Thanh D. Do

Ion mobility spectrometry reveals the mechanism of amyloid formation of Aβ(25-35) and its modulation by inhibitors at the molecular level: epigallocatechin gallate and scyllo-inositol.

Amyloid cascades leading to peptide β-sheet fibrils and plaques are central to many important diseases. Recently, intermediate assemblies of these cascades were identified as the toxic agents that interact with the cellular machinery. The relationship between the transformation from natively unstructured assembly to the β-sheet oligomers to disease is important in understanding disease onset and the development of therapeutic agents. Research on this early oligomeric region has largely been unsuccessful since traditional techniques measure only ensemble average oligomer properties. Here, ion mobility methods are utilized to deduce the modulation of peptide self-assembly pathways in the amyloid-β protein fragment Aβ(25-35) by two amyloid inhibitors (epigallocatechin gallate and scyllo-inositol) that are currently in clinical trials for Alzheimers Disease. We provide evidence that suppression of β-extended oligomers from the onset of the conversion into β-oligomer conformations is essential for effective attenuation of β-structured amyloid oligomeric species often associated with oligomer toxicity. Furthermore, we demonstrate the ease with which ion mobility spectrometry-mass spectrometry can guide the development of therapeutic agents and drug evaluation by providing molecular level insight into the amyloid formation process and its modulation by small molecule assembly modulators.

Amyloid β-Protein Assembly and Alzheimer’s Disease: Dodecamers of Aβ42, but Not of Aβ40, Seed Fibril Formation

Evidence suggests that oligomers of the 42-residue form of the amyloid β-protein (Aβ), Aβ42, play a critical role in the etiology of Alzheimers disease (AD). Here we use high resolution atomic force microscopy to directly image populations of small oligomers of Aβ42 that occur at the earliest stages of aggregation. We observe features that can be attributed to a monomer and to relatively small oligomers, including dimers, hexamers, and dodecamers. We discovered that Aβ42 hexamers and dodecamers quickly become the dominant oligomers after peptide solubilization, even at low (1 μM) concentrations and short (5 min) incubation times. Soon after (≥10 min), dodecamers are observed to seed the formation of extended, linear preprotofibrillar β-sheet structures. The preprotofibrils are a single Aβ42 layer in height and can extend several hundred nanometers in length. To our knowledge this is the first report of structures of this type. In each instance the preprotofibril is associated off center with a single layer of a dodecamer. Protofibril formation continues at longer times, but is accompanied by the formation of large, globular aggregates. Aβ40, by contrast, does not significantly form the hexamer or dodecamer but instead produces a mixture of smaller oligomers. These species lead to the formation of a branched chain-like network rather than discrete structures.

Phenylalanine Oligomers and Fibrils: The Mechanism of Assembly and the Importance of Tetramers and Counterions.

Phenylalanine is the only amino acid known to self-assemble into toxic fibrillar aggregates. An elevated concentration of phenylalanine in the blood can result in Phenylketonuria, a progressive mental retardation. Ion-mobility mass spectrometry is employed to investigate the structure and distribution of phenylalanine oligomers formed in the early stage of the aggregation cascade. The experimental cross sections indicate that phenyl-alanine self-assembles at neutral pH into oligomers composed of multiple layers of four monomers. The monomers arrange themselves to create a hydrophilic core made of zwitterionic termini and expose hydrophobic aromatic side chains to the outside. At high pH, the interactions between the neutral amino and negatively charged carboxylate of phenylalanine allow a minor population of ladder-like oligomers to be formed and detected in ion-mobility experiments. However, counterions such as ammonium rearrange those structures into the same structures observed at neutral pH. The cytotoxicity of Phe oligomers and fibrils may be due to favorable interactions between the hydrophobic exterior and the cell membrane and strong interactions between the hydrophilic core of Phe oligomers and ions, resulting in ion leakage and cellular damage.

Amyloid β-Protein C-Terminal Fragments: Formation of Cylindrins and β-Barrels.

In order to evaluate potential therapeutic targets for treatment of amyloidoses such as Alzheimers disease (AD), it is essential to determine the structures of toxic amyloid oligomers. However, for the amyloid β-protein peptide (Aβ), thought to be the seminal neuropathogenetic agent in AD, its fast aggregation kinetics and the rapid equilibrium dynamics among oligomers of different size pose significant experimental challenges. Here we use ion-mobility mass spectrometry, in combination with electron microscopy, atomic force microscopy, and computational modeling, to test the hypothesis that Aβ peptides can form oligomeric structures resembling cylindrins and β-barrels. These structures are hypothesized to cause neuronal injury and death through perturbation of plasma membrane integrity. We show that hexamers of C-terminal Aβ fragments, including Aβ(24-34), Aβ(25-35) and Aβ(26-36), have collision cross sections similar to those of cylindrins. We also show that linking two identical fragments head-to-tail using diglycine increases the proportion of cylindrin-sized oligomers. In addition, we find that larger oligomers of these fragments may adopt β-barrel structures and that β-barrels can be formed by folding an out-of-register β-sheet, a common type of structure found in amyloid proteins.

Tau Assembly: The Dominant Role of PHF6 (VQIVYK) in Microtubule Binding Region Repeat R3

Self-aggregation of the microtubule-binding protein Tau reduces its functionality and is tightly associated with Tau-related diseases, termed tauopathies. Tau aggregation is also strongly associated with two nucleating six-residue segments, namely PHF6 (VQIVYK) and PHF6* (VQIINK). In this paper, using experiments and computational modeling, we study the self-assembly of individual and binary mixtures of Tau fragments containing PHF6* (R2/wt; (273)GKVQIINKKLDL(284)) and PHF6 (R3/wt; (306)VQIVYKPVDLSK(317)) and a mutant R2/ΔK280 associated with a neurodegenerative tauopathy. The initial stage of aggregation is probed by ion-mobility mass spectrometry, the kinetics of aggregation monitored with Thioflavin T assays, and the morphology of aggregates visualized by transmission electron microscopy. Insights into the structure of early aggregates and the factors stabilizing the aggregates are obtained from replica exchange molecular dynamics simulations. Our data suggest that R3/wt has a much stronger aggregation propensity than either R2/wt or R2/ΔK280. Heterodimers containing R3/wt are less stable than R3/wt homodimers but much more stable than homodimers of R2/wt and R2/ΔK280, suggesting a possible role of PHF6*-PHF6 interactions in initiating the aggregation of full-length Tau. Lastly, R2/ΔK280 binds more strongly to R3/wt than R2/wt, suggesting a possible mechanism for a pathological loss of normal Tau function.

Interactions between Amyloid-β and Tau Fragments Promote Aberrant Aggregates: Implications for Amyloid Toxicity

We have investigated at the oligomeric level interactions between Aβ(25–35) and Tau(273–284), two important fragments of the amyloid-β and Tau proteins, implicated in Alzheimer’s disease. We are able to directly observe the coaggregation of these two peptides by probing the conformations of early heteroligomers and the macroscopic morphologies of the aggregates. Ion-mobility experiment and theoretical modeling indicate that the interactions of the two fragments affect the self-assembly processes of both peptides. Tau(273–284) shows a high affinity to form heteroligomers with existing Aβ(25–35) monomer and oligomers in solution. The configurations and characteristics of the heteroligomers are determined by whether the population of Aβ(25–35) or Tau(273–284) is dominant. As a result, two types of aggregates are observed in the mixture with distinct morphologies and dimensions from those of pure Aβ(25–35) fibrils. The incorporation of some Tau into β-rich Aβ(25–35) oligomers reduces the aggregation propensity of Aβ(25–35) but does not fully abolish fibril formation. On the other hand, by forming complexes with Aβ(25–35), Tau monomers and dimers can advance to larger oligomers and form granular aggregates. These heteroligomers may contribute to toxicity through loss of normal function of Tau or inherent toxicity of the aggregates themselves.

Initiation of assembly of tau(273-284) and its ΔK280 mutant: an experimental and computational study

The microtubule associated protein tau is essential for the development and maintenance of the nervous system. Tau dysfunction is associated with a class of diseases called tauopathies, in which tau is found in an aggregated form. This paper focuses on a small aggregating fragment of tau, (273)GKVQIINKKLDL(284), encompassing the (PHF6*) region that plays a central role in tau aggregation. Using a combination of simulations and experiments, we probe the self-assembly of this peptide, with an emphasis on characterizing the early steps of aggregation. Ion-mobility mass spectrometry experiments provide a size distribution of early oligomers, TEM studies provide a time course of aggregation, and enhanced sampling molecular dynamics simulations provide atomistically detailed structural information about this intrinsically disordered peptide. Our studies indicate that a point mutation, as well the addition of heparin, lead to a shift in the conformations populated by the earliest oligomers, affecting the kinetics of subsequent fibril formation as well as the morphology of the resulting aggregates. In particular, a mutant associated with a K280 deletion (a mutation that causes a heritable form of neurodegeneration/dementia in the context of full length tau) is seen to aggregate more readily than its wild-type counterpart. Simulations and experiment reveal that the ΔK280 mutant peptide adopts extended conformations to a greater extent than the wild-type peptide, facilitating aggregation through the pre-structuring of the peptide into a fibril-competent structure.

Effects of pH and charge state on peptide assembly: the YVIFL model system.

Peptide oligomerization is necessary but not sufficient for amyloid fibril formation. Here, we use a combination of experiments and simulations to understand how pH influences the aggregation properties of a small hydrophobic peptide, YVIFL, which is a mutant form of [Leu-5]-Enkephalin. Transmission electron microscopy and atomic force microscopy measurements reveal that this peptide forms small aggregates under acidic conditions (pH = 2), but that extensive fibrillization only occurs under basic conditions (pH = 9 and 11). Ion-mobility mass spectrometry identifies key oligomers in the oligomerization process, which are further characterized at an atomistic level by molecular dynamics simulations. These simulations suggest that terminal charges play a critical role in determining aggregation propensity and aggregate morphology. They also reveal the presence of steric zipper oligomers under basic conditions, a possible precursor to fibril formation. Our experiments suggest that multiple aggregation pathways can lead to YVIFL fibrils, and that cooperative and multibody interactions are key mechanistic elements in the early stages of aggregation.

Diphenylalanine Self Assembly: Novel Ion Mobility Methods Showing the Essential Role of Water

The mechanism and driving forces behind the formation of diphenylalanine (FF) nanotubes have attracted much attention in the past decades. The hollow structure of the nanotubes suggests a role for water during the self-assembly process. Here, we use novel ion-mobility mass spectrometry methods to probe the early oligomers formed by diphenylalanine peptides. Interestingly, water-bound oligomers are observed in nano-electrospray ionization (ESI) mass spectra in the absence of bulk solvent. In addition, ligated water clusters transit the ion mobility cell but (often) dissociate before detection. These water molecules are shown to be essential for the formation of diphenylalanine oligomers larger than the dimer. The ligated water molecules exist in the solvent free environment either as neutral water or as protonated water clusters, depending on the composition of solvent from which they are sprayed. Water adduction helps stabilize conformers that are otherwise energetically unstable ultimately leading to the assembly of FF nanotubes.

Factors That Drive Peptide Assembly from Native to Amyloid Structures: Experimental and Theoretical Analysis of [Leu-5]-Enkephalin Mutants

Five different mutants of [Leu-5] Enkephalin YGGFL peptide have been investigated for fibril formation propensities. The early oligomer structures have been probed with a combination of ion-mobility mass spectrometry and computational modeling. The two peptides YVIFL and YVVFL form oligomers and amyloid-like fibrils. YVVFV shows an early stage oligomer distribution similar to those of the previous two, but amyloid-like aggregates are less abundant. Atomic resolution X-ray structures of YVVFV show two different modes of interactions at the dry interface between steric zippers and pairs of antiparallel β-sheets, but both are less favorable than the packing motif found in YVVFL. Both YVVFV and YVVFL can form a Class 6 steric zipper. However, in YVVFV, the strands between mating sheets are parallel to each other and in YVVFL they are antiparallel. The overall data highlight the importance of structurally characterizing high order oligomers within oligomerization pathways in studies of nanostructure assembly.