Debanjan Bhowmik

Significant Structural Differences between Transient Amyloid‐β Oligomers and Less‐Toxic Fibrils in Regions Known To Harbor Familial Alzheimer′s Mutations

Small oligomers of the amyloid β (Aβ) peptide, rather than the monomers or the fibrils, are suspected to initiate Alzheimers disease (AD). However, their low concentration and transient nature under physiological conditions have made structural investigations difficult. A method for addressing such problems has been developed by combining rapid fluorescence techniques with slower two-dimensional solid-state NMR methods. The smallest Aβ40 oligomers that demonstrate a potential sign of toxicity, namely, an enhanced affinity for cell membranes, were thus probed. The two hydrophobic regions (residues 10-21 and 30-40) have already attained the conformation that is observed in the fibrils. However, the turn region (residues 22-29) and the N-terminal tail (residues 1-9) are strikingly different. Notably, ten of eleven known Aβ mutants that are linked to familial AD map to these two regions. Our results provide potential structural cues for AD therapeutics and also suggest a general method for determining transient protein structures.

Curcumin Alters the Salt Bridge-containing Turn Region in Amyloid β(1–42) Aggregates

Background: Curcumin reduces the risk of Alzheimer disease via an unknown mechanism. Results: Curcumin-incubated Aβ42 aggregates retain the hairpin architecture but have disruptions in the turn region (surprising similarity with Zn2+ incubation). Conclusion: Salt bridge-containing turn region is a major determinant of morphology and toxicity. Significance: Identification of crucial structural changes provides a checkpoint for developing effective AD therapeutics. Amyloid β (Aβ) fibrillar deposits in the brain are a hallmark of Alzheimer disease (AD). Curcumin, a common ingredient of Asian spices, is known to disrupt Aβ fibril formation and to reduce AD pathology in mouse models. Understanding the structural changes induced by curcumin can potentially lead to AD pharmaceutical agents with inherent bio-compatibility. Here, we use solid-state NMR spectroscopy to investigate the structural modifications of amyloid β(1–42) (Aβ42) aggregates induced by curcumin. We find that curcumin induces major structural changes in the Asp-23–Lys-28 salt bridge region and near the C terminus. Electron microscopy shows that the Aβ42 fibrils are disrupted by curcumin. Surprisingly, some of these alterations are similar to those reported for Zn2+ ions, another agent known to disrupt the fibrils and alter Aβ42 toxicity. Our results suggest the existence of a structurally related family of quasi-fibrillar conformers of Aβ42, which is stabilized both by curcumin and by Zn2+.

An early folding contact between Phe19 and Leu34 is critical for amyloid-β oligomer toxicity.

Small hydrophobic oligomers of aggregation-prone proteins are thought to be generically toxic. Here we examine this view by perturbing an early folding contact between Phe19 and Leu34 formed during the aggregation of Alzheimers amyloid-β (Aβ40) peptide. We find that even conservative single mutations altering this interaction can abolish Aβ40 toxicity. Significantly, the mutants are not distinguishable either by the oligomers size or by the end-state fibrillar structure from the wild type Aβ40. We trace the change in their toxicity to a drastic lowering of membrane affinity. Therefore, nonlocal folding contacts play a key role in steering the oligomeric intermediates through specific conformations with very different properties and toxicity levels. Our results suggest that engineering the folding energy landscape may provide an alternative route to Alzheimer therapeutics.

Rapid, cell-free assay for membrane-active forms of amyloid-β.

Small oligomers of amyloid beta (Aβ) are suspected to be the key to Alzheimers disease (AD). However, identifying these toxic species in the background of other similar but nontoxic Aβ aggregates has remained a challenge. Recent studies indicate that Aβ undergoes a global structural transition in an early step of aggregation. This transition is marked by a strong increase in its affinity for cell membranes, which suggests that the resultant oligomers could be the key to Aβ toxicity. Here we use this increased membrane affinity to develop a rapid, quantitative, cell-free assay for these bioactive oligomers. It uses fluorescence correlation spectroscopy of fluorescently labeled Aβ and requires only 30 s of measurement time. We also describe a simpler (though less rapid) assay based on the same principles, which uses a dialysis step followed by conventional fluorescence spectroscopy. Our results potentially provide a much-needed high-throughput assay for AD drug development.

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-β18–35 and Makes It Soluble

Aβ self-assembles into parallel cross-β fibrillar aggregates, which is associated with Alzheimers disease pathology. A central hairpin turn around residues 23–29 is a defining characteristic of Aβ in its aggregated state. Major biophysical properties of Aβ, including this turn, remain unaltered in the central fragment Aβ18–35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aβ18–35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aβ18–35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe19 and Leu34 residues, observed in full-length Aβ and Aβ18–35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel β-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel β-sheet. We conclude that the self-assembly of Aβ is critically dependent on the hairpin turn and on the contact between the Phe19 and Leu34 regions, making them potentially sensitive targets for Alzheimers therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.

Major Reaction Coordinates Linking Transient Amyloid-β Oligomers to Fibrils Measured at Atomic Level

The structural underpinnings for the higher toxicity of the oligomeric intermediates of amyloidogenic peptides, compared to the mature fibrils, remain unknown at present. The transient nature and heterogeneity of the oligomers make it difficult to follow their structure. Here, using vibrational and solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that freely aggregating Aβ40 oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel β-sheet structure observed in mature fibrils. The intramolecular hydrogen-bonding network flips nearly 90°, and the two β-strands of each monomeric unit move apart, to give rise to the well-known intermolecular in-register parallel β-sheet structure in the mature fibrils. Solid-state nuclear magnetic resonance distance measurements capture the interstrand separation within monomer units during the transition from the oligomer to the fibril form. We further find that the D23-K28 salt-bridge, a major feature of the Aβ40 fibrils and a focal point of mutations linked to early onset Alzheimers disease, is not detectable in the small oligomers. Molecular dynamics simulations capture the correlation between changes in the D23-K28 distance and the flipping of the monomer secondary structure between antiparallel and parallel β-sheet architectures. Overall, we propose interstrand separation and salt-bridge formation as key reaction coordinates describing the structural transition of the small Aβ40 oligomers to fibrils.

Curcumin Dictates Divergent Fates for the Central Salt Bridges in Amyloid-β40 and Amyloid-β42

There are three specific regions in the Amyloid beta (Aβ) peptide sequence where variations cause enhanced toxicity in Alzheimers disease: the N-terminus, the central salt bridge, and the C-terminus. Here, we investigate if there is a close conformational connection between these three regions, which may suggest a concerted mechanism of toxicity. We measure the effects of Zn2+ and curcumin on Aβ40, and compare these with their previously reported effects on Aβ42. Aβ42 and Aβ40 differ only near the C-terminus, where curcumin interacts, while Zn2+ interacts near the N-terminus. Therefore, this comparison should help us differentiate the effect of modulating the C- and the N-termini. We find that curcumin allows fibril-like structures containing the salt bridge to emerge in the mature Aβ40 aggregates, but not in Aβ42. In contrast, we find no difference in the effects of Zn+2 on Aβ40 and Aβ42. In the presence of Zn+2, both of these fail to form proper fibrils, and the salt bridge remains disrupted. These results indicate that modulations of the Aβ termini can determine the fate of a salt bridge far away in the sequence, and this has significant consequences for Aβ toxicity. We also infer that small molecules can alter oligomer-induced toxicity by modulating the aggregation pathway, without substantially changing the final product of aggregation.

Amyloid Aggregation of Amylin: Gain of Function along Aggregation Pathway?

Abstract: Aggregation of human amylin, a 37 amino acid residue neuropeptide of pancreatic origin, into amyloid aggregates is implicated in the etiology of diabetes mellitus type II. Despite its clinical significance, details of progression of nontoxic amylin monomers into cytotoxic oligomers are not very clear. Studies based on Amyloid-β (Aβ), a peptide with similar size, have suggested that a conformational transition along the aggregation pathway makes the oligomers of Aβ membrane-binding competent (1). We have explored the possibility of amylin undergoing a similar transition during aggregation as it could shed light on potential commonalities in the conformation and function of different toxic amyloid species. Using fluorescence correlation spectroscopy, we identify two distinct oligomers of amylin along the aggregation pathway having hydrodynamic radius of 0.90 nm and 1.6 nm respectively. The membrane affinity of amylin increases remarkably from ∼15 % in smaller species to ∼ 85% in the larger one as assessed by an in vitro membrane-binding assay developed in our lab (2). We observe similar difference in the cell membrane attachment ability of these two species in RIN5mf cell lines using confocal microscopy. A preliminary conformational study in artificial lipid bilayers using a SERS based methodology (3) suggests a temporal conformational reorganization in the peptide backbone. Our data suggest that amylin might acquire toxic function by a mechanism which depends on similar conformational features as Aβ in presence of membranes. Further studies aimed at obtaining high-resolution structural details of amylin oligomers in solution and in membranes are currently in progress.References:1. Nag S, et al. (2013) Phys. Chem. Chem. Phys. 15:19129-33.2. Bhowmik D, et al. (2015) Langmuir. 31(14):4049-53.3. Bhowmik D, et al. (2015) ACS Nano. 9(9):9070-7.