Tuning β-sheet peptide self-assembly and hydrogelation behavior by modification of sequence hydrophobicity and aromaticity

Peptide self-assembly leading to cross-β amyloid structures is a widely studied phenomenon because of its role in amyloid pathology and the exploitation of amyloid as a functional biomaterial. The self-assembly process is governed by hydrogen bonding, hydrophobic, aromatic π-π, and electrostatic Coulombic interactions. A role for aromatic π-π interactions in peptide self-assembly leading to amyloid has been proposed, but the relative contributions of π-π versus general hydrophobic interactions in these processes are poorly understood. The Ac-(XKXK)(2)-NH(2) peptide was used to study the contributions of aromatic and hydrophobic interactions to peptide self-assembly. Position X was globally replaced by valine (Val), isoleucine (Ile), phenylalanine (Phe), pentafluorophenylalanine (F(5)-Phe), and cyclohexylalanine (Cha). At low pH, these peptides remain monomeric because of repulsion of charged lysine (Lys) residues. Increasing the solvent ionic strength to shield repulsive charge-charge interactions between protonated Lys residues facilitated cross-β fibril formation. It was generally found that as peptide hydrophobicity increased, the required ionic strength to induce self-assembly decreased. At [NaCl] ranging from 0 to 1000 mM, the Val sequence failed to assemble. Assembly of the Phe sequence commenced at 700 mM NaCl and at 300 mM NaCl for the less hydrophobic Ile variant, even though it displayed a mixture of random coil and β-sheet secondary structures over all NaCl concentrations. β-Sheet formation for F(5)-Phe and Cha sequences was observed at only 20 and 60 mM NaCl, respectively. Whereas self-assembly propensity generally correlated to peptide hydrophobicity and not aromatic character the presence of aromatic amino acids imparted unique properties to fibrils derived from these peptides. Nonaromatic peptides formed fibrils of 3-15 nm in diameter, whereas aromatic peptides formed nanotape or nanoribbon architectures of 3-7 nm widths. In addition, all peptides formed fibrillar hydrogels at sufficient peptide concentrations, but nonaromatic peptides formed weak gels, whereas aromatic peptides formed rigid gels. These findings clarify the influence of aromatic amino acids on peptide self-assembly processes and illuminate design principles for the inclusion of aromatic amino acids in amyloid-derived biomaterials.     

Probing aromatic, hydrophobic, and steric effects on the self-assembly of an amyloid-β fragment peptide

Aromatic amino acids have been shown to promote self-assembly of amyloid peptides, although the basis for this amyloid-inducing behavior is not understood. We adopted the amyloid-β 16-22 peptide (Aβ(16-22), Ac-KLVFFAE-NH(2)) as a model to study the role of aromatic amino acids in peptide self-assembly. Aβ(16-22) contains two consecutive Phe residues (19 and 20) in which Phe 19 side chains form interstrand contacts in fibrils while Phe 20 side chains interact with the side chain of Va l18. The kinetic and thermodynamic effect of varying the hydrophobicity and aromaticity at positions 19 and 20 by mutation with Ala, Tyr, cyclohexylalanine (Cha), and pentafluorophenylalanine (F(5)-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F(5)-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F(5)-Phe variants self-assembled at dramatically enhanced rates relative to wild-type. Cha mutation was thermodynamically stabilizing at position 20 (ΔΔG = -0.2 kcal mol(-1) relative to wild-type) and destabilizing at position 19 (ΔΔG = +0.2 kcal mol(-1)). Conversely, F(5)-Phe mutations were strongly stabilizing at both positions (ΔΔG = -1.3 kcal mol(-1) at 19, ΔΔG = -0.9 kcal mol(-1) at 20). The double Cha and F(5)-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol(-1) for Cha 19,20 and -2.1 kcal mol(-1) for F(5)-Phe 19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.     

Evidence of π‐stacking interactions in the self‐assembly of hIAPP22‐29

The role aromatic amino acids play in the formation of amyloid is a subject of controversy. In an effort to clarify the contribution of aromaticity to the self‐assembly of human islet amyloid polypeptide (hIAPP)_22‐29, peptide analogs containing electron donating groups (EDGs) or electron withdrawing groups (EWGs) as substituents on the aromatic ring of Phe‐23 at the para position have been synthesized and characterized using turbidity measurements in conjunction with Raman and fluorescence spectroscopy. Results indicate the incorporation of EDGs on the aromatic ring of Phe‐23 virtually abolish the ability of hIAPP_22‐29 to form amyloid. Peptides containing EWGs were still capable of forming aggregates. These aggregates were found to be rich in β‐sheet secondary structure. Transmission electron microscopy images of the aggregates confirm the presence of amyloid fibrils. The observed difference in amyloidogenic propensity between peptides containing EDGs and those with EWGs appears not to be based on differences in peptide hydrophobicity. Fluorescence and Raman spectroscopic investigations reveal that the environment surrounding the aromatic ring becomes more hydrophobic and ordered upon aggregation. Furthermore, Raman measurements of peptide analogs containing EWGs, conclusively demonstrate a distinct downshift in the ? C?C? ring mode (ca. 1600 cm^?1) upon aggregation that has previously been shown to be indicative of π‐stacking. While previous work has demonstrated that π‐stacking is not an absolute requirement for fibrillization, our findings indicate that Phe‐23 also contributes to fibril formation through π‐stacking interactions and that it is not only the hydrophobic nature of this residue that is relevant in the self‐assembly of hIAPP_22‐29. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Clarifying the influence of core amino acid hydrophobicity, secondary structure propensity, and molecular volume on amyloid-β 16-22 self-assembly

The self-assembly of amyloid peptides is influenced by hydrophobicity, charge, secondary structure propensity, and sterics. Previous experiments have shown that increasing hydrophobicity at the aromatic positions of the amyloid-β 16-22 fragment (Aβ(16-22)) without introducing steric restraints greatly increases the rate of self-assembly and thermodynamically stabilizes the resulting fibrils [Senguen et al., Mol. BioSyst., 2011, DOI: 10.1039/c0mb00080a]. Conversely, when increasing side chain hydrophobicity coincides with an increase in side chain volume, the increase in the rate of self-assembly is offset by a thermodynamic destabilization of the resulting amyloid fibrils when direct cross-strand side chain interactions occur. These findings indicate that steric effects also influence the self-assembly of amyloidogenic peptides. Herein, the aromatic Phe residues at positions 19, 20, and 19,20 of Aβ(16-22) have been systematically replaced by Val, Leu, Ile, or hexafluoroleucine (Hfl) and amyloid formation has been characterized. The Val variants, despite the high β-sheet propensity of Val, were thermodynamically destabilized (ΔΔG = +0.1-0.4 kcal mol(-1)) relative to the wild-type with the double mutant failing to self-assemble at the concentrations studied. Conversely, the Leu and Ile variants formed fibrils at enhanced rates relative to wild-type and exhibited similar, or in some cases enhanced thermodynamic stabilities relative to the wild-type (ΔΔG = 0-0.6 kcal mol(-1)). The more hydrophobic Hfl variants were greatly stabilized (ΔΔG = -0.3-2.1 kcal mol(-1)) relative to the wild-type. These data indicate that hydrophobicity and steric effects both influence peptide self-assembly processes, including nucleation and fibrillization rates and the thermodynamic stability of the resulting fibrils.     

Porcine islet amyloid polypeptide fragments are refractory to amyloid formation

Of 10 variation sites between sequences of amyloid-resistant porcine islet amyloid polypeptide (pIAPP) and amyloid-prone human IAPP (hIAPP), seven locate within residues 17–29, the most amyloidogenic fragment within hIAPP. To investigate how these variations affect amyloidogenicity, 26 IAPP(17–29) or IAPP(20–29) variants were synthesized and their secondary structures, amyloidogenicity, oligomerization and cytotoxicity were studied. Our results indicated that pIAPP fragments are refractory to amyloid formation and significantly less cytotoxic compared with hIAPP fragments. A novel stable dimer was observed in pIAPP(20–29) solution, whereas hIAPP(20–29) exists mostly as monomers and trimers. Among all human to porcine substitutions, S20R caused the most prolonged lag time and significantly attenuated cytotoxicity. The different oligomerization and amyloidogenic properties of hIAPP and pIAPP fragments are discussed.

Structured summary

pIAPP and pIAPPbind: shown by molecular sieving (view interactions 1, 2)hIAPP and hIAPPbind: shown by molecular sieving (view interactions 1, 2)     

Structure-based design and study of non-amyloidogenic, double N-methylated IAPP amyloid core sequences as inhibitors of IAPP amyloid formation and cytotoxicity.

Pancreatic amyloid is formed by the aggregation of the 37-residue islet amyloid polypeptide (IAPP) in type II diabetes patients and is cytotoxic. Pancreatic amyloid deposits are found in more than 95 % of type II diabetes patients and their formation is strongly associated with disease progression. IAPP amyloid forms via a conformational transition of soluble IAPP into aggregated beta-sheets. We recently identified IAPP(22-27) (NFGAIL) as a minimum length sequence sufficient to self-associate into beta-sheet-containing amyloid fibrils. Here, we have used the NFGAIL model of the IAPP amyloid core as a structural template to design non-amyloidogenic derivatives of amyloidogenic sequences of IAPP that are able to interact with the native sequences and inhibit amyloid formation. The design of the derivatives was based on a simple, structure-based minimalistic and selective N-methylation approach. Accordingly, a minimum number of two amide bonds on the same side of the beta-strand of the amyloid core was N-methylated. This was expected to eliminate the two intermolecular backbone NH to CO hydrogen bonds which are critical for the extension of the beta-sheet dimers into multimers and amyloid. Other beta-strand "contact sides" remained intact allowing for the derivatives to interact with the native sequences. Double N-methylated derivatives of amyloidogenic and cytotoxic partial IAPP sequences generated included F(N-Me)GA(N-Me)IL, NF(N-Me)GA(N-Me)IL, SNNF(N-Me)GA(N-Me)IL, and SNNF(N-Me)GA(N-Me)ILSS and were found to be devoid of beta-sheet structure, amyloidogenicity and cytotoxicity according to Fourier transform-infrared spectroscopy (FT-IR), Congo red (CR) staining, electron microscopy (EM), and cell viability tests. The derivatives were able to interact with the native sequences and inhibit amyloid formation as shown by circular dichroism spectroscopy (CD), FT-IR and EM. Moreover, SNNF(N-Me)GA(N-Me)ILSS inhibited cytotoxicity of SNNFGAILSS and is thus the first reported inhibitor of IAPP amyloid formation and cytotoxicity. Our results demonstrate the validity of the design approach for IAPP and suggest that it may find application in understanding the structural features of amyloid formation and in the development of inhibitors of amyloid formation and cytotoxicity of other amyloidogenic polypeptides as well.     

Investigation of the nature of the methionine-pi interaction in beta-hairpin peptide model systems

There are frequent contacts between aromatic rings and sulfur atoms in proteins. However, it is unclear to what degree this putative interaction is stabilizing and what the nature of the interaction is. We have investigated the aryl-sulfur interaction by placing a methionine residue diagonal to an aromatic ring on the same face of a beta-hairpin, which places the methionine side chain in close proximity to the aryl side chain. The methionine (Met)-aryl interaction was compared with an equivalent hydrophobic and cation-pi interaction in the context of the beta-hairpin. The interaction between phenylalanine (Phe), tryptophan (Trp), or cyclohexylalanine (Cha) and Met stabilized the beta-hairpin by -0.3 to -0.5 kcal mole(-1), as determined by double-mutant cycles. The peptides were subjected to thermal denaturations that suggest a hydrophobic driving force for the interactions between Met and Trp or Cha. The observed interaction of Met or norleucine (Nle) with Trp or Cha are quite similar, implying a hydrophobic driving force for the Met-pi interaction. However, the thermodynamic data suggest that there may be some differences between the interaction of Met with Trp and Phe and that there may be a small thermodynamic component to the Met...Phe interaction.     

Evaluation of "click" binaphthyl chiral stationary phases by liquid chromatography

Two "click" binaphthyl chiral stationary phases were synthesized and evaluated by liquid chromatography. Their structures incorporate S-(-)-1,1'-binaphthyl moiety as the chiral selector and 1,2,3-triazole ring as the spacer. These chiral stationary phases (CSPs) allowed the efficient resolution for a wide range of racemic BINOL derivatives, particularly for nonpolar diether derivatives and 3-phenyl indolin-2-one analogs. The chromatographic data showed that the π-π interaction was crucial for enantiorecognition of these CSPs. Loss of enantioselectivity observed on CSP3, which are lacking the triazole ring linkage, indicated that the triazole ring linkage took part in the enantioseparation process, although it was remote from the chiral selector of the CSP. The substitution of the phenyl group at 6 and 6' positions can significantly improve the separation ability of the CSP. The chiral recognition mechanism was also investigated by tracking the elution orders and studying the thermodynamic parameters.     

Differential effects of serine side chain interactions in amyloid formation by islet amyloid polypeptide

Islet amyloid polypeptide (IAPP), a 37 residue polypeptide, is the main protein component of islet amyloid deposits produced in the pancreas in Type 2 diabetes. Human IAPP contains five serine residues at positions 19, 20, 28, 29, and 34. Models of the IAPP amyloid fibril indicate a structure composed of two closely aligned columns of IAPP monomers with each monomer contributing to two intermolecular β‐strands. Ser 19 and Ser 20 are in the partially ordered β‐turn region, which links the two strands, whereas Ser 28, Ser 29, and Ser 34 are in the core region of the amyloid fibril. Ser 29 is involved in contacts between the two columns of monomers and is the part of the steric zipper interface. We undertook a study of individual serine substitutions with the hydrophobic isostere 2‐aminobutyric acid (2‐Abu) to examine the site‐specific role of serine side chains in IAPP amyloid formation. All five variants formed amyloid. The Ser 19 to 2‐Abu mutant accelerates amyloid formation by a factor of 3 to 4, while the Ser 29 to 2‐Abu mutation modestly slows the rate of amyloid formation. 2‐Abu replacements at the other sites had even smaller effects. The data demonstrate that the cross‐column interactions made by residue 29 are not essential for amyloid formation and also show that cross‐strand networks of hydrogen‐bonded Ser side chains, so called Ser‐ladders, are not required for IAPP amyloid formation. The effect of the Ser 19 to 2‐Abu mutant suggests that residues in this region are important for amyloid formation by IAPP.     

A mechanistic approach for islet amyloid polypeptide aggregation to develop anti-amyloidogenic agents for type-2 diabetes

Type-2 diabetes mellitus (DM-2) is a conformational disease involving intrinsically disordered islet amyloid polypeptide (IAPP), in which a structural transition from physiological polypeptide to pathological deposits takes place. Different factors acquired or inherited, contribute to endoplasmic reticulum stress and/or impair mitochondrial function which leads to conformational changes in IAPP intermediates and ultimately produces oligomers of an anti-parallel crossed β-pleated sheets that eventually accumulate as space-occupying lesions within the islets. Clusters of IAPP monomers form a pore which is linked to channel-like behavior in planar bilayers, indicating that these oligomeric IAPP pores could become incorporated into membranes and alter its barrier properties. Identification of nucleating residues and the residues responsible for this oligomeric tendency could improve understanding of structure-function relationships as well as the molecular mechanism of folding and aggregation of IAPP contributing to the onset of DM-2. A combination of biological, chemical or physical approaches is required to be extensively pursued for the development of a successful anti-amyloidogenic agent to prevent this malady. Exploring the hypothesis of π-stacking may be a better option to control IAPP aggregation if researchers go through the mechanism of π-π interaction, which provides entropy driven energy and direction for self-assembly to control amyloidogenic aggregation.     

Functional Amyloids: Where Supramolecular Amyloid Assembly Controls Biological Activity or Generates New Functionality

Functional amyloids are a rapidly expanding class of fibrillar protein structures, with a core cross-β scaffold, where novel and advantageous biological function is generated by the assembly of the amyloid. The growing number of amyloid structures determined at high resolution reveal how this supramolecular template both accommodates a wide variety of amino acid sequences and also imposes selectivity on the assembly process. The amyloid fibril can no longer be considered a generic aggregate, even when associated with disease and loss of function. In functional amyloids the polymeric β-sheet rich structure provides multiple different examples of unique control mechanisms and structures that are finely tuned to deliver assembly or disassembly in response to physiological or environmental cues. Here we review the range of mechanisms at play in natural, functional amyloids, where tight control of amyloidogenicity is achieved by environmental triggers of conformational change, proteolytic generation of amyloidogenic fragments, or heteromeric seeding and amyloid fibril stability. In the amyloid fibril form, activity can be regulated by pH, ligand binding and higher order protofilament or fibril architectures that impact the arrangement of associated domains and amyloid stability. The growing understanding of the molecular basis for the control of structure and functionality delivered by natural amyloids in nearly all life forms should inform the development of therapies for amyloid-associated diseases and guide the design of innovative biomaterials.     

Mutations that replace aromatic side chains promote aggregation of the Alzheimer's Aβ peptide

The aggregation of polypeptides into amyloid fibrils is associated with a number of human diseases. Because these fibrils--or intermediates on the aggregation pathway--play important roles in the etiology of disease, considerable effort has been expended to understand which features of the amino acid sequence promote aggregation. One feature suspected to direct aggregation is the π-stacking of aromatic residues. Such π-stacking interactions have also been proposed as the targets for various aromatic compounds that are known to inhibit aggregation. In the case of Alzheimer's disease, the aromatic side chains Phe19 and Phe20 in the wild-type amyloid beta (Aβ) peptide have been implicated. To explicitly test whether the aromaticity of these side chains plays a role in aggregation, we replaced these two phenylalanine side chains with leucines or isoleucines. These residues have similar sizes and hydrophobicities as Phe but are not capable of π-stacking. Thioflavin-T fluorescence and electron microscopy demonstrate that replacement of residues 19 and 20 by Leu or Ile did not prevent aggregation, but rather enhanced amyloid formation. Further experiments showed that aromatic inhibitors of aggregation are as effective against Ile- and Leu-substituted versions of Aβ42 as they are against wild-type Aβ. These results suggest that aromatic π-stacking interactions are not critical for Aβ aggregation or for the inhibition of Aβ aggregation.     

Destabilization of human IAPP amyloid fibrils by proline mutations outside of the putative amyloidogenic domain: is there a critical amyloidogenic domain in human IAPP?

Islet amyloid polypeptide (IAPP; amylin) is responsible for amyloid formation in type-2 diabetes. Not all organisms form islet amyloid, and amyloid formation correlates strongly with variations in primary sequence. Studies of human and rodent IAPP have pointed to the amino acid residues 20-29 region as the important amyloid-modulating sequence. The rat 20-29 sequence contains three proline residues and does not form amyloid, while the human sequence contains no proline and readily forms amyloid. This has led to the view that the 20-29 region constitutes a critical amyloidogenic domain that dictates the properties of the entire sequence. The different behavior of human and rat IAPP could be due to differences in the 20-29 region or due simply to the fact that multiple proline residues destabilize amyloid fibrils. We tested how critical the 20-29 region is by studying a variant identical with the human peptide in this segment but with three proline residues outside this region. We designed a variant of the amyloidogenic 8-37 region of human IAPP (hIAPP(8-37) 3xP) with proline substitutions at positions 17, 19 and 30. Compared to the wild-type, the 3xP variant was much easier to synthesize and had dramatically greater solubility. Fourier transform infra red spectroscopy, transmission electron microscopy, Congo red staining and thioflavin-T binding indicate that this variant has a reduced tendency to form beta-sheet structure and forms deposits with much less structural order than the wild-type. Far-UV CD studies show that the small amount of beta-sheet structure developed by hIAPP(8-37) 3xP after long periods of incubation dissociates readily into random-coil structure upon dilution into Tris buffer. The observation that proline substitutions outside the putative core domain effectively abolish amyloid formation indicates that models of IAPP aggregation must consider contributions from other regions.     

The Sulfated Triphenyl Methane Derivative Acid Fuchsin Is a Potent Inhibitor of Amyloid Formation by Human Islet Amyloid Polypeptide and Protects against the Toxic Effects of Amyloid Formation

Islet amyloid polypeptide (IAPP), also known as amylin, is responsible for amyloid formation in type 2 diabetes. The formation of islet amyloid is believed to contribute to the pathology of the disease by killing β-cells, and it may also contribute to islet transplant failure. The design of inhibitors of amyloid formation is an active area of research, but comparatively little attention has been paid to inhibitors of IAPP in contrast to the large body of work on β-amyloid, and most small-molecule inhibitors of IAPP amyloid are generally effective only when used at a significant molar excess. Here we show that the simple sulfonated triphenyl methane derivative acid fuchsin, 3-(1-(4-amino-3-methyl-5-sulfonatophenyl)-1-(4-amino-3-sulfonatophenyl) methylene) cyclohexa-1,4-dienesulfonic acid, is a potent inhibitor of in vitro amyloid formation by IAPP at substoichiometric levels and protects cultured rat INS-1 cells against the toxic effects of human IAPP. Fluorescence-detected thioflavin-T binding assays, light-scattering, circular dichroism, two-dimensional IR, and transmission electron microscopy measurements confirm that the compound prevents amyloid fibril formation. Ionic-strength-dependent studies show that the effects are mediated in part by electrostatic interactions. Experiments in which the compound is added at different time points during the lag phase after amyloid formation has commenced reveal that it arrests amyloid formation by trapping intermediate species. The compound is less effective against the β-amyloid peptide, indicating specificity in its ability to inhibit amyloid formation by IAPP. The work reported here provides a new structural class of IAPP amyloid inhibitors and demonstrates the power of two-dimensional infrared spectroscopy for characterizing amyloid inhibitor interactions.     

On the origin of the stronger binding of PIB over thioflavin T to protofibrils of the Alzheimer amyloid-β peptide: a molecular dynamics study

Pittsburgh compound B (PIB) is a neutral derivative of the fluorescent dye Thioflavin T (ThT), which displays enhanced hydrophobicity and binding affinity to amyloid fibrils. We present molecular dynamics simulations of binding of PIB and ThT to a common cross-β-subunit of the Alzheimer Amyloid-β peptide (Aβ). Our simulations of binding to Aβ(9-40) protofibrils show that PIB, like ThT, selectively binds to the hydrophobic or aromatic surface grooves on the β-sheet surface along the fibril axis. The lack of two methyl groups and charge in PIB not only improves its hydrophobicity but also leads to a deeper insertion of PIB compared to ThT into the surface grooves. This significantly increases the steric, aromatic, and hydrophobic interactions, and hence leads to stronger binding. Simulations on protofibrils consisting of the more-toxic Aβ(17-42) revealed an additional binding mode in which PIB and ThT insert into the channel that forms in the loop region of the protofibril, sandwiched between two sheet layers. Our simulations indicate that the rotation between the two ring parts of the dyes is significantly more restricted when the dyes are bound to the surface of the cross-β-subunits or to the channel inside the Aβ(17-42) cross-β-subunit, compared with free solution. The specific conformations of the dyes are influenced by small chemical modifications (ThT versus PIB) and by the environment in which the dye is placed.     

The FGFR D3 domain determines receptor selectivity for fibroblast growth factor 21

Islet amyloid polypeptide (IAPP; also known as amylin) is responsible for islet amyloid formation in type 2 diabetes, and IAPP-induced toxicity is believed to contribute to the loss of β-cell mass associated with the late stages of type 2 diabetes. Islet amyloid formation may also play a role in graft failure after transplantation. IAPP is produced as a prohormone, pro-islet amyloid polypeptide (proIAPP), and processed in the secretory granules of the pancreatic β-cells. Partially processed forms of proIAPP are found in amyloid deposits; most notable is a 48-residue intermediate, proIAPP_1-48, which includes the N-terminal pro-extension, but which has been properly processed at the C-terminus. Incomplete processing may play a role in islet amyloid formation by promoting interactions with sulfated proteoglycans of the extracellular matrix, which, in turn, promote amyloid formation. We show that acid fuchsin (3-(1-(4-amino-3-methyl-5-sulphonatophenyl)-1-(4-amino-3-sulphonatophenyl)methylene)cyclohexa-1,4-dienesulphonic acid), a simple sulfonated triphenyl methyl derivative, is a potent inhibitor of amyloid formation by the proIAPP_1-48 intermediate. The more complicated triphenyl methane derivative fast green FCF {ethyl-[4-[[4-[ethyl-[(3-sulfophenyl)methyl]amino]phenyl]-(4-hydroxy-2-sulfophenyl)methylidene]-1-cyclohexa-2,5-dienylidene]-[(3-sulfophenyl)methyl]azanium} also inhibits amyloid formation by IAPP and the proIAPP processing intermediate. Both compounds inhibit amyloid formation by mixtures of the proIAPP intermediate and the model glycosaminoglycan heparan sulfate. Acid fuchsin also inhibits glycosaminoglycan-mediated amyloid formation by mature IAPP. The ability to inhibit amyloid formation is not simply due to the compounds being sulfonated, since the sulfonated inhibitor of amyloid-β, tramiprosate, is not an inhibitor of amyloid formation by proIAPP_1-48.     

Influence of the germline sequence on the thermodynamic stability and fibrillogenicity of human lambda 6 light chains

Light chain-associated amyloidosis is a fatal disease characterized by the aggregation and pathologic deposition of monoclonal light chain-related fragments as amyloid fibrils in organs or tissues throughout the body. Notably, it has been observed that proteins encoded by the lambda variable light chain (V(L)) gene segment 6a are invariably associated with amyloid deposition; however, the contribution of the gene to this phenomenon has not been established. In this regard, we have determined the thermodynamic stability and kinetics of in vitro fibrillogenesis of a recombinant (r) V(L) protein, designated 6aJL2, which contains the predicted sequences encoded by the 6a and JL2 germline genes. Additionally, we studied a 6a mutant (6aJL2-Arg25Gly), that is present in approximately 25% of all amyloid-associated lambda6 light chains. Remarkably, the wild-type 6aJL2 protein was more stable than were all known amyloidogenic kappa and lambda light chains for which stability parameters are available; more importantly, it was even more so (and less fibrillogenic) than the only clinically proven nonamyloidogenic lambda6 protein, Jto. Conversely, the mutated 6aJL2-R25G molecule was considerably less stable and more fibrillogenic than was the native 6aJL2. Our data indicate that the propensity of lambda6 light chains to form amyloid can not be attributed to thermodynamic instability of the germline-encoded Vlambda6 domain, but rather, is dependent on sequence alterations that render such proteins amyloidogenic.     

Design of peptide-based inhibitors of human islet amyloid polypeptide fibrillogenesis

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. We have generated a series of overlapping hexapeptides to target an amyloidogenic region of IAPP (residues 20-29) and examined their effects on fibril assembly. Peptide fragments corresponding to SNNFGA (residues 20-25) and GAILSST (residues 24-29) were strong inhibitors of the beta-sheet transition and amyloid aggregation. Circular dichroism indicated that even at 1:1 molar ratios, these peptides maintained full-length IAPP (1-37) in a largely random coil conformation. Negative stain electron microscopy revealed that co-incubation of these peptides with IAPP resulted in the formation of only semi-fibrous aggregates and loss of the typical high density and morphology of IAPP fibrils. This inhibitory activity, particularly for the SNNFGA sequence, also correlated with a reduction in IAPP-induced cytotoxicity as determined by cell culture studies. In contrast, the peptide NFGAIL (residues 22-27) enhanced IAPP fibril formation. Conversion to the amyloidogenic beta-sheet was immediate and the accompanying fibrils were more dense and complex than IAPP alone. The remaining peptide fragments either had no detectable effects or were only weakly inhibitory. Specificity of peptide activity was illustrated by the fragments, SSNNFG and AILSST. These differed from the most active inhibitors by only a single amino acid residue but delayed the random-to-beta conformational change only when used at higher molar ratios. This study has identified internal IAPP peptide fragments which can regulate fibrillogenesis and may be of therapeutic use for the treatment of type-2 diabetes.     

The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12–18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.     

Morin hydrate inhibits amyloid formation by islet amyloid polypeptide and disaggregates amyloid fibers

The polypeptide hormone Islet Amyloid Polypeptide (IAPP, amylin) is responsible for islet amyloid formation in type-2 diabetes and in islet cell transplants, where it may contribute to graft failure. Human IAPP is extremely amyloidogenic and fewer inhibitors of IAPP amyloid formation have been reported than for the Alzheimer's Aβ peptide or for α-synuclein. The ability of a set of hydroxyflavones to inhibit IAPP amyloid formation was tested. Fluorescence detected thioflavin-T-binding assays are the most popular methods for measuring the kinetics of amyloid formation and for screening potential inhibitors; however, we show that they can lead to false positives with hydroxyflavones. Several of the compounds inhibit thioflavin-T fluorescence, but not amyloid formation; a result which highlights the hazards of relying solely on thioflavin-T assays to screen potential inhibitors. Transmission electron microscopy (TEM) and right-angle light scattering show that Morin hydrate (2',3,4',5,7-Pentahydroxyflavone) inhibits amyloid formation by human IAPP and disaggregates preformed IAPP amyloid fibers. In contrast, Myricetin, Kaempferol, and Quercetin, which differ only in hydroxyl groups on the B-ring, are not effective inhibitors. Morin hydrate represents a new type of IAPP amyloid inhibitor and the results with the other compounds highlight the importance of the substitution pattern on the B-ring.