Solution NMR studies of Aβ monomer dynamics

Aβ is widely recognized as a key molecule in Alzheimer's disease, causing neurotoxicity through Aβ aggregates such as Aβ oligomers and fibrils. Aβ40 and Aβ42, composed of 40 and 42 residues, respectively, are the major Aβ species in human brain. Aβ42 aggregates much faster than Aβ40 but the mechanism of such difference in aggregation propensity is poorly understood. Using NMR spin relaxation, we have shown that Aβ40 and Aβ42 monomers have different dynamics in both backbone and sidechain on the ps-ns time scale. Aβ42 is more rigid in C-terminus in both backbone and sidechain while Aβ40 has more rigid methyl groups in the central hydrophobic cluster (CHC: Aβ17-21). These observations are consistent with differences in the major conformations of Aβ40 and Aβ42 monomers derived from replica exchange MD (REMD). To further demonstrate the relevance of dynamics in aggregation mechanism, a perturbation was introduced to Aβ42 in the form of M35 oxidation. After M35 side chain oxidation to sulfoxide, Aβ42 experiences Aβ40-like changes in dynamics. At the same time, M35 oxidation causes dramatic reduction in Aβ42 aggregation rate. These data have thus established an important role for protein dynamics in the mechanism of Aβ aggregation.     

Structural dynamics of the amyloid β-protein monomer folding nucleus

Alzheimer's disease (AD) is linked to the aberrant assembly of the amyloid β-protein (Aβ). The (21)AEDVGSNKGA(30) segment, Aβ(21-30), forms a turn that acts as a monomer folding nucleus. Amino acid substitutions within this nucleus cause familial forms of AD. To determine the biophysical characteristics of the folding nucleus, we studied the biologically relevant acetyl-Aβ(21-30)-amide peptide using experimental techniques (limited proteolysis, thermal denaturation, urea denaturation followed by pulse proteolysis, and electron microscopy) and computational methods (molecular dynamics). Our results reveal a highly stable foldon and suggest new strategies for therapeutic drug development.     

Aβ peptide fibrillar architectures controlled by conformational constraints of the monomer

Anomalous self-assembly of the Aβ peptide into fibrillar amyloid deposits is strongly correlated with the development of Alzheimer's disease. Aβ fibril extension follows a template guided "dock and lock" mechanism where polymerisation is catalysed by the fibrillar ends. Using surface plasmon resonance (SPR) and quenched hydrogen-deuterium exchange NMR (H/D-exchange NMR), we have analysed the fibrillar structure and polymerisation properties of both the highly aggregation prone Aβ1-40 Glu22Gly (Aβ(40Arc)) and wild type Aβ1-40 (Aβ(40WT)). The solvent protection patterns from H/D exchange experiments suggest very similar structures of the fibrillar forms. However, through cross-seeding experiments monitored by SPR, we found that the monomeric form of Aβ(40WT) is significantly impaired to acquire the fibrillar architecture of Aβ(40Arc). A detailed characterisation demonstrated that Aβ(40WT) has a restricted ability to dock and isomerise with high binding affinity onto Aβ(40Arc) fibrils. These results have general implications for the process of fibril assembly, where the rate of polymerisation, and consequently the architecture of the formed fibrils, is restricted by conformational constraints of the monomers. Interestingly, we also found that the kinetic rate of fibril formation rather than the thermodynamically lowest energy state determines the overall fibrillar structure.     

Cholesterol promotes the interaction of Alzheimer β-amyloid monomer with lipid bilayer

Cholesterol in the plasma membrane plays an important role in the pathogenesis of Alzheimer's disease, but the exact function of cholesterol in the regulation of amyloid-β (Aβ) generation, aggregation, and toxicity remains elusive. To gain insight into the bioactivity of cholesterol, we investigate the effect of cholesterol levels on the interaction of Aβ(1-42) monomer with the zwitterionic 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayer containing different mole fractions of cholesterol from χ=0, 0.2, to 0.4 using all-atom molecular dynamics simulations. Simulation results show that an increased cholesterol level alters the structure, dynamics, and surface chemistry of the lipid bilayer, leading to increased bilayer thickness, hydrophobic chain order, surface hydrophobicity, and decreased lipid mobility. All these effects significantly promote the binding of Aβ to the lipid bilayer. Mechanistically, the adsorption of Aβ on the bilayer is a cooperative process. First, charged residues act as anchors to establish the initial binding of Aβ to phosphate headgroups of the bilayer driven by electrostatic interactions, which further facilitates hydrophobic residues to reside on the bilayer. Once hydrophobic residues especially from C-terminus are locked on the bilayer, the interactions among charged residues, lipid bilayer, and calcium ions are optimized to provide additional attractive forces to stabilize Aβ adsorbed on or inserted into the lipid bilayer. Inclusion of cholesterol makes this binding process more energetically favorable. Upon adsorption on the bilayer, Aβ appears to preferentially adopt α-helical or unstructured conformation. This work supports that cholesterol acts as a promoter for Aβ--membrane interactions, which would facilitate Aβ aggregation and membrane insertion.     

Production of two monomer structures containing medium-chain-length polyhydroxyalkanoates by β-oxidation-impaired mutant of Pseudomonas putida KT2442

Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) from fatty acids. When gene encoding 3-hydroxyacyl-CoA dehydrogenase which catalyzes long-chain-3-hydroxyacyl-CoA to 3-ketoacyl-CoA, was partially or completely deleted in P. putida KTOY08, the PHA accumulated was shown to contain only two different monomer structures dominated by a monomer of the same chain length as that of the fatty acids fed and another monomer two carbon atoms shorter. Among the PHA copolymers, P(44% 3HD-co-3HDD) containing 44% 3HD and 56% 3HDD was demonstrated to have a melting temperature T_m, an apparent heat of fusion △H_m and a Young’s modulus E of 75 °C, 51 J g^?1 and 2.0 MPa, respectively, the highest among all the MCL PHA studied.     

Characterization of the response of primary cells relevant to dialysis-related amyloidosis to β2-microglobulin monomer and fibrils

The formation of insoluble amyloid fibrils is associated with an array of devastating human diseases. Dialysis-related amyloidosis (DRA) is a severe complication of hemodialysis that results in the progressive destruction of the bones and joints. Elevated concentrations of β(2)-microglobulin (β(2)m) in the serum of subjects on hemodialysis promote the formation of amyloid fibrils in the osteoarticular tissues, but the cellular basis for the destruction of these tissues in DRA is poorly understood. In this study we performed a systematic analysis of the interaction of monomeric and fibrillar β(2)m with primary human cells of the types present in the synovial joints of subjects with DRA. Building upon observations that macrophages infiltrate β(2)m amyloid deposits in vivo we demonstrate that monocytes, the precursors of macrophages, cannot degrade β(2)m fibrils, and that both monomeric β(2)m and fibrillar β(2)m are cytotoxic to these cells. β(2)m fibrils also impair the formation of bone resorbing osteoclasts from monocytes and reduce the viability of osteoblasts, the cell type that produces bone. As a consequence, we predict that β(2)m amyloid will disrupt the remodelling of the bone, which is critical for the maintenance of this tissue. Moreover, we show that β(2)m fibrils reduce the viability of chondrocytes, rationalizing the loss of cartilage in DRA. Together, our observations demonstrate that β(2)m cytotoxicity has multiple cellular targets in the osteoarticular tissues and is likely to be a key factor in the bone and joint destruction characteristic of DRA.     

Scrutiny of the mechanism of small molecule inhibitor preventing conformational transition of amyloid-β42 monomer: insights from molecular dynamics simulations

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by loss of intellectual functioning of brain and memory loss. According to amyloid cascade hypothesis, aggregation of amyloid-β₄₂ (Aβ₄₂) peptide can generate toxic oligomers and their accumulation in the brain is responsible for the onset of AD. In spite of carrying out a large number of experimental studies on inhibition of Aβ₄₂ aggregation by small molecules, the detailed inhibitory mechanism remains elusive. In the present study, comparable molecular dynamics (MD) simulations were performed to elucidate the inhibitory mechanism of a sulfonamide inhibitor C1 (2,5-dichloro-N-(4-piperidinophenyl)-3-thiophenesulfonamide), reported for its in vitro and in vivo anti-aggregation activity against Aβ₄₂. MD simulations reveal that C1 stabilizes native α-helix conformation of Aβ₄₂ by interacting with key residues in the central helix region (13–26) with hydrogen bonds and π–π interactions. C1 lowers the solvent-accessible surface area of the central hydrophobic core (CHC), KLVFF (16–20), that confirms burial of hydrophobic residues leading to the dominance of helical conformation in the CHC region. The binding free energy analysis with MM–PBSA demonstrates that Ala2, Phe4, Tyr10, Gln15, Lys16, Leu17, Val18, Phe19, Phe20, Glu22, and Met35 contribute maximum to binding free energy (?43.1 kcal/mol) between C1 and Aβ₄₂ monomer. Overall, MD simulations reveal that C1 inhibits Aβ₄₂ aggregation by stabilizing native helical conformation and inhibiting the formation of aggregation-prone β-sheet conformation. The present results will shed light on the underlying inhibitory mechanism of small molecules that show potential in vitro anti-aggregation activity against Aβ₄₂.     

Prolines in βA-sheet of neural cadherin act as a switch to control the dynamics of the equilibrium between monomer and dimer

Neural cadherins dimerize through the formation of calcium-dependent strand-crossover structures. Dimerization of cadherins leads to cell-cell adhesion in multicellular organisms. Strand-crossover dimer forms exclusively between the first N-terminal extracellular modules (EC1) of the adhesive partners via swapping of their βA-sheets and docking of tryptophan-2 in the hydrophobic pocket. In the apo-state wild-type cadherin is predominantly monomer, which indicates that the dimerization is energetically unfavorable in the absence of calcium. Addition of calcium favors dimer formation by creating strain in the monomer and lowering the energetic barrier between monomer and dimer. Dynamics of the monomer-dimer equilibrium is vital for plasticity of synapses. Prolines recurrently occur in proteins that form strand-crossover dimer and are believed to be the source of the strain in the monomer. N-cadherins have two proline residues in the βA-sheet. We focused our studies on the role of these two prolines in calcium-dependent dimerization. Spectroscopic, electrophoretic, and chromatopgraphic studies showed that mutations of both prolines to alanines increased the dimerization affinity by ~20-fold and relieved the requirement of calcium in dimerization. The P5A and P6A mutant formed very stable dimers that required denaturation of protein to disassemble in the apo conditions. In summary, the proline residues act as a switch to control the dynamics of the equilibrium between monomer and dimer which is crucial for the plasticity of synapses.     

'Click peptide' using production of monomer Aβ from the O-acyl isopeptide: application to assay system of aggregation inhibitors and cellular cytotoxicity

The O-acyl isopeptide of Aβ1-42 (1), possessing an ester bond at the Gly(25)-Ser(26) sequence, is a water-soluble and non-aggregative precursor molecule and is capable of production of monomer Aβ1-42. The SDS-PAGE result showed that the Aβ1-42, produced from 1, adopted monomeric state at first and then self-assembled to oligomer. The oligomeric state was stabilized by nordihydroguaiaretic acid. The Thioflavin-T (ThT) fluorescence intensity derived from Aβ1-42 (generated from 1) was suppressed by various aggregation inhibitors. Finally, 1 could generate Aβ1-42 via the O-to-N acyl migration under cellular medium conditions and the produced Aβ1-42 exhibited cytotoxicity against PC12 cells. These results suggest that the click peptide system, which enables us to predominantly produce monomer Aβ1-42 under physiological conditions, would be adoptable to various biochemical and biophysical experiments including cellular system to investigate the functions of Aβ1-42.     

The 1.6Å resolution structure of activated D138L mutant of catabolite gene activator protein with two cAMP bound in each monomer

The X-ray crystal structure of the cAMP-liganded D138L mutant of Escherichia coli catabolite gene activator protein (CAP) was determined at a resolution of 1.66?. This high resolution crystal structure reveals four cAMP binding sites in the homodimer. Two anti conformations of cAMPs (anti-cAMP) locate between the β-barrel and the C-helix of each subunit; two syn conformations of cAMPs (syn-cAMP) bind on the surface of the C-terminal domain. With two syn-cAMP molecules bound, the D138L CAP is highly symmetrical with both subunits assuming a "closed" conformation. These differences make the hinge region of the mutant more flexible. Protease susceptibility measurements indicate that D138L is more susceptible to proteases than that of wild type (WT) CAP. The results of protein dynamic experiments (H/D exchange measurements) indicate that the structure of D138L mutant is more dynamic than that of WT CAP, which may impact the recognition of specific DNA sequences.     

NMR studies on the monomer–tetramer transition of melittin in an aqueous solution at high and low temperatures

Melittin, a peptide of 26 amino acid residues, has been used as a model peptide for protein folding and unfolding, and extensive research has been done into its structure and conformational stability. Circular dichroism (CD) studies have demonstrated that melittin in an aqueous solution undergoes a transition from a helical tetramer to a random coil monomer not only by heating but also by cooling from room temperature (i.e., heat- and cold-denaturation, respectively). The heat-denaturation has been also examined by nuclear magnetic resonance (NMR) experiments, however, no NMR data have been presented on the cold-denaturation. In this paper, using proton ((1)H) NMR spectroscopy, we show that melittin undergoes conformational transitions from the monomer to the tetramer to the monomer by elevating temperature from 2 to 70 °C. Only melittin including a trans proline peptide bond participates in the transitions, whereas melittin including a cis proline one does not. The tetramer has maximum conformation stability at around 20 °C, and cooperativity of the heat-denaturation is extremely low.     

N-terminal acetylation of α-synuclein induces increased transient helical propensity and decreased aggregation rates in the intrinsically disordered monomer

The conformational properties of soluble α-synuclein, the primary protein found in patients with Parkinson's disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N-terminal acetylated α-synuclein purified under mild physiological conditions presents as a primarily monomeric protein, and that the N-terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue-specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first 9 N-terminal residues, as well as smaller long-range changes in residues 28-31, 43-46, and 50-66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition, we show that the N-terminal acetylated protein forms fibrils that are morphologically similar to those formed from nonacetylated α-synuclein, but that their growth rates are slower. Our results highlight that N-terminal acetylation does not form significant numbers of dimers, tetramers, or higher molecular weight species, but does alter the conformational distributions of monomeric α-synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.     

Fibril formation from the amyloid-β peptide is governed by a dynamic equilibrium involving association and dissociation of the monomer

Here I review the molecular mechanisms by which water-soluble monomeric amyloid-β (Aβ) peptides are transformed into well-organized supramolecular complexes called amyloid fibrils. The mechanism of amyloid formation is considered theoretically on the basis of experimental results, and the structural and mechanistic similarities of amyloid fibrils to three-dimensional crystals are highlighted. A number of important results from the literature are described. These include the observation that a correct ratio of monomer association and dissociation rate constants is key for formation of well-organized amyloid fibrils. The dynamic nature of the amyloid-β structure is discussed, along with the possibly obligate requirement of the transient formation of a hairpin-like fold prior to its incorporation into amyloid fibrils. Many rounds of monomer association and dissociation events may be present during an apparently silent lag-period. Amongst these association/dissociation events, interaction between the C-terminal regions of the Aβ peptide seems to be more favored. Such association and dissociation events occurring in a “trial-and-error” fashion may be an important requirement for the formation of well-organized amyloid fibrils.     

ε-Viniferin is more effective than its monomer resveratrol in improving the functions of vascular endothelial cells and the heart

The present study compared the effects of resveratrol and its dimer ε-viniferin on vascular endothelial cells (VECs) functions, and on the blood pressure and cardiac mass of spontaneously hypertensive rats (SHRs). Treatment of VECs with these compounds enhanced cell proliferation via nitric oxide generation and protected the cells from oxidative stress by suppressing increases in intracellular oxygen species. ε-Viniferin was more potent than resveratrol in most of these effects. ε-Viniferin, but not resveratrol inhibited angiotensin-converting enzyme activity in vitro. Three weeks of ε-viniferin treatment (5 mg/kg) reduced the systolic blood pressure and improved the whole cardiac mass and left ventricle mass indexes in SHRs. In contrast, resveratrol administration (2.5 mg/kg) failed to lower the blood pressure and significantly improve these mass indexes. These data suggest that ε-viniferin as well as resveratrol may be involved in protecting the functions of VECs and the heart.