␤-(25-35) is a synthetic derivative of ␤-amyloid, the peptide that is believed to cause Alzheimer's disease. As it is highly toxic and forms fibrillar aggregates typical of ␤-amyloid, it is suitable as a model for testing inhibitors of aggregation and toxicity. We demonstrate that N-methylated derivatives of ␤-(25-35), which in isolation are soluble and non-toxic, can prevent the aggregation and inhibit the resulting toxicity of the wild type peptide. N-Methylation can block hydrogen bonding on the outer edge of the assembling amyloid. The peptides are assayed by Congo red and thioflavin T binding, electron microscopy, and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) toxicity assay on PC12 cells. One peptide (Gly 25 N-methylated) has properties similar to the wild type, whereas five have varying effects on prefolded fibrils and fibril assembly. In particular, ␤-(25-35) with Gly 33 N-methylated is able to completely prevent fibril assembly and to reduce the toxicity of prefolded amyloid. With Leu 34 N-methylated, the fibril morphology is altered and the toxicity reduced. We suggest that the use of N-methylated derivatives of amyloidogenic peptides and proteins could provide a general solution to the problem of amyloid deposition and toxicity.Alzheimer's disease (AD) 1 is the most common form of senile dementia. ␤-Amyloid (A␤), a 39 -43-amino acid ␤-sheet peptide, aggregates in the brain to form the major component of characteristic deposits known as senile plaques (1-4). X-ray diffraction data have shown that the conformation of A␤ is characterized by an antiparallel cross-␤-pleated sheet (5), although more recent solid state NMR evidence suggests that the peptide has a parallel ␤-sheet structure (6). Nevertheless, aggregation occurs because of hydrogen bonding between ␤-strands, and the resulting fibrils have axes perpendicular to the ␤-strand and parallel to the cross-linking hydrogen bonds (5).Of all of the A␤ derivatives studied so far, ␤-(25-35), sequence GSNKGAIIGLM, is the shortest fragment that exhibits large ␤-sheet fibrils and retains the toxicity of the full-length peptide (2,(7)(8)(9). It has been proposed that ␤-(25-35) represents the biologically active region of A␤. In vitro studies have shown that it does not require aging to aggregate and become toxic (8 -10), unlike the full-length peptide. As with A␤-␤, toxicity is dependent on the aggregation state of the peptide, because ␤-(25-35) that has been solubilized and unfolded in 35% acetonitrile (AcN), 0.1% trifluoroacetic acid is nontoxic (8,11). In this study, ␤-(25-35) has been chosen as a model for full-length A␤ because it retains both its physical and biological properties, while its short length readily allows derivatives to be synthesized and studied. A great deal of evidence, much of which comes from studying hereditary forms of the disease, supports the view that A␤ aggregation is implicated in AD (1-4, 12-16). Controversy has raged, however, over whether these fibrils are actually a cause or a consequence of the ...

No abstract

The amyloid plaques associated with Alzheimer's disease (AD) comprise fibrillar amyloid-β (Aβ) peptides as well as non-protein factors including glycosaminoglycan (GAG) polysaccharides. GAGs affect the kinetics and pathway of Aβ self-assembly and can impede fibril clearance; thus, they may be accessory molecules in AD. Here we report the first high-resolution details of GAG-Aβ fibril interactions from the perspective of the saccharide. Binding analysis indicated that the GAG proxy heparin has a remarkably high affinity for Aβ fibrils with 3-fold cross-sectional symmetry (3Q). Chemical synthesis of a uniformly (13)C-labeled octasaccharide heparin analogue enabled magic-angle spinning solid-state NMR of the GAG bound to 3Q fibrils, and measurements of dynamics revealed a tight complex in which all saccharide residues are restrained without undergoing substantial conformational changes. Intramolecular (13)C-(15)N dipolar dephasing is consistent with close (<5 Å) contact between GAG anomeric position(s) and one or more histidine residues in the fibrils. These data provide a detailed model for the interaction between 3Q-seeded Aβ40 fibrils and a major non-protein component of AD plaques, and they reveal that GAG-amyloid interactions display a range of affinities that critically depend on the precise details of the fibril architecture.

Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca 2؉ -ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca 2؉ -ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. switches from an ␣-helix in pure lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca 2؉ -ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins. Phospholamban (PLB)1 is a 52-amino acid membrane-spanning protein, which is expressed predominantly in the sarcoplasmic reticulum of cardiac myocytes (1). The primary physiological function of PLB is to regulate the active transport of calcium ions into the sarcoplasmic reticulum lumen via an inhibitory association with SERCA2a, the cardiac isoform of Ca 2ϩ -ATPase (2, 3). PLB is believed to bind to the calcium-free conformation of SERCA2a (4), and exerts its inhibitory action by reducing the apparent calcium affinity of the enzyme (5). In response to ␤-adrenergic stimulation, PLB is phosphorylated at Ser 16 and Thr 17 by cAMP-dependent kinase and calcium/calmodulin-dependent kinase, respectively. PLB phosphorylation relieves SERCA2a inhibition, which increases the rate of calcium uptake into the sarcoplasmic reticulum and results in the accelerated relaxation of cardiac muscle (2). Mechanistic failures in the reversibility of SERCA2a inhibition, or overexpression of PLB relative to SERCA2a, are linked to the disruption of calcium homeostasis in cardiac cells and may contribute to cardiovascular disorders such as congestive heart failure (6).The exact nature of the interaction between PLB and SERCA enzymes is a subject of debate. PLB readily self-associates to form a homopentamer within the lipid bilayer (7,8), with a dynamic equilibrium believed to exist between the oligomeric and monomeric state of the protein (9). Most evidence suggests that it is the monomeric form of PLB that binds to and inhibits SERCA, with the pentamer acting as a reservoir from which monomers dissociate (5, 8, 10 -12). In reconstitution studies, the molar stoichiometry of PLB to SERCA required for maximal regulation of calcium transport is between 3:1 and 15:1 (12, 13), whereas a fluorescence energy transfer study has suggested that two PLB monomers interact with two Ca 2ϩ ATPase molecules to form a heterodimer (14). In the latter study, it was found that both PLB molecules must be phosphorylated in order t...

Phospholamban (PLB) and phospholemman (PLM, also called FXYD1) are small transmembrane proteins that interact with P-type ATPases and regulate ion transport in cardiac cells and other tissues. This work has investigated the hypothesis that the cytoplasmic domains of PLB and PLM, when not interacting with their regulatory targets, are stabilized through associations with the surface of the phospholipid membrane. Peptides representing the 35 C-terminal cytoplasmic residues of PLM (PLM(37-72)), the 23 N-terminal cytoplasmic residues of PLB (PLB(1-23)), and the same sequence phosphorylated at Ser-16 (P-PLB(1-23)) were synthesized to examine their interactions with model membranes composed of zwitterionic phosphatidylcholine (PC) lipids alone or in admixture with anionic phosphatidylglycerol (PG) lipids. Wide-line 2H NMR spectra of PC/PG membranes, with PC deuterated in the choline moiety, indicated that all three peptides interacted with the membrane surface and perturbed the orientation of the choline headgroups. Fluorescence and 31P magic-angle spinning (MAS) NMR measurements indicated that PLB(1-23) and P-PLB(1-23) had a higher affinity for PC/PG membranes, which carry an overall negative surface charge, than for PC membranes, which have no net surface charge. The 31P MAS NMR spectra of the PC/PG membranes in the presence of PLM(37-72), PLB(1-23), and P-PLB(1-23) indicated that all three peptides induced clustering of the lipids into PC-enriched and PG-enriched regions. These findings support the theory that the cytoplasmic domains of PLB and PLM are stabilized by interacting with lipid headgroups at the membrane surface, and it is speculated that such interactions may modulate the functional properties of biological membranes.

Peptides derived from apolipoprotein A-I (apoA-I), the main component of high-density lipoprotein (HDL), constitute the main component of amyloid deposits that colocalize with atherosclerotic plaques. Here we investigate the molecular details of full-length, lipid-deprived apoA-I after assembly into insoluble aggregates under physiologically relevant conditions known to induce aggregation in vitro. Unmodified apoA-I is shown to remain soluble at pH 7 for at least 3 days, retaining its native α-helical-rich structure. Upon acidification to pH 4, apoA-I rapidly assembles into insoluble nonfibrillar aggregates lacking the characteristic cross-β features of amyloid. In the presence of heparin, the rate and thioflavin T responsiveness of the aggregates formed at pH 4 increase and short amyloid-like fibrils are observed, which give rise to amyloid-characteristic X-ray reflections at 4.7 and 10 Å. Solid-state nuclear magnetic resonance (SSNMR) and synchrotron radiation circular dichroism spectroscopy of fibrils formed in the presence of heparin show they retain some α-helical characteristics together with new β-sheet structures. Interestingly, SSNMR indicates a similar molecular structure of aggregates formed in the absence of heparin at pH 6 after oxidation of the three methionine residues, although their morphology is rather different from that of the heparin-derived fibrils. We propose a model for apoA-I aggregation in which perturbations of a four-helix bundle-like structure, induced by interactions of heparin or methionine oxidation, cause the partially helical N-terminal residues to disengage from the remaining, intact helices, thereby allowing self-assembly via β-strand associations.

The Aβ peptide forms extracellular plaques associated with Alzheimer's disease. In addition to protein fibrils, amyloid plaques also contain non-proteinaceous components, including glycosaminoglycans (GAGs). We have shown previously that the GAG low-molecular-weight heparin (LMWH) binds to Aβ40 fibrils with a three-fold-symmetric (3Q) morphology with higher affinity than Aβ40 fibrils in alternative structures, Aβ42 fibrils, or amyloid fibrils formed from other sequences. Solid-state NMR analysis of the GAG–3Q fibril complex revealed an interaction site at the corners of the 3Q fibril structure, but the origin of the binding specificity remained obscure. Here, using a library of short heparin polysaccharides modified at specific sites, we show that the N-sulfate or 6-O-sulfate of glucosamine, but not the 2-O-sulfate of iduronate within heparin is required for 3Q binding, indicating selectivity in the interactions of the GAG with the fibril that extends beyond general electrostatic complementarity. By creating 3Q fibrils containing point substitutions in the amino acid sequence, we also show that charged residues at the fibril three-fold apices provide the majority of the binding free energy, while charged residues elsewhere are less critical for binding. The results indicate, therefore, that LMWH binding to 3Q fibrils requires a precise molecular complementarity of the sulfate moieties on the GAG and charged residues displayed on the fibril surface. Differences in GAG binding to fibrils with distinct sequence and/or structure may thus contribute to the diverse etiology and progression of amyloid diseases.

Cardiotonic steroids (CTS) are clinically important drugs for the treatment of heart failure owing to their potent inhibition of cardiac Na+, K+-ATPase (NKA). Bufadienolides constitute one of the two major classes of CTS, but little is known about how they interact with NKA. We report a remarkable stereoselectivity of NKA inhibition by native 3β-hydroxy bufalin over the 3α-isomer, yet replacing the 3β-hydroxy group with larger polar groups in the same configuration enhances inhibitory potency. Binding of the two 13C-labelled glycosyl diastereomers to NKA were studied by solid-state NMR (SSNMR), which revealed interactions of the glucose group of the 3β- derivative with the inhibitory site, but much weaker interactions of the 3α- derivative with the enzyme. Molecular docking simulations suggest that the polar 3β-groups are closer to the hydrophilic amino acid residues in the entrance of the ligand-binding pocket than those with α-configuration. These first insights into the stereoselective inhibition of NKA by bufadienolides highlight the important role of the hydrophilic moieties at C3 for binding, and may explain why only 3β-hydroxylated bufadienolides are present as a toxic chemical defence in toad venom.