Fluorenylmethoxycarbonyl-protected phenylalanine (Fmoc-Phe) derivatives are a privileged class of molecule that spontaneously self-assemble into hydrogel fibril networks. Fmoc-Phe-derived hydrogels are typically formed by dilution of the hydrogelator from an organic cosolvent into water, by dissolution of the hydrogelator under basic aqueous conditions followed by adjustment of the pH with acid, or by other external triggering forces, including sonication and heating. These conditions complicate biological applications of these hydrogels. Herein, we report C-terminal cation-modified Fmoc-Phe derivatives that are positively charged across a broad range of pH values and that can self-assemble and form hydrogel networks spontaneously without the need to adjust pH or to use an organic cosolvent. In addition, these cationic Fmoc-Phe derivatives are found to self-assemble into novel sheet-based nanotube structures at higher concentrations. These nanotube structures are unique to C-terminal cationic Fmoc-Phe derivatives; the parent Fmoc-Phe carboxylic acids form only fibril or worm-like micelle structures. Nanotube formation by the cationic Fmoc-Phe molecules is dependent on positive charge at the C-terminus, since at basic pH where the positive charge is reduced only fibrils/worm-like micelles are formed and nanotube formation is suppressed. These studies provide an important example of Fmoc-Phe derivatives that can elicit hydrogelation without organic cosolvent or pH modification and also provide insight into how subtle modification of structure can perturb the self-assembly pathways of Fmoc-Phe derivatives.

The promise of oligonucleotide therapeutic agents to perturb expression of disease-related genes remains unrealized, in part due to challenges with functional cellular delivery of these agents. Herein, we describe disulfide-constrained cyclic amphipathic peptides that complex with short-interfering RNA (siRNA) and affect functional cytosolic delivery and knockdown of target gene products in cell culture and in vivo to mouse lung. Reduction of the constraining disulfide bond and subsequent proteolytic clearance of the peptide are key design features that allow unmasking of the siRNA cargo and presentation to the RNA interference machinery.Keywords: cell-penetrating peptides; cyclic peptides; siRNA delivery

Supramolecular hydrogels derived from the self-assembly of organic molecules have been exploited for applications ranging from drug delivery to tissue engineering. The relationship between the structure of the assembly motif and the emergent properties of the resulting materials is often poorly understood, impeding rational approaches for the creation of next-generation materials. Aromatic π–π interactions play a significant role in the self-assembly of many supramolecular hydrogelators, but the exact nature of these interactions lacks definition. Conventional models that describe π–π interactions rely on quadrupolar electrostatic interactions between neighboring aryl groups in the π-system. However, recent experimental and computational studies reveal the potential importance of local dipolar interactions between elements of neighboring aromatic rings in stabilizing π–π interactions. Herein, we examine the nature of π–π interactions in the self- and coassembly of Fmoc-Phe-derived hydrogelators by systematically varying the electron-donating or electron-withdrawing nature of the side chain benzyl substituents and correlating these effects to the emergent assembly and gelation properties of the systems. These studies indicate a significant role for stabilizing dipolar interactions between neighboring benzyl groups in the assembled materials. Additional evidence for specific dipolar interactions is provided by high-resolution crystal structures obtained from dynamic transition of gel fibrils to crystals for several of the self-assembled/coassembled Fmoc-Phe derivatives. In addition to electronic effects, steric properties also have a significant effect on the interaction between neighboring benzyl groups in these assembled systems. These findings provide significant insight into the structure–function relationship for Fmoc-Phe-derived hydrogelators and give cues for the design of next-generation materials with desired emergent properties.

Fmoc-3F-Phe-Arg-NH2 and Fmoc-3F-Phe-Asp-OH dipeptides undergo coassembly to form two-component nanofibril hydrogels. These hydrogels support the viability and growth of NIH 3T3 fibroblast cells. The supramolecular display of Arg and Asp at the nanofibril surface effectively mimics the integrin-binding RGD peptide of fibronectin, without covalent connection between the Arg and Asp functionality.

Hydrogel fibril and crystal formation are related self-assembly processes that provide materials with distinct emergent properties. The relationship between fibril and crystal growth is poorly understood, and efforts to engineer controlled hydrogelation vs crystallization via small molecule self-assembly currently depend on empirical approaches. Herein, we report the dynamic transition of self-assembled hydrogel fibrils of a phenylalanine (Phe) derivative, Fmoc-p-nitrophenylalanine (Fmoc-4-NO2-Phe), to crystalline microtubes. As has been shown with other Fmoc-Phe derivatives, Fmoc-4-NO2-Phe spontaneously self-assembles into amyloid-like fibrils that form an entangled hydrogel network when suspended in water. However, Fmoc-4-NO2-Phe fibrils uniquely transform over time into crystalline microtubes. Hydrogel fibrils appear to be a kinetic state with microtube crystals more thermodynamically favored. This dynamic transition from fibril to crystal has enabled a high-resolution structural analysis of the packing orientation of these self-assembled materials. Taking cues from this structural analysis, we demonstrate a rational strategy for stabilization of the kinetic Fmoc-4-NO2-Phe hydrogel fibrils. These results represent significant advances in our understanding of the dynamic nature of self-assembly processes and in our ability to rationally engineer these processes to provide materials with desired emergent properties.

Peptide hydrogels are promising biomaterials for applications ranging from drug delivery to tissue engineering. Peptide hydrogels that change their physical properties in response to an exogenous stimulus are advantageous as biomaterials that can be temporally controlled. Herein, we report the use of an azobenzene turn mimetic, [3-(3-aminomethylphenylazo)phenyl]acetic acid (AMPP), to engineer a light-responsive β-hairpin into the center of a hydrogel-forming peptide. In the trans state, AMPP exists in a β-arc conformation, and the peptide forms a rigid self-supporting gel. The peptide hydrogel rigidity is reduced upon trans–cis azobenzene isomerization, which promotes formation of putative β-hairpin assemblies. This process is reversible in that hydrogel viscoelasticity is restored upon reverse cis–trans photoisomerization. TEM imaging and spectroscopic data reveal that the loss in rigidity is a result of disruption of the well-ordered macromolecular structure and not due to disassembly of the constituent self-assembled β-sheet fibrils. These findings provide insight into the effect of β-arc and β-hairpin turns on the emergent properties of self-assembled peptide hydrogels and provide a basis for temporal control of hydrogel rigidity using near-UV light.

Amphipathic peptides have an increased propensity to self-assemble into amyloid-like β-sheet fibrils when their primary sequence pattern consists of alternating hydrophobic and hydrophilic amino acids. These fibrils adopt a bilayer architecture composed of two β-sheets laminated to bury the hydrophobic side chains of the β-sheet in the bilayer interior, leaving the hydrophilic side chains exposed at the bilayer surface. In this study, the effects of altering the sequence pattern of amphipathic peptides from strictly alternating hydrophobic/hydrophilic repeats to more complex patterning of hydrophobic and hydrophilic residues on self-assembly of the resulting sequences is reported. Self-assembly of the Ac-(FKFE)2-NH2 peptide was compared to that of four related sequences with varied amino acid sequence patterning: Ac-(FK)2(FE)2-NH2, Ac-KEFFFFKE-NH2, Ac-(KFFE)2-NH2, and Ac-FFKEKEFF-NH2. The Ac-(FKFE)2-NH2 and Ac-(FK)2(FE)2-NH2 peptides effectively self-assembled at high (1.0 mM) and low (0.2 mM) concentrations (pH 3–4) into β-sheet nanoribbons that were 8 and 4 nm wide, respectively. The Ac-KEFFFFKE-NH2 peptide failed to self-assemble at low concentration (pH 3–4), but self-assembled into distinct nanotapes that were ∼20 nm in width at high concentration. Ac-(KFFE)2-NH2 and Ac-FFKEKEFF-NH2 failed to self-assemble into fibril/tape-like materials at either high or low concentration at pH 3–4, although Ac-FFKEKEFF-NH2 formed micelle-like aggregates at higher concentrations. At neutral pH, similar self-assembly behavior was observed for each peptide as was observed at acidic pH. An exception was the Ac-FFKEKEFF-NH2 peptide, which formed ∼20 nm nanotapes at neutral pH. These results indicate that amino acid sequence patterns exert a profound influence on self-assembly propensity and morphology of the resulting materials even when the overall hydrophobicity or charge of the related peptides are identical. Sequence pattern variation can thus be exploited as a variable in the creation of novel materials composed of self-assembled peptides.

Amphipathic peptides composed of alternating hydrophobic and hydrophilic amino acids self-assemble into amyloid-inspired, β-sheet nanoribbon fibrils. Herein, we report a new fibril type that is formed from equimolar mixtures of enantiomeric amphipathic peptides (l- and d-(FKFE)2). Spectroscopic analysis indicates that these peptides do not self-sort and assemble into enantiomeric fibrils composed of all-l and all-d peptides, but rather coassemble into fibrils that contain alternating l- and d-peptides in a “rippled β-sheet” orientation. Isothermal titration calorimetry indicates an enthalpic advantage for rippled β-sheet coassembly compared to self-sorted β-sheet assembly of enantiomeric peptides.

Noncovalent hydrogels derived from the self-assembly of peptides and proteins have demonstrated advantages over covalent hydrogels for three-dimensional cell scaffolding applications. There is growing interest in exploiting minimal, self-assembling dipeptides and amino acids as hydrogel networks that support cell culture applications, but significant questions persist concerning the mechanism of self-assembly and the relationship between the molecular structure of the assembled materials and their emergent viscoelastic and biochemical properties. This review will critically assess current progress in the use of minimal self-assembling peptides and functionalized amino acids to create hydrogels, with a focus on the challenges of understanding the structure and function of these materials and on the outlook for the use of these modular and dynamic materials as robust networks for tissue engineering.

Amyloid-β (Aβ) self-assembly into cross-β amyloid fibrils is implicated in a causative role in Alzheimer’s disease pathology. Uncertainties persist regarding the mechanisms of amyloid self-assembly and the role of metastable prefibrillar aggregates. Aβ fibrils feature a sheet-turn-sheet motif in the constituent β-strands; as such, turn nucleation has been proposed as a rate-limiting step in the self-assembly pathway. Herein, we report the use of an azobenzene β-hairpin mimetic to study the role turn nucleation plays on Aβ self-assembly. [3-(3-Aminomethyl)phenylazo]phenylacetic acid (AMPP) was incorporated into the putative turn region of Aβ42 to elicit temporal control over Aβ42 turn nucleation; it was hypothesized that self-assembly would be favored in the cis-AMPP conformation if β-hairpin formation occurs during Aβ self-assembly and that the trans-AMPP conformer would display attenuated fibrillization propensity. It was unexpectedly observed that the trans-AMPP Aβ42 conformer forms fibrillar constructs that are similar in almost all characteristics, including cytotoxicity, to wild-type Aβ42. Conversely, the cis-AMPP Aβ42 congeners formed nonfibrillar, amorphous aggregates that exhibited no cytotoxicity. Additionally, cis-trans photoisomerization resulted in rapid formation of native-like amyloid fibrils and trans–cis conversion in the fibril state reduced the population of native-like fibrils. Thus, temporal photocontrol over Aβ turn conformation provides significant insight into Aβ self-assembly. Specifically, Aβ mutants that adopt stable β-turns form aggregate structures that are unable to enter folding pathways leading to cross-β fibrils and cytotoxic prefibrillar intermediates.Keywords: Alzheimer’s disease; amyloid fibrils; Amyloid-β; azobenzene photoswitch; turn nucleation; β-turn

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.

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-NH2) 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 Phe19 side chains form interstrand contacts in fibrils while Phe20 side chains interact with the side chain of Val18. 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 (F5-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F5-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F5-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, F5-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 F5-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol−1 for Cha19,20 and −2.1 kcal mol−1 for F5-Phe19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.

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-NH2 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 (F5–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 F5–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.

Noncovalent self-assembled materials inspired by amyloid architectures are useful for biomedical applications ranging from regenerative medicine to drug delivery. The selective coassembly of complementary monomeric units to provide ordered multicomponent fibrils is a possible strategy for enhancing the sophistication of these noncovalent materials. Herein we report that complementary π–π interactions can be exploited to promote the coassembly of phenylalanine (Phe) derivatives that possess complementary aromatic side-chain functionality. Specifically, equimolar mixtures of Fmoc-Phe and Fmoc-F5-Phe, which possess side-chain groups with complementary quadrupole electronics, readily coassemble to form two-component fibrils and hydrogels under conditions where Fmoc-Phe alone fails to self-assemble. In addition, it was found that equimolar mixtures of Fmoc-Phe with monohalogenated (F, Cl, and Br) Fmoc-Phe derivatives also coassembled into two-component fibrils. These results collectively indicate that face-to-face quadrupole stacking between benzyl side-chain groups does not account for the molecular recognition between Phe and halogenated Phe derivatives that promote cofibrillization but that coassembly is mediated by more subtle π–π effects arising from the halogenation of the benzyl side chain. The use of complementary π–π interactions to promote the coassembly of two distinct monomeric units into ordered two-component fibrils dramatically expands the repertoire of noncovalent interactions that can be used in the development of sophisticated noncovalent materials.

The development of hydrogels resulting from the self-assembly of low molecular weight (LMW) hydrogelators is a rapidly expanding area of study. Fluorenylmethoxycarbonyl (Fmoc) protected aromatic amino acids derived from phenylalanine (Phe) have been shown to be highly effective LMW hydrogelators. It has been found that side chain functionalization of Fmoc-Phe exerts a significant effect on the self-assembly and hydrogelation behavior of these molecules; fluorinated derivatives, including pentafluorophenylalanine (F5-Phe) and 3-F-phenylalanine (3-F-Phe), spontaneously self-assemble into fibrils that form a hydrogel network upon dissolution into water. In this study, Fmoc-F5-Phe-OH and Fmoc-3-F-Phe-OH were used to characterize the role of the C-terminal carboxylic acid on the self-assembly and hydrogelation of these derivatives. The C-terminal carboxylic acid moieties of Fmoc-F5-Phe-OH and Fmoc-3-F-Phe-OH were converted to C-terminal amide and methyl ester groups in order to perturb the hydrophobicity and hydrogen bond capacity of the C-terminus. Self-assembly and hydrogelation of these derivatives was investigated in comparison to the parent carboxylic acid compounds at neutral and acidic pH. It was found that hydrogelation of the C-terminal acids was highly sensitive to solvent pH, which influences the charge state of the terminal group. Rigid hydrogels form at pH 3.5, but at pH 7 hydrogel rigidity is dramatically weakened. C-terminal esters self-assembled into fibrils only slowly and failed to form hydrogels due to the higher hydrophobicity of these derivatives. C-terminal amide derivatives assembled much more rapidly than the parent carboxylic acids at both acidic and neutral pH, but the resultant hydrogels were unstable to shear stress as a function of the lower water solubility of the amide functionality. Co-assembly of acid and amide functionalized monomers was also explored in order to characterize the properties of hybrid hydrogels; these gels were rigid in unbuffered water but significantly weaker in phosphate buffered saline. These results highlight the complex nature of monomer/solvent interactions and their ultimate influence on self-assembly and hydrogelation, and provide insight that will facilitate the development of optimal amino acid LMW hydrogelators for gelation of complex buffered media.

Stimulus-responsive peptide self-assembly provides a powerful method for controlling self-assembly as a function of environment. The development of a reductive trigger for peptide self-assembly and subsequent hydrogelation is described herein. A self-assembling peptide sequence, Ac-C(FKFE)2CG-NH2, was cyclized via disulfide bonding of the flanking cysteine residues. The macrocyclic form of this peptide enforces a conformational restraint that prevents adoption of the β-sheet conformation that is required for self-assembly. Upon reduction of this disulfide bond, the peptide relaxes into the preferred β-sheet conformation, and immediate self-assembly into fibrillar superstructures occurs. At sufficient peptide concentration, self-assembly is accompanied by the formation of rigid, viscoelastic hydrogels.

Fmoc-protected aromatic amino acids, including Fmoc-phenylalanine (Fmoc-Phe), Fmoc-tyrosine (Fmoc-Tyr), and Fmoc-pentafluorophenylalanine (Fmoc-F5-Phe), have been shown to undergo efficient self-assembly and to promote hydrogelation in aqueous solvents. In order to probe the electronic and steric role of the benzyl side-chain in hydrophobic and π–π interactions during self-assembly, the hydrogelation behavior of monohalogenated (F, Cl, Br) Fmoc-Phe side-chain derivatives was assessed. Incorporation of single halogen substituents on the aromatic side-chain of Fmoc-Phe dramatically enhances the efficient self-assembly of these amino acid derivatives (relative to Fmoc-Phe) into amyloid-like fibrils that promote hydrogelation in aqueous solvents. The position of halogen substitution (ortho, meta, para) and the halogen itself (F, Cl, Br) exert a strong influence on the self-assembly rate and on the bulk rheological properties of the resultant hydrogel. These results demonstrate that minimal atomic substitutions can be used to tune self-assembly and gelation of small molecule hydrogelators.

Phenylalanine (Phe)-derived molecules have been exploited as low molecular weight hydrogelators. Perturbing the hydrophobic and π–π interactions that promote self-assembly and hydrogelation of these derivatives will facilitate improved understanding of hydrogelation phenomena and the design of small molecule hydrogelators with novel properties. The efficient self-assembly and hydrogelation of Fmoc-protected pentafluorophenylalanine (Fmoc-F5-Phe) are reported herein. Suspensions of Fmoc-F5-Phe in water undergo rapid self-assembly to entangled fibrillar structures within minutes, giving rise to rigid supramolecular gels. Self-assembly occurs at concentrations as low as 2 mM (0.1 wt%). Variation of the fluorinated aromatic side chain or N-terminal functionalization perturbs hydrogelation, implicating fluorous and π–π interactions as the primary determinants for molecular recognition and self-assembly. The hydrophobic and electronic properties of F5-Phe provide remarkable potential for functional self-assembly in a minimal amino acid scaffold.

Peptide self-assembly processes are central to the etiology of amyloid diseases. Much effort has been devoted to characterizing amyloid structure and the mechanisms of peptideself-assembly leading to amyloid. It has been proposed that aromatic side-chain interactions play a central role in early self-assembly recognition events, but this contention remains somewhat controversial. Recent studies have indicated that in some amyloid peptides, aromatic residues can be exchanged for other hydrophobic residues and these nonaromatic variant peptides still retain competency to form amyloid, although with attenuated kinetics. In an effort to understand the relative contributions of aromatic versus generic hydrophobic interactions, studies to quantify the self-assembly properties of amyloid peptides as a function of increasing hydrophobicity and altered aromatic character have been undertaken. In the present study, the amphipathic (FKFE)2peptide has been chosen as a model system. The aromatic phenylalanine residues have been globally replaced with nonaromatic natural residues with lower hydrophobicity (alanine, valine, and leucine) and a nonnatural residue with greater hydrophobicity (cyclohexylalanine). The self-assembly properties of these peptides have been characterized by secondary structure analysis and microscopic analysis of the resulting aggregate structures. These studies confirm that aromatic interactions are not strictly required for amyloid formation and that the nonaromatic, but highly hydrophobic, cyclohexylalanine appears to have unique self-assembly characteristics and enhanced hydrogelation properties. The aromatic phenylalanine-containing peptide displays intriguing solvent- and concentration-dependent polymorphism, suggesting that aromatic interactions, while not essential for self-assembly, may give rise to unique structural features.

Semen-derived enhancer of viral infection (SEVI), an amyloid fibril formed from a cationic peptide fragment of prostatic acidic phosphatase (PAP), dramatically enhances the infectivity of human immunodeficiency virus type 1 (HIV-1). Insoluble, sedimentable fibrils contribute to SEVI-mediated enhancement of virus infection. However, the SEVI-forming PAP(248–286) peptide is able to produce infection-enhancing structures much more quickly than it forms amyloid fibrils. This suggests that soluble supramolecular assemblies may enhance HIV-1 infection. To address this question, non-SEVI amyloid-like fibrils were derived from general amphipathic peptides of sequence Ac-Kn(XKXE)2-NH2. These cationic peptides efficiently self-assembled to form soluble, fibril-like structures that were, in some cases, able to enhance HIV-1 infection even more efficiently than SEVI. Experiments were also performed to determine whether agents that efficiently shield the charged surface of SEVI fibrils block SEVI-mediated infection-enhancement. To do this, we generated self-assembling anionic peptides of sequence Ac-En(XKXE)2-NH2. One of these peptides completely abrogated SEVI-mediated enhancement of HIV-1 infection, without altering HIV-1 infectivity in the absence of SEVI. Collectively, these data suggest that soluble SEVI assemblies may mediate infection-enhancement, and that anionic peptide supramolecular assemblies have the potential to act as anti-SEVI microbicides.

The accumulation of senile plaques composed of amyloid β (Aβ) fibrils is a hallmark of Alzheimer's disease, although prefibrillar oligomeric species are believed to be the primary neurotoxic congeners in the pathogenesis of Alzheimer's disease. Uncertainty regarding the mechanistic relationship between Aβ oligomer and fibril formation and the cytotoxicity of these aggregate species persists. β-Turn formation has been proposed to be a potential rate-limiting step during Aβ fibrillogenesis. The effect of turn nucleation on Aβ self-assembly was probed by systematically replacing amino acid pairs in the putative turn region of Aβ (residues 24–27) with d-ProGly (DPG), an effective turn-nucleating motif. The kinetic, thermodynamic, and cytotoxic effects of these mutations were characterized. It was found that turn formation dramatically accelerated Aβ fibril self-assembly dependent on the site of turn nucleation. The cytotoxicity of the three DPG-containing Aβ variants was significantly lower than that of wild-type Aβ40, presumably due to decreased oligomer populations as a function of a more rapid progression to mature fibrils; oligomer populations were not eliminated, however, suggesting that turn formation is also a feature of oligomer structures. These results indicate that turn nucleation is a critical step in Aβ40 fibril formation.Download high-res image (218KB)Download full-size imageHighlights► β-Hairpin nucleation was modeled in Aβ using DPG mutations in order to probe the influence of turn formation on Aβ amyloid self-assembly. ► Incorporation of DPG into the putative turn region of the Aβ peptide sequence (residues 24–27) enhanced cross-β amyloid self-assembly rates in a position-dependent manner. ► Aggregates formed by Aβ variants containing DPG were cytotoxic but at lower levels compared to aggregates of the wild-type sequence. ► These findings indicate that turn nucleation is a relevant step in Aβ oligomer and fibril formation.

•Tau facilitates KA-induced seizures and hippocampal superoxide production in mice in vivo.•Tau does not facilitate KA-induced Ca2+ influx in rat primary neurons cultures on its own or in cooperation with Aβ42.•Aβ42 biphasically modulates KA-induced Ca2+ influx in rat neurons: acute Aβ42 enhances, while chronic Aβ42 decreases it.•Aβ42 modulation of KA-induced Ca2+ influx is not mediated by group 1 mGluRs.Excitotoxicity was originally postulated to be a late stage side effect of Alzheimer׳s disease (AD)-related neurodegeneration, however more recent studies indicate that it may occur early in AD and contribute to the neurodegenerative process. Tau and amyloid beta (Aβ), the main components of neurofibrillary tangles (NFTs) and amyloid plaques, have been implicated in cooperatively and independently facilitating excitotoxicity. Our study investigated the roles of tau and Aβ in AD-related excitotoxicity. In vivo studies showed that tau knockout (tau−/−) mice were significantly protected from seizures and hippocampal superoxide production induced with the glutamate analog, kainic acid (KA). We hypothesized that tau accomplished this by facilitating KA-induced Ca2+ influx into neurons, however lentiviral tau knockdown failed to ameliorate KA-induced Ca2+ influx into primary rat cortical neurons. We further investigated if tau cooperated with Aβ to facilitate KA-induced Ca2+ influx. While Aβ biphasically modulated the KA-induced Cacyt2+ responses, tau knockdown continued to have no effect. Therefore, tau facilitates KA-induced seizures and superoxide production in a manner that does not involve facilitation of Ca2+ influx through KA receptors (KAR). On the other hand, acute pretreatment with Aβ (10 min) enhanced KA-induced Ca2+ influx, while chronic Aβ (24 h) significantly reduced it, regardless of tau knockdown. Given previously published connections between Aβ, group 1 metabotropic glutamate receptors (mGluRs), and KAR regulation, we hypothesized that Aβ modulates KAR via a G-protein coupled receptor pathway mediated by group 1 mGluRs. We found that Aβ did not activate group 1 mGluRs and inhibition of these receptors did not reverse Aβ modulation of KA-induced Ca2+ influx. Therefore, Aβ biphasically regulates KAR via a mechanism that does not involve group 1 mGluR activation.

Hydrogel networks composed of rippled β-sheet fibrils of coassembled D- and L-Ac-(FKFE)2-NH2 amphipathic peptides exhibit proteolytic stability and increased rheological strength compared to networks of self-assembled L-Ac-(FKFE)2-NH2 pleated β-sheet fibrils. Modifying the ratios of L and D peptides in the coassembled rippled β-sheet fibrils alters the degradation profiles of these hydrogel networks.

Fmoc-3F-Phe-Arg-NH2 and Fmoc-3F-Phe-Asp-OH dipeptides undergo coassembly to form two-component nanofibril hydrogels. These hydrogels support the viability and growth of NIH 3T3 fibroblast cells. The supramolecular display of Arg and Asp at the nanofibril surface effectively mimics the integrin-binding RGD peptide of fibronectin, without covalent connection between the Arg and Asp functionality.