Joan-Emma Shea

Amyloid-β protein oligomerization and the importance of tetramers and dodecamers in the aetiology of Alzheimer’s disease

In recent years, small protein oligomers have been implicated in the aetiology of a number of important amyloid diseases, such as type 2 diabetes, Parkinsons disease and Alzheimers disease. As a consequence, research efforts are being directed away from traditional targets, such as amyloid plaques, and towards characterization of early oligomer states. Here we present a new analysis method, ion mobility coupled with mass spectrometry, for this challenging problem, which allows determination of in vitro oligomer distributions and the qualitative structure of each of the aggregates. We applied these methods to a number of the amyloid-β protein isoforms of Aβ40 and Aβ42 and showed that their oligomer-size distributions are very different. Our results are consistent with previous observations that Aβ40 and Aβ42 self-assemble via different pathways and provide a candidate in the Aβ42 dodecamer for the primary toxic species in Alzheimers disease.

Amyloid β-protein monomer structure: A computational and experimental study

The structural properties of the Aβ42 peptide, a main constituent of the amyloid plaques formed in Alzheimers disease, were investigated through a combination of ion‐mobility mass spectrometry and theoretical modeling. Replica exchange molecular dynamics simulations using a fully atomic description of the peptide and implicit water solvent were performed on the −3 charge state of the peptide, its preferred state under experimental conditions. Equilibrated structures at 300 K were clustered into three distinct families with similar structural features within a family and with significant root mean square deviations between families. An analysis of secondary structure indicates the Aβ42 peptide conformations are dominated by loops and turns but show some helical structure in the C‐terminal hydrophobic tail. A second calculation on Aβ42 in a solvent‐free environment yields compact structures turned “inside out” from the solution structures (hydrophobic parts on the outside, polar parts on the inside). Ion mobility experiments on the Aβ42 −3 charge state electrosprayed from solution yield a bimodal arrival time distribution. This distribution can be quantitatively fit using cross‐sections from dehydrated forms of the three families of calculated solution structures and the calculated solvent‐free family of structures. Implications of the calculations on the early stages of aggregation of Aβ42 are discussed.

Human Islet Amyloid Polypeptide Monomers Form Ordered β-hairpins: A Possible Direct Amyloidogenic Precursor

Oligomerization of human islet amyloid polypeptide (IAPP) has been increasingly considered a pathogenic process in type II diabetes. Here structural features of the IAPP monomer have been probed using a combination of ion mobility mass spectrometry (IMS-MS) and all-atom replica exchange molecular dynamics (REMD) simulations. Three distinct conformational families of human IAPP monomer are observed in IMS experiments, and two of them are identified as dehydrated solution structures on the basis of our simulation results: one is an extended beta-hairpin structural family, and the second is a compact helix-coil structural family. The extended beta-hairpin family is topologically similar to the peptide conformation in the solid-state NMR fibril structure published by Tycko and co-workers. It is absent in both experiments and simulations performed on the non-amyloidogenic rat IAPP, suggesting it may play an important role in the fibrillation pathway of human IAPP. In addition, pH dependence studies show that the relative abundance of the beta-hairpin structural family is significantly enhanced at pH 8.0. This observation is consistent with the increased rate of fibrillation at high pH in vitro and offers a possible explanation of the pH dependent fibrillation in vivo. This paper, to the best of our knowledge, presents the first experimental evidence of a significant population of beta-hairpin conformers for the IAPP peptide. It is consistent with a previous suggestion in the literature that beta-sheet-rich oligomers are assembled from ordered beta-hairpins rather than from coiled structures.

Role of Water in Mediating the Assembly of Alzheimer Amyloid-β Aβ16–22 Protofilaments

The role of water in promoting the formation of protofilaments (the basic building blocks of amyloid fibrils) is investigated using fully atomic molecular dynamics simulations. Our model protofilament consists of two parallel beta-sheets of Alzheimer Amyloid-beta 16-22 peptides (Ac-K(16)-L(17)-V(18)-F(19)-F(20)-A(21)-E(22)-NH2). Each sheet presents a distinct hydrophobic and hydrophilic face and together self-assemble to a stable protofilament with a core consisting of purely hydrophobic residues (L(17), F(19), A(21)), with the two charged residues (K(16), E(22)) pointing to the solvent. Our simulations reveal a subtle interplay between a water mediated assembly and one driven by favorable energetic interactions between specific residues forming the interior of the protofilament. A dewetting transition, in which water expulsion precedes hydrophobic collapse, is observed for some, but not all molecular dynamics trajectories. In the trajectories in which no dewetting is observed, water expulsion and hydrophobic collapse occur simultaneously, with protofilament assembly driven by direct interactions between the hydrophobic side chains of the peptides (particularly between F-F residues). For those same trajectories, a small increase in the temperature of the simulation (on the order of 20 K) or a modest reduction in the peptide-water van der Waals attraction (on the order of 10%) is sufficient to induce a dewetting transition, suggesting that the existence of a dewetting transition in simulation might be sensitive to the details of the force field parametrization.

The Amyloid Formation Mechanism in Human IAPP: Dimers Have β-Strand Monomer-Monomer Interfaces

Early oligomerization of human IAPP (hIAPP) is responsible for β-cell death in the pancreas and is increasingly considered a primary pathological process linked to Type II Diabetes (T2D). Yet, the assembly mechanism remains poorly understood, largely due to the inability of conventional techniques to probe distributions or detailed structures of early oligomeric species. Here, we describe the first experimental data on the isolated and unmodified dimers of human (hIAPP) and nonamyloidogenic rat IAPP (rIAPP). The experiments reveal that the human IAPP dimers are more extended than those formed by rat IAPP and likely descend from extended monomers. Independent all-atom molecular dynamics simulations show that rIAPP forms compact helix and coil rich dimers, whereas hIAPP forms β-strand rich dimers that are generally more extended. Also, the simulations reveal that the monomer-monomer interfaces of the hIAPP dimers are dominated by β-strands and that β-strands can recruit coil or helix structured regions during the dimerization process. Our β-rich interface contrasts with an N-terminal helix-to-helix interface proposed in the literature but is consistent with existing experimental data on the self-interaction pattern of hIAPP, mutation effects, and inhibition effects of the N-methylation in the mutation region.

Probing the folding free energy landscape of the Src-SH3 protein domain.

The mechanism and thermodynamics of folding of the Src homology 3 (SH3) protein domain are characterized at an atomic level through molecular dynamics with importance sampling. This methodology enables the construction of the folding free energy landscape of the protein as a function of representative reaction coordinates. We observe that folding proceeds in a downhill manner under native conditions, with early compaction and structure formation in the hydrophobic sheet consisting of the three central β strands of the protein. This state bears considerable resemblance to the experimentally determined transition state for folding. Folding proceeds further with the formation of the second hydrophobic sheet consisting of the terminal strands and the RT loop. The final stages of folding appear to involve the formation of the hydrophobic core through the expulsion of water molecules bridging the two hydrophobic sheets. This work sheds new light on the complementary roles of sequence and topology in governing the folding mechanism of small proteins and provides further support for the role of water in facilitating the late stages in folding.

Structure of the 21-30 fragment of amyloid β-protein

Folding and self‐assembly of the 42‐residue amyloid β‐protein (Aβ) are linked to Alzheimers disease (AD). The 21–30 region of Aβ, Aβ(21–30), is resistant to proteolysis and is believed to nucleate the folding of full‐length Aβ. The conformational space accessible to the Aβ(21–30) peptide is investigated by using replica exchange molecular dynamics simulations in explicit solvent. Conformations belonging to the global free energy minimum (the “native” state) from simulation are in good agreement with reported NMR structures. These conformations possess a bend motif spanning the central residues V24–K28. This bend is stabilized by a network of hydrogen bonds involving the side chain of residue D23 and the amide hydrogens of adjacent residues G25, S26, N27, and K28, as well as by a salt bridge formed between side chains of K28 and E22. The non‐native states of this peptide are compact and retain a native‐like bend topology. The persistence of structure in the denatured state may account for the resistance of this peptide to protease degradation and aggregation, even at elevated temperatures.

Effect of β-sheet propensity on peptide aggregation

The effect of beta-sheet propensity on the structural features of peptide aggregates was investigated using an off-lattice coarse-grained peptide model. A phase diagram as a function of temperature and beta-sheet propensity reveals a diverse family of supramolecular assemblies. Highly rigid peptides (peptides with high beta-sheet propensity) are seen to assemble predominantly into fibrillar structures. Increasing the flexibility of the peptide (reducing beta-sheet propensity) leads to a variety of structures, including fibrils, beta-barrel structures, and amorphous aggregates. Nonfibrillar entities have been suggested as primary causative agents in amyloid diseases and our simulations indicate that mutations that decrease beta-sheet propensity will decrease fibril formation and favor the formation of such toxic oligomers. Parallels between beta-sheet aggregates and nematic liquid crystals are discussed.

Effects of confinement in chaperonin assisted protein folding: rate enhancement by decreasing the roughness of the folding energy landscape.

Chaperonins, such as the GroE complex of the bacteria Escherichia coli, assist the folding of proteins under non-permissive folding conditions by providing a cavity in which the newly translated or translocated protein can be encapsulated. Whether the chaperonin cage plays a passive role in protecting the protein from aggregation, or an active role in accelerating folding rates, remains a matter of debate. Here, we investigate the role of confinement in chaperonin mediated folding through molecular dynamics simulations. We designed a substrate protein with an alpha/beta sandwich fold, a common structural motif found in GroE substrate proteins and confined it to a spherical hydrophilic cage which mimicked the interior of the GroEL/ES cavity. The thermodynamics and kinetics of folding were studied over a wide range of temperature and cage radii. Confinement was seen to significantly raise the collapse temperature, T(c), as a result of the associated entropy loss of the unfolded state. The folding temperature, T(f), on the other hand, remained unaffected by encapsulation, a consequence of the folding mechanism of this protein that involves an initial collapse to a compact misfolded state prior to rearranging to the native state. Folding rates were observed to be either accelerated or retarded compared to bulk folding rates, depending on the temperature of the simulation. Rate enhancements due to confinement were observed only at temperatures above the temperature T(m), which corresponds to the temperature at which the protein folds fastest. For this protein, T(m) lies above the folding temperature, T(f), implying that encapsulation alone will not lead to a rate enhancement under conditions where the native state is stable (T<T(f)). For confinement to positively impact folding rates under physiological conditions, it is hence necessary for the protein to exhibit a folding transition above the temperature at which it exhibits its fastest folding rate (T(m)<T(f)). We designed a protein with this property by reducing the energetic frustration in the original alpha/beta sandwich substrate protein. The modified protein exhibited a twofold acceleration in folding rates upon encapsulation. This rate enhancement is due to a mechanistic change in folding involving the elimination, upon encapsulation, of accessible local energy minima corresponding to structures with large radii of gyration. For this protein, confinement hence plays more than the role of a passive cage, but rather adopts an active role, accelerating folding rates by decreasing the roughness of the energy landscape of the protein.

Effects of Familial Alzheimer's Disease Mutations on the Folding Nucleation of the Amyloid β-Protein

The effect of single amino acid substitutions associated with the Italian (E22K), Arctic (E22G), Dutch (E22Q) and Iowa (D23N) familial forms of Alzheimers disease and cerebral amyloid angiopathy on the structure of the 21-30 fragment of the Alzheimer amyloid beta-protein (Abeta) is investigated by replica-exchange molecular dynamics simulations. The 21-30 segment has been shown in our earlier work to adopt a bend structure in solution that may serve as the folding nucleation site for Abeta. Our simulations reveal that the 24-28 bend motif is retained in all E22 mutants, suggesting that mutations involving residue E22 may not affect the structure of the folding nucleation site of Abeta. Enhanced aggregation in Abeta with familial Alzheimers disease substitutions may result from the depletion of the E22-K28 salt bridge, which destabilizes the bend structure. Alternately, the E22 mutations may affect longer-range interactions outside the 21-30 segment that can impact the aggregation of Abeta. Substituting at residue D23, on the other hand, leads to the formation of a turn rather than a bend motif, implying that in contrast to E22 mutants, the D23N mutant may affect monomer Abeta folding and subsequent aggregation. Our simulations suggest that the mechanisms by which E22 and D23 mutations affect the folding and aggregation of Abeta are fundamentally different.