Unmodified and pyroglutamylated amyloid ß peptides form hypertoxic hetero-oligomers of unique secondary structure.

Amyloid ß (Aß) peptide plays a major role in Alzheimer's disease (AD) and occurs in multiple forms, including pyroglutamylated Aß (AßpE). Identification and characterization of the most cytotoxic Aß species is necessary for advancement in AD diagnostics and therapeutics. While in brain tissue multiple Aß species act in combination, structure/toxicity studies and immunotherapy trials have been focused on individual forms of Aß. As a result, the molecular composition and the structural features of "toxic Aß oligomers" have remained unresolved. Here, we have used a novel approach, hydration from gas phase coupled with isotope-edited Fourier transform infrared (FTIR) spectroscopy, to identify the prefibrillar assemblies formed by Aß and AßpE and to resolve the structures of both peptides in combination. The peptides form unusual ß-sheet oligomers stabilized by intramolecular H-bonding as opposed to intermolecular H-bonding in the fibrils. Time-dependent morphological changes in peptide assemblies have been visualized by atomic force microscopy. Aß/AßpE hetero-oligomers exert unsurpassed cytotoxic effect on PC12 cells as compared to oligomers of individual peptides or fibrils. These findings lead to a novel concept that Aß/AßpE hetero-oligomers, not just Aß or AßpE oligomers, constitute the main neurotoxic conformation. The hetero-oligomers thus present a new biomarker that may be targeted for development of more efficient diagnostic and immunotherapeutic strategies to combat AD.

Isotope-edited FTIR reveals distinct aggregation and structural behaviors of unmodified and pyroglutamylated amyloid ß peptides.

Amyloid ß peptide (Aß) is causatively associated with Alzheimer's disease (AD), and N-terminally truncated and pyroglutamylated Aß peptides (AßpE) exert hypertoxic effect by an unknown mechanism. Recent evidence has identified the prefibrillar oligomers of Aß, not the fibrils, as the prevalent cytotoxic species. Structural characterization of Aß and AßpE oligomers is therefore important for better understanding of their toxic effect. Here we have used isotope-edited Fourier transform infrared (FTIR) spectroscopy to identify the conformational changes in Aß(1-42) and AßpE(3-42) upon aggregation, individually and in 1 : 1 molar combination. During the first two hours of exposure to aqueous buffer, the peptides undergo transition from mostly α-helical to mostly ß-sheet structure. Data on peptides (13)C,(15)N-labeled at K(16)L(17)V(18) or V(36)G(37)G(38)V(39) allowed construction of structural models for the monomer and early oligomers. The peptide monomer comprises a ß-hairpin that involves residues upstream of the K(16)L(17)V(18) sequence and an N-terminal α-helix. The oligomers form by non-H-bonding interactions between the ß-strands of neighboring ß-hairpins, in lateral or staggered manner, with the strands running parallel or antiparallel. Relative α-helical and ß-sheet propensities of Aß(1-42) and AßpE(3-42) depend on the ionic strength of the buffer, emphasizing the importance of ionic interactions in Aß peptide structure and aggregation. It is inferred that N-terminal modification of AßpE(3-42) affects the helix stability and thereby modulates ß-sheet oligomer formation. The data thus provide new insight into the molecular mechanism of Aß oligomerization by emphasizing the role of the N-terminal transient α-helical structure and by identifying structural constraints for molecular organization of the oligomers.

Morphology-Dependent HIV-Enhancing Effect of Semen-Derived Enhancer of Viral Infection.

PAP248-286 is a 39-residue fragment (residues 248 to 286) derived from protease cleavage of prostatic acidic phosphatase in semen. The amyloid fibrils formed in vitro by PAP248-286 can dramatically enhance human immunodeficiency virus (HIV) infection. To our knowledge, we present the first report that the HIV-enhancing potency of fibrils formed by PAP248-286 is morphology dependent. We identified pleomorphic fibrils by transmission electron microscopy in two buffer conditions. Our solid-state NMR data showed that these fibrils consist of molecules in distinct conformations. In agreement with NMR, fluorescence measurements confirmed that they are assembled along different pathways, with distinct molecular structures. Furthermore, our cell-based infectivity tests detected distinct HIV-enhancing potencies for fibrils in distinct morphologies. In addition, our transmission electron microscopy and NMR results showed that semen-derived enhancer of viral infection fibrils formed in sodium bicarbonate buffer remain stable over time, but semen-derived enhancer of viral infection fibrils formed in phosphate buffered saline keep evolving after the initial 7 days incubation period. Given time, most of the assemblies in phosphate buffered saline will turn into elongated thin fibrils. They have similar secondary structure but different packing than thin fibrils formed initially after 7 days incubation.