Nanodroplet Oligomers (NanDOs) of Aß40.

Using atomic force microscopy (AFM) and nuclear magnetic resonance (NMR), we describe small Aß40 oligomers, termed nanodroplet oligomers (NanDOs), which form rapidly and at Aß40 concentrations too low for fibril formation. NanDOs were observed in putatively monomeric solutions of Aß40 (e.g., by size exclusion chromatography). Video-rate scanning AFM shows rapid fusion and dissolution of small oligomer-sized particles, of which the median size increases with peptide concentration. In NMR (13C HSQC), a small number of chemical shifts changed with a change in peptide concentration. Paramagnetic relaxation enhancement NMR experiments also support the formation of NanDOs and suggest prominent interactions in hydrophobic domains of Aß40. Addition of Zn2+ to Aß40 solutions caused flocculation of NanDO-containing solutions, and selective loss of signal intensity in NMR spectra from residues in the N-terminal domain of Aß40. NanDOs may represent the earliest aggregated form of Aß40 in the aggregation pathway and are akin to premicelles in solutions of amphiphilies.

A versatile method for producing labeled or unlabeled Aß55, Aß40, and other ß-amyloid family peptides.

We present a straightforward, versatile method for expressing and purifying ß-amyloid (Aß40) and transmembrane peptides derived from ß-amyloid precursor protein (Aß55). In principle, these methods should be applicable to other types of strongly aggregating peptides. We start with a DNA plasmid encoding a HexaHis tag with a flexible, hydrophilic linker sequence, followed by a cleavage site, and then Aß peptides. The HexaHis tag rather than a protein fusion partner (e.g., GST) obviates the need for a folded protein in affinity purification. Second, we present two cleavage methods, using either Factor Xa or BNPS-Skatole. Although the latter procedure requires subsequent reduction of the product, we describe methods for minimizing side reactions. Because the use of BNPS-Skatole obviates the need for a folded protein in the cleavage reaction, it is compatible with harsh conditions (e.g., inclusion of detergents and denaturants) needed to solubilize the fusion proteins; such conditions tend to inactivate Factor Xa. Finally, we also describe purification strategies for Aß40 and Aß55 using FPLC and/or reverse phase HPLC. Yields of peptide after these BNPS-Skatole cleavage and peptide reduction, though subquantitative, greatly exceed those obtained using Factor Xa cleavage, as the reaction of BNPS-Skatole is insensitive to the presence of detergents and denaturants, and therefore can be used to produce highly aggregative and low solubility peptides such as Aß55. Trp is a low abundance amino acid in proteins generally, and for peptides like Aß55, and other transmembane peptides lacking Trp in relevant positions, this cleavage method remains a useful option.

2H-NMR and MD Simulations Reveal Membrane-Bound Conformation of Magainin 2 and Its Synergy with PGLa.

Magainin 2 (MAG2) and PGLa are two α-helical antimicrobial peptides found in the skin of the African frog Xenopus laevis. They act by permeabilizing bacterial membranes and exhibit an exemplary synergism. Here, we determined the detailed molecular alignment and dynamical behavior of MAG2 in oriented lipid bilayers by using 2H-NMR on Ala-d3-labeled peptides, which yielded orientation-dependent quadrupolar splittings of the labels. The amphiphilic MAG2 helix was found to lie flat on the membrane surface in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC)/1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol (DMPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), as expected, with a tilt angle close to 90°. This orientation fits well with all-atom molecular-dynamics simulations of MAG2 performed in DMPC and DMPC/DMPG. In the presence of an equimolar amount of PGLa, the NMR analysis showed that MAG2 becames tilted at an angle of 120°, and its azimuthal rotation angle also changes. Since this interaction was found to occur in a concentration range where the peptides per se do not interact with their own type, we propose that MAG2 forms a stable heterodimer with PGLa. Given that the PGLa molecules in the complex are known to be flipped into a fully upright orientation, with a helix tilt close to 180°, they must make up the actual transmembrane pore. We thus suggest that the two negative charges on the C-terminus of the obliquely tilted MAG2 peptides neutralize some of the cationic groups on the upright PGLa helices. This would stabilize the assembly of PGLa into a toroidal pore with an overall reduced charge density, which could explain the mechanism of synergy.

Homo- and heteromeric interaction strengths of the synergistic antimicrobial peptides PGLa and magainin 2 in membranes.

PGLa and magainin 2 (MAG2) are amphiphilic α-helical frog peptides with synergistic antimicrobial activity. In vesicle leakage assays we observed the strongest synergy for equimolar mixtures of PGLa and MAG2. This result was consistent with solid-state (15)N-NMR data on the helix alignment in model membranes. The Hill coefficients determined from the vesicle leakage data showed that the heterodimeric (PGLa-MAG2) interactions were stronger than the homodimeric (PGLa-PGLa and MAG2-MAG2) interactions. This result was also reflected in the free energy of dimerization determined from oriented circular dichroism and quantitative solid-state (19)F-NMR analysis.

Influence of hydrophobic residues on the activity of the antimicrobial peptide magainin 2 and its synergy with PGLa.

Magainin 2 (MAG2) and PGLa are two related antimicrobial peptides found in the skin of the African frog Xenopus laevis with a pronounced synergistic activity, which act by permeabilizing bacterial membranes. To probe the influence of hydrophobic peptide-lipid and peptide-peptide interactions on the antimicrobial activity and on synergy, the sequence of MAG2 was modified by replacing single amino acids either with a small alanine or with the stiff and bulky hydrophobic 3-(trifluoromethyl)-L-bicyclopent-[1.1.1]-1-ylglycine side chain. The minimum inhibitory concentration of 14 MAG2 analogs was strongly influenced by these single substitutions: the antimicrobial activity was consistently improved when the hydrophobicity was increased on the hydrophobic face of the amphiphilic helix, while the activity decreased when the hydrophobicity was reduced. The synergy with PGLa, on the other hand, was rather insensitive to mutations of hydrophobic residues. It thus seems that the antimicrobial effect of MAG2 on its own depends strongly on the hydrophobicity of the peptide, while the synergy with PGLa does not depend on the overall hydrophobicity of MAG2.

Synergistic insertion of antimicrobial magainin-family peptides in membranes depends on the lipid spontaneous curvature.

PGLa and magainin 2 (MAG2) are amphiphilic antimicrobial peptides from frog skin with known synergistic activity. The orientation of the two helices in membranes was studied using solid-state (15)N-NMR, for each peptide alone and for a 1:1 mixture of the peptides, in a range of different lipid systems. Two types of orientational behavior emerged. 1), In lipids with negative spontaneous curvature, both peptides remain flat on the membrane surface, when assessed both alone and in a 1:1 mixture. 2), In lipids with positive spontaneous curvature, PGLa alone assumes a tilted orientation but inserts into the bilayer in a transmembrane alignment in the presence of MAG2, whereas MAG2 stays on the surface or gets only slightly tilted, when observed both alone and in the presence of PGLa. The behavior of PGLa alone is identical to that of another antimicrobial peptide, MSI-103, in the same lipid systems, indicating that the curvature-dependent helix orientation is a general feature of membrane-bound peptides and also influences their synergistic intermolecular interactions.

ß-amyloid model core peptides: Effects of hydrophobes and disulfides.

The mechanism by which a disordered peptide nucleates and forms amyloid is incompletely understood. A central domain of ß-amyloid (Aß21-30) has been proposed to have intrinsic structural propensities that guide the limited formation of structure in the process of fibrillization. In order to test this hypothesis, we examine several internal fragments of Aß, and variants of these either cyclized or with an N-terminal Cys. While Aß21-30 and variants were always monomeric and unstructured (circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMRS)), we found that the addition of flanking hydrophobic residues in Aß16-34 led to formation of typical amyloid fibrils. NMR showed no long-range nuclear overhauser effect (nOes) in Aß21-30, Aß16-34, or their variants, however. Serial 1 H-15 N-heteronuclear single quantum coherence spectroscopy, 1 H-1 H nuclear overhauser effect spectroscopy, and 1 H-1 H total correlational spectroscopy spectra were used to follow aggregation of Aß16-34 and Cys-Aß16-34 at a site-specific level. The addition of an N-terminal Cys residue (in Cys-Aß16-34) increased the rate of fibrillization which was attributable to disulfide bond formation. We propose a scheme comparing the aggregation pathways for Aß16-34 and Cys-Aß16-34, according to which Cys-Aß16-34 dimerizes, which accelerates fibril formation. In this context, cysteine residues form a focal point that guides fibrillization, a role which, in native peptides, can be assumed by heterogeneous nucleators of aggregation.

ß-Amyloid aggregation and heterogeneous nucleation.

In this article, we consider the role of heterogeneous nucleation in ß-amyloid aggregation. Heterogeneous nucleation is more common and occurs at lower levels of supersaturation than homogeneous nucleation. The nucleation period is also the stage at which most of the polymorphism of amyloids arises, this being one of the defining features of amyloids. We focus on several well-known heterogeneous nucleators of ß-amyloid, including lipid surfaces, especially those enriched in gangliosides and cholesterol, and divalent metal ions. These two broad classes of nucleators affect ß-amyloid particularly in light of the amphiphilicity of these peptides: the N-terminal region, which is largely polar and charged, contains the metal binding site, whereas the C-terminal region is aliphatic and is important in lipid binding. Notably, these two classes of nucleators can interact cooperatively, aggregation begetting greater aggregation.

Multiple cancer-specific antigens are targeted by a chimeric antigen receptor on a single cancer cell.

Human cancer cells were eradicated by adoptive transfer of T cells transduced with a chimeric antigen receptor (CAR) made from an antibody (237Ab) that is highly specific for the murine Tn-glycosylated podoplanin (Tn-PDPN). The objectives were to determine the specificity of these CAR-transduced T (CART) cells and the mechanism for the absence of relapse. We show that although the 237Ab bound only to cell lines expressing murine Tn-PDPN, the 237Ab-derived 237CART cells lysed multiple different human and murine cancers not predicted by the 237Ab binding. Nevertheless, the 237CART cell reactivities remained cancer specific because all recognitions were dependent on the Tn glycosylation that resulted from COSMC mutations that were not present in normal tissues. While Tn was required for the recognition by 237CART, Tn alone was not sufficient for 237CART cell activation. Activation of 237CART cells required peptide backbone recognition but tolerated substitutions of up to 5 of the 7 amino acid residues in the motif recognized by 237Ab. Together, these findings demonstrate what we believe is a new principle whereby simultaneous recognition of multiple independent Tn-glycopeptide antigens on a cancer cell makes tumor escape due to antigen loss unlikely.

Molecular mechanism of synergy between the antimicrobial peptides PGLa and magainin 2.

PGLa and magainin 2 (MAG2) are amphiphilic α-helical membranolytic peptides from frog skin with known synergistic antimicrobial activity. By systematically mutating residues in the two peptides it was possible to identify the ones crucial for the synergy, as monitored by biological assays, fluorescence vesicle leakage, and solid-state 15N-NMR. Electrostatic interactions between anionic groups in MAG2 and cationic residues in PGLa enhance synergy but are not necessary for the synergistic effect. Instead, two Gly residues (7 and 11) in a so-called GxxxG motif in PGLa are necessary for synergy. Replacing either of them with Ala or another hydrophobic residue completely abolishes synergy according to all three methods used. The designer-made peptide MSI-103, which has a similar sequence as PGLa, shows no synergy with MAG2, but by introducing two Gly mutations it was possible to make it synergistic. A molecular model is proposed for the functionally active PGLa-MAG2 complex, consisting of a membrane-spanning antiparallel PGLa dimer that is stabilized by intimate Gly-Gly contacts, and where each PGLa monomer is in contact with one MAG2 molecule at its C-terminus.