bonding. Curcumin was also found to acquire disaggregating ability on preformed lysozyme fibrils. Finally, we observed that curcumin pre-incubated at 25 °C for at least 7 days inhibited lysozyme fibrillogenesis better than untreated curcumin and the enhanced inhibition against HEWL fibrillation might be attributed to the presence of dimeric species.     

Effects of p-benzoquinone and melatonin on amyloid fibrillogenesis of hen egg-white lysozyme

More than 20 different human proteins can fold abnormally resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained far from complete. In this work, utilizing hen egg-white lysozymes as a model system, two objectives were pursued: (1) to search for suitable conditions for producing amyloid fibrils and (2) to investigate inhibitory activities of two potential molecules against lysozyme fibril formation. Via numerous spectroscopic analyses and electron microscopy, our results showed that the formation of lysozyme amyloid fibrils at pH 2.0 was considerably increased by the addition of salt. Moreover, the inhibition of lysozyme amyloid formation by either p-benzoquinone or melatonin followed a concentration-dependent fashion. Furthermore, p-benzoquinone, in comparison with melatonin, served as a more effective inhibitor against amyloid fibril formation of lysozyme. We believe that a better understanding of how hen egg-white lysozymes aggregate will not only aid in deciphering the molecular mechanism of amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.     

Change and stabilization of the amyloid-β(1–40) secondary structure by fluorocompounds

The misfolding of the amyloid peptide, which is the result of a well-known α-to-β transition, causes neurodegenerative disorder. Fluorinated alcohols have been described in the literature as potent solvents which can refold the β-conformation. The present studies demonstrate the effectiveness of differently fluorinated alcohols for the β-to-α refolding process on fibrillar aggregated amyloid β(1–40). The regenerated helical structure is shown to be maintained in the absence of the fluoroalcohols, a behaviour which was found to contrast with immunoglobulin. We interpret this difference on the basis of the hydrophilic/hydrophobic domains in the amyloid sequence and present some speculations regarding the free-energy levels of the folded states of both proteins. The effect of the –CF₃ group on the observed conformational changes is interpreted as a result of alterations of the hydration shell of the peptides. Moreover, based on the results achieved with fluoroalcohols, we have used novel fluorinated amphiphiles possessing blood-compatibility properties and studied their effect on amyloid β(1–40). First results point in the direction of a β-to-α transition. Therefore, the use of fluorine groups in the development of new drugs is considered a new possibility requiring further investigation for the prevention of amyloidosis.     

Quantitative determination of the topological propensities of amyloidogenic peptides

One of the interesting puzzles of amyloid beta-peptide of Alzheimer's disease (Abeta) is that it appears to polymerize into amyloid fibrils in a parallel beta sheet topology, while smaller subsets of the peptide produce anti-parallel beta sheets. In order to target potential weak points of amyloid fibrils in a rational drug design effort, it would be helpful to understand the forces that drive this change. We have designed two peptides CHQKLVFFAEDYNGKDEAFFVLKQHW and CHQKLVFFAEDYNGKHQKLVFFAEDW that join the significant amyloidogenic Abeta (14-23) sequence HQKLVFFAED in parallel and anti-parallel topologies, respectively. (Here, the word "parallel" refers only to residue sequence and not backbone topology). The N-termini of the hairpins were labeled with the fluorescent dye 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), forming a fluorescence energy transfer donor-acceptor pair with the C-terminus tryptophan. Circular dichroism results show that the anti-parallel hairpin adopts a beta-sheet conformation, while the parallel hairpin is disordered. Fluorescent Resonance Energy Transfer (FRET) results show that the distance between the donor and the acceptor is significantly shorter in the anti-parallel topology than in the parallel topology. The fluorescence intensity of anti-parallel hairpin also displays a linear concentration dependence, indicating that the FRET observed in the anti-parallel hairpin is from intra-molecular interactions. The results thus provide a quantitative estimate of the relative topological propensities of amyloidogenic peptides. Our FRET and CD results show that beta sheets involving the essential Abeta (14-23) fragment, strongly prefer the anti-parallel topology. Moreover, we provide a quantitative estimate of the relative preference for these two topologies. Such analysis can be repeated for larger subsets of Abeta to determine quantitatively the relative degree of preference for parallel/anti-parallel topologies in given fragments of Abeta.     

Inhibitors of islet amyloid polypeptide fibrillogenesis, and the treatment of type-2 diabetes

Human islet amyloid polypeptide (IAPP) is the major component of amyloid deposits found in the pancreas of over 90% of all cases of type-2 diabetes. Although it may be a secondary event in the etiology of diabetes, the accumulation of insoluble IAPP fibrils is considered to be a primary cause of β-cell failure in affected individuals. A possible means of inhibiting this process is through the use of small peptides that bind to IAPP and prevent fibril polymerization. This approach has been examined using a series of overlapping hexamers that target the known amyloidogenic regions of IAPP. Peptides were examined usingin vitroassays and active inhibitors were identified by their ability to prevent amyloid-related conformational transitions and IAPP aggregation. Fragments such as those corresponding to the IAPP-derived sequences, SNNFGA (residues 20–25) and GAILSS (residues 24–29), were potent inhibitors ofβ-sheet folding and amyloid fibril formation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP significantly decreased the density of fibrils and any remaining structures displayed altered morphology. In some, but not all cases, inhibition of amyloid fibrils also correlated with an ability to reduce IAPP-mediated cytotoxicity as determined in cell culture studies. The results from these studies suggest that these two peptide inhibitors differ in their mechanisms of action possibly due to unique interactions with the full-length IAPP molecule. These inhibitors form the basis of a therapeutic strategy to prevent amyloid accumulation leading to improved islet survival and a potentially novel treatment for type-2 diabetes.     

Influence of dendrimer's structure on its activity against amyloid fibril formation

Inhibition of fibril assembly is a potential therapeutic strategy in neurodegenerative disorders such as prion and Alzheimer's diseases. Highly branched, globular polymers-dendrimers-are novel promising inhibitors of fibril formation. In this study, the effect of polyamidoamine (PAMAM) dendrimers (generations 3rd, 4th, and 5th) on amyloid aggregation of the prion peptide PrP 185-208 and the Alzheimer's peptide Abeta 1-28 was examined. Amyloid fibrils were produced in vitro and their formation was monitored using the dye thioflavin T (ThT). Fluorescence studies were complemented with electron microscopy. The results show that the higher the dendrimer generation, the larger the degree of inhibition of the amyloid aggregation process and the more effective are dendrimers in disrupting the already existing fibrils. A hypothesis on dendrimer-peptide interaction mechanism is presented based on the dendrimers' molecular structure.     

Structural characterization of the lipovitellin from the shrimp Macrobrachium borellii

In oviparous species, proteins and lipids are found in the vitellus forming lipoproteins called lipovitellins. They are an important energy source for embryos development and larvae growth and survival. We have previously isolated and partially characterized the sole egg cytosolic lipovitellin from the freshwater shrimp Macrobrachium borellii. It is a native protein of 440 kDa, composed of two subunits of 94 and 112 kDa. In the present work we studied size, shape and structure of M. borellii lipovitellin using electron microscopy, crosslinking reagents, MALDI-TOF, circular dichroism, fluorescence and partial proteolysis. The results showed that lipovitellin has a quasi spherical morphology with an estimated diameter of 18.5 ± 3.5 nm. It appears to be composed of two subunits of 94 kDa, and one of 112 kDa. The larger subunit is more susceptible to trypsinolysis, indicating that it is less compactly folded and/or more exposed to the aqueous medium than the 94 kDa subunits. The hetero-trimer is held together by non-covalent interactions. Peptide mass fingerprinting by MALDI-TOF, produced 42 polypeptides matching to a vitellogenin of a related species (Macrobrachium rosenbergii). Circular dichroism indicated that this protein contains 35.7% α-helix, 16.6% β-sheet and 20% turns. Tryptophan fluorescence emission, at a maximum of 334 nm, indicated that the environment polarity of these aromatic residues is similar to that of other crustacean lipoproteins.     

Helix-loop-helix peptide foldamers and their use in the construction of hydrolase mimetics

De novo designed helix-loop-helix peptide foldamers containing cis-2-aminocyclopentanecarboxylic acid residues were evaluated for their conformational stability and possible use in enzyme mimetic development. The correlation between hydrogen bond network size and conformational stability was demonstrated through CD and NMR spectroscopies. Molecules incorporating a Cys/His/Glu triad exhibited enzyme-like hydrolytic activity.     

Structure and dynamics of a DNA-based model system for the study of electron spin-spin interactions

We report on the structure and dynamics of a model system for measuring long-range distances in biological macromolecules by saturation-recovery EPR. Four DNA duplexes that incorporate a paramagnetic dysprosium ion (Dy(III)) and a nitroxide spin-label were examined by electron paramagnetic resonance (EPR), circular dichroism (CD), and ultra-violet absorbance (UV) spectroscopy. Dy(III) is chelated by the modified base deoxythymidine-EDTA, (dT-EDTA). Electron spin-spin interactions between the Dy(III) ion and the nitroxide radical are observed at distances as great as ∼5.3 nm. A slight change in the conformation of those nucleotides lying between the EDTA(Dy(III)) complex and the nitroxide spin-label results in a “stiffening” of the DNA helix on the EPR time scale. Changes in conformation and helix dynamics are due to the binding of the EDTA(Dy(III)) complex to the phosphodiester backbone of the complementary strand. Molecular mechanics calculations indicate that binding occurs in the 5′ direction on the complementary strand, at a position 3 or 4 phosphates distant from the dT-EDTA(Dy(III)) * dA base pair.     

New lipid formulation of amphotericin B: spectral and microscopic analysis

UV-visible and dichroic spectrum analysis and electron microscopy have been used to characterize a new amphotericin B (AmB) lipid formulation prepared by a solvent displacement process. The composition was dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) in molar ratio DMPC/DMPG/AmB 7:3:5, a similar composition to that of Abelcet®. Although the latter has a “ribbon-like” structure, our process gave a thin disc-like structure. Analysis of circular dichroism (CD) and UV-visible spectra of formulations containing different percentages of AmB revealed that a minimum of AmB self-association was observed with 7:3:5 molar ratio. Varying the lipid ratio (DMPC/DMPG) while maintaining the fixed ratio of AmB yielded similar results when DMPC was in excess (DMPC/DMPG from 10:0 to 6:4). However, when the ratio was between 5:5 to 3:7, AmB self-aggregation increased. For compositions rich in DMPG (2:8 and 0:10), inversion of the CD spectrum was observed. The influence of the lipid composition on the morphology of the complex was also evident in electron microscopy. DMPC/DMPG/AmB (10:0:5) gave large unfracturable lamellae. The presence of DMPG shortened the lamellae, which often appeared as disc-like structures. AmB content, the presence of DMPG and the preparation process all contribute to generating these original structures with particular CD spectra.     

Stabilization of the fibrous structure of an alpha-helix-forming peptide by sequence reversal

The alpha3-peptide, which comprises three repeats of the sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala and forms an amphipathic alpha-helix, is unique among various alpha-helix-forming peptides in that it assembles into fibrous structures that can be observed by transmission electron microscopy. As part of our investigation of the structure-stability relationships of the alpha3-peptide, we synthesized the r3-peptide, whose amino acid sequence is the reverse of that of the alpha3-peptide, and we investigated the effects of sequence reversal on alpha-helix stability and the formation of fibrous structures. Unexpectedly, the r3-peptide formed a more-stable alpha-helix and longer fibers than did the alpha3-peptide. The stability of the r3-peptide helix decreased when the ionic strength of the buffer was increased and when the pH of the buffer was adjusted to 2 or 12. These results suggest that the r3-peptide underwent a "magnet-like" oligomerization and that an increase in the charge-distribution inequality may be the driving force for the formation of fibrous structures.     

Circular dichroism investigation of the effect of plasmid DNA structure on retention in histidine chromatography

The effect of temperature-induced changes in secondary and tertiary structures of plasmid DNA (pDNA) and on the retention behaviour of open circular (oc) and supercoiled (sc) isoforms in histidine-agarose chromatography was investigated by Circular dichroism (CD) spectroscopy. Chromatographic experiments performed with three plasmids (2.7, 6.1 and 7.4 kbp) and with a decreasing ammonium sulphate gradient (2.3--2.0 M) showed that the retention of sc pDNA increased as temperature decreased from 24 to 5 °C. Such behaviour was attributed to the temperature-induced removal of negative superhelical turns in sc pDNA which is accompanied by a decrease in the number of dissociated base pairs responsible for interaction with the histidine ligands. CD spectroscopy showed that temperature has an important effect on plasmid secondary structure if adenine-rich inserts are present in the plasmid structure. Chromatographic experiments also suggested that base composition could also be responsible for the induction of specific interactions with histidine ligands.     

First Results of the RAMBAS Experiment on Investigation of the Radiation Mechanism of Chiral Influence

The first results of the RAdiation Mechanism of Biomolecular ASymmetry (RAMBAS) experiment on investigation of the radiation mechanism of the influence on chiral molecules, as a factor leading to origination of chiral asymmetry are presented. It was found that irradiation of simple achiral materials by a flux of electrons from radioactive source initiated the synthesis of amino acids, and it resulted in asymmetric degradation and chiral asymmetry in a racemic mixture of amino acids. The results obtained can be important for the solution of the origin-of-life and biological homochirality problems.     

Synthesis, DNA interaction and cytotoxicity studies of cis-{[1, 2-bis(aminomethyl)cyclohexane]dihalo}platinum(II) complexes

A series of platinum compounds with an analogue structure to cisplatin have been synthesized and their biological activity against HL-60 cancer cell line has been studied. The interaction with DNA was evaluated by circular dichroism (CD), electrophoresis and atomic force microscopy (AFM) techniques showing slight but significant structure-dependent differences among the evaluated complexes. The cytotoxicity assays afforded interesting relationships between the structure and the biological activity, thus, a better antiproliferative activity was observed for the complexes with higher hydrophobicity: the methoxylated complexes showed better activity than the hydroxylated ones (17versus20 and 19versus21). Especially compound 22 having a fatty acid subunit presented a promising cytotoxic activity. On the other hand, dichloro complexes 12 and 13 had better activities than the diiodo complexes, probably due to their better metabolic stability. Between both dichloro complexes the aromatic one showed much higher activity, which could be rationalized on the basis of the intercalating ability of the benzene ring. The flow cytometry assays indicated that most of the complexes induced the cell death by apoptosis except for aromatic compound 12 and the lipophilic compound 22 that induced preferably a mechanism of necrosis.     

Method of addition of acetonitrile influences the structure and stability of collagen

Collagen has been extensively used as a biomaterial in many biomedical applications. Recently, collagen based biomaterials were prepared using organic solvents. In this context, the method of addition of organic solvent described in the present study will be an important contribution in the preparation of collagen-based biomaterials. The effect of acetonitrile on collagen structure and stability was investigated using biophysical methods. Collagen undergoes solvent-induced denaturation with increasing concentration of acetonitrile. It was observed that addition of acetonitrile (50–90%) to a collagen solution in a single shot (method 1) led to precipitation. Contrary, collagen remained in the solution when acetonitrile content was increased to 90% in a collagen solution that had been formerly equilibrated with 20% acetonitrile (method 2). Interestingly, triple helical structure was retained when precipitated collagen, obtained from method 1, was re-dissolved in acetic acid solution. The re-dissolved collagen exhibits comparable melting temperature as that of native collagen. Re-dissolved collagen also showed fibril formation, but with decreased rate. The soluble collagen in 90% acetonitrile, prepared by method 2, is found to be unordered. The above results thus suggest that the method of addition of acetonitrile plays an important role in the folding and unfolding of collagen.