Jüri Jarvet

The Alzheimer β-peptide shows temperature-dependent transitions between left-handed 31-helix, β-strand and random coil secondary structures

The temperature‐induced structural transitions of the full length Alzheimer amyloid β‐peptide [Aβ(1–40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left‐handed 31 (polyproline II; PII)‐helix at 0 °C to a random coil state at 60 °C, with an average ΔH of 6.8 ± 1.4 kJ·mol−1 per residue, obtained by fitting a Zimm–Bragg model to the CD data. The transition is noncooperative for the shortest N‐terminal fragment Aβ(1–9) and weakly cooperative for Aβ(1–40) and the longer fragments. By analysing the temperature‐dependent 3JHNHα couplings and hydrodynamic radii obtained by NMR for Aβ(1–9) and Aβ(12–28), we found that the structure transition includes more than two states. The N‐terminal hydrophilic Aβ(1–9) populates PII‐like conformations at 0 °C, then when the temperature increases, conformations with dihedral angles moving towards β‐strand at 20 °C, and approaches random coil at 60 °C. The residues in the central hydrophobic (18–28) segment show varying behaviour, but there is a significant contribution of β‐strand‐like conformations at all temperatures below 20 °C. The C‐terminal (29–40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of Aβ may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques.

Secondary structure conversions of Alzheimer’s Aβ(1–40) peptide induced by membrane‐mimicking detergents

The amyloid β peptide (Aβ) with 39–42 residues is the major component of amyloid plaques found in brains of Alzheimer’s disease patients, and soluble oligomeric peptide aggregates mediate toxic effects on neurons. The Aβ aggregation involves a conformational change of the peptide structure to β‐sheet. In the present study, we report on the effect of detergents on the structure transitions of Aβ, to mimic the effects that biomembranes may have. In vitro, monomeric Aβ(1–40) in a dilute aqueous solution is weakly structured. By gradually adding small amounts of sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate to a dilute aqueous solution, Aβ(1–40) is converted to β‐sheet, as observed by CD at 3 °C and 20 °C. The transition is mainly a two‐state process, as revealed by approximately isodichroic points in the titrations. Aβ(1–40) loses almost all NMR signals at dodecyl sulfate concentrations giving rise to the optimal β‐sheet content (approximate detergent/peptide ratio = 20). Under these conditions, thioflavin T fluorescence measurements indicate a maximum of aggregated amyloid‐like structures. The loss of NMR signals suggests that these are also involved in intermediate chemical exchange. Transverse relaxation optimized spectroscopy NMR spectra indicate that the C‐terminal residues are more dynamic than the others. By further addition of SDS or lithium dodecyl sulfate reaching concentrations close to the critical micellar concentration, CD, NMR and FTIR spectra show that the peptide rearranges to form a micelle‐bound structure with α‐helical segments, similar to the secondary structures formed when a high concentration of detergent is added directly to the peptide solution.

Cell membrane translocation of the N-terminal (1-28) part of the prion protein.

The N-terminal (1-28) part of the mouse prion protein (PrP) is a cell penetrating peptide, capable of transporting large hydrophilic cargoes through a cell membrane. Confocal fluorescence microscopy shows that it transports the protein avidin (67kDa) into several cell lines. The (1-28) peptide has a strong tendency for aggregation and beta-structure formation, particularly in interaction with negatively charged phospholipid membranes. The findings have implications for how prion proteins with uncleaved signal peptides in the N-termini may enter into cells, which is important for infection. The secondary structure conversion into beta-structure may be relevant as a seed for the conversion into the scrapie (PrP(Sc)) form of the protein and its amyloidic transformation.

Structural characterization of inter-alpha-inhibitor. Evidence for an extended shape.

Inter-α-inhibitor (IαI) is a 180-kDa serum protein consisting of three polypeptides. Two of these, the heavy chains 1 and 2 (H1 and H2), are of 75–80 kDa and have similar amino acid sequences. The third polypeptide, bikunin, has a molecular mass of 25 kDa and contains a 7-kDa chondroitin sulfate chain that is covalently linked to the C-terminal amino acid residues of H1 and H2. IαI has been shown to be required for the formation of the hyaluronan-containing extracellular matrix of certain cell types. How IαI exerts this function is not known, but it appears that upon interaction with cells, the heavy chains are released and become covalently linked to hyaluronan. Our results indicate that IαI and its heavy chains are extended molecules; thus, upon electron microscopy, IαI appeared to consist of two globular domains connected by a thin structure 31-nm long and the isolated heavy chains of a globular domain and a “tail” about 15-nm long. Analysis of the heavy chains by partial proteolysis showed that the C-terminal halves are particularly sensitive to hydrolysis indicating that they are loosely folded. Furthermore, electron microscopy showed that partially degraded heavy chains lacked the extended regions. Taken together, these results suggest that the N-terminal half of the heavy chains forms a globular domain, whereas the other half has an extended and loosely folded structure.

A left-handed 31 helical conformation in the Alzheimer Aβ(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer β‐peptide is in a temperature‐dependent equilibrium between an extended left‐handed 31 helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left‐handed 31 helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Aβ(12–28) in aqueous solution at 0°C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well‐ordered secondary structure. The solvent exchange rates of the amide protons at 0°C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03–0.04 min−1) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.

The hairpin conformation of the amyloid β peptide is an important structural motif along the aggregation pathway

The amyloid β (Aβ) peptides are 39–42 residue-long peptides found in the senile plaques in the brains of Alzheimer’s disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the Aβ peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the Aβ peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of Aβ complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.

The role of pro-inflammatory S100A9 in Alzheimer’s disease amyloid-neuroinflammatory cascade

Pro-inflammatory S100A9 protein is increasingly recognized as an important contributor to inflammation-related neurodegeneration. Here, we provide insights into S100A9 specific mechanisms of action in Alzheimer’s disease (AD). Due to its inherent amyloidogenicity S100A9 contributes to amyloid plaque formation together with Aβ. In traumatic brain injury (TBI) S100A9 itself rapidly forms amyloid plaques, which were reactive with oligomer-specific antibodies, but not with Aβ and amyloid fibrillar antibodies. They may serve as precursor-plaques for AD, implicating TBI as an AD risk factor. S100A9 was observed in some hippocampal and cortical neurons in TBI, AD and non-demented aging. In vitro S100A9 forms neurotoxic linear and annular amyloids resembling Aβ protofilaments. S100A9 amyloid cytotoxicity and native S100A9 pro-inflammatory signaling can be mitigated by its co-aggregation with Aβ, which results in a variety of micron-scale amyloid complexes. NMR and molecular docking demonstrated transient interactions between native S100A9 and Aβ. Thus, abundantly present in AD brain pro-inflammatory S100A9, possessing also intrinsic amyloidogenic properties and ability to modulate Aβ aggregation, can serve as a link between the AD amyloid and neuroinflammatory cascades and as a prospective therapeutic target.

Characterization of Mn(II) ion binding to the amyloid-β peptide in Alzheimers disease

Growing evidence links neurodegenerative diseases to metal exposure. Aberrant metal ion concentrations have been noted in Alzheimers disease (AD) brains, yet the role of metals in AD pathogenesis remains unresolved. A major factor in AD pathogenesis is considered to be aggregation of and amyloid formation by amyloid-β (Aβ) peptides. Previous studies have shown that Aβ displays specific binding to Cu(II) and Zn(II) ions, and such binding has been shown to modulate Aβ aggregation. Here, we use nuclear magnetic resonance (NMR) spectroscopy to show that Mn(II) ions also bind to the N-terminal part of the Aβ(1-40) peptide, with a weak binding affinity in the milli- to micromolar range. Circular dichroism (CD) spectroscopy, solid state atomic force microscopy (AFM), fluorescence spectroscopy, and molecular modeling suggest that the weak binding of Mn(II) to Aβ may not have a large effect on the peptides aggregation into amyloid fibrils. However, identification of an additional metal ion displaying Aβ binding reveals more complex AD metal chemistry than has been previously considered in the literature.

Ionic Strength Modulation of the Free Energy Landscape of Aβ40 Peptide Fibril Formation

Protein misfolding and formation of cross-β structured amyloid fibrils are linked to many neurodegenerative disorders. Although recently developed quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid β peptide (Aβ40) self-association, implicated in Alzheimers disease. We found that physiological ionic strength accelerates Aβ40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different β-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (Fβ*) and a mature stable state (Fβ), where salt favors formation of the Fβ fibril morphology. Addition of salt to preformed Fβ* accelerates transition to Fβ, underlining the dynamic nature of Aβ40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aβ40 folding to the Fβ state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

Regulation of GTPase and adenylate cyclase activity by amyloid β-peptide and its fragments in rat brain tissue

Modulation of GTPase and adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) activity by Alzheimers disease related amyloid beta-peptide, A beta (1-42), and its shorter fragments, A beta (12-28), A beta (25-35), were studied in isolated membranes from rat ventral hippocampus and frontal cortex. In both tissues, the activity of GTPase and adenylate cyclase was upregulated by A beta (25-35), whereas A beta (12-28) did not have any significant effect on the GTPase activity and only weakly influenced adenylate cyclase. A beta (1-42), similar to A beta (25-35), stimulated the GTPase activity in both tissues and adenylate cyclase activity in ventral hippocampal membranes. Surprisingly, A beta (1-42) did not have a significant effect on adenylate cyclase activity in the cortical membranes. At high concentrations of A beta (25-35) and A beta (1-42), decreased or no activation of adenylate cyclase was observed. The activation of GTPase at high concentrations of A beta (25-35) was pertussis toxin sensitive, suggesting that this effect is mediated by Gi/G(o) proteins. Addition of glutathione and N-acetyl-L-cysteine, two well-known antioxidants, at 1.5 and 0.5 mM, respectively, decreased A beta (25-35) stimulated adenylate cyclase activity in both tissues. Lys-A beta (16-20), a hexapeptide shown previously to bind to the same sequence in A beta-peptide, and prevent fibril formation, decreased stimulation of adenylate cyclase activity by A beta (25-35), however, NMR diffusion measurements with the two peptides showed that this effect was not due to interactions between the two and that A beta (25-35) was active in a monomeric form. Our data strongly suggest that A beta and its fragments may affect G-protein coupled signal transduction systems, although the mechanism of this interaction is not fully understood.