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The brains of Alzheimer's disease (AD) patients contain numerous amyloid plaques that are diagnostic of the disease. The plaques are primarily composed of the amyloidogenic peptides proteins Aβ40 and Aβ42, which are derived by the processing of the amyloid pre-cursor protein (APP) by two proteases called β-secretase and γ-secretase. Aβ42 differs from Aβ40 in having two additional hydrophobic amino acids, ILE and ALA, at the C-terminus. A small percentage of AD is autosomal dominant (ADAD) and linked either to the genes for the presenilins, which are part of γ-secretase, or APP. Because ADAD shares most pathogenic features with widespread late-onset AD, Aβ peptides have become the focus of AD research. Fibrils formed by the aggregation of these peptides are the major component of plaques and were initially targeted in AD therapy. However, the fact that the abundance of plaques does not correlate well with cognitive decline in AD patients has led investigators to examine smaller Aβ aggregates called oligomers. The low levels and heterogeneity of Aβ oligomers have made the determination of their structures difficult, but recent structure determinations of oligomers either formed or initiated in detergents have been achieved. We report here on the structures of these oligomers and suggest how they may be involved in AD. [ABSTRACT FROM AUTHOR]

Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. In this report, we review recent data showing that decarbamoylation rate constants are independent of the ester leaving group for a series of carbamic acid esters with the same carbamoyl group and that decarbamoylation rate constants decreased by 800-fold when the alkyl substituents on the carbamoyl group increased in size from N -monomethyl- to N , N -diethyl-. We also review data showing that solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N -monomethylcarbamoyl AChE to 1.1 for N , N -diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site in N , N -diethylcarbamoyl AChE. The nature of such a conformational change is suggested from X-ray crystal structures of AChE phosphorylated by paraoxon. • Kinetic study of hydrolysis of carbamoylated acetylcholinesterase (AChE). • Decarbamoylation rate constants k21 decreased with increased carbamoyl group size. • Solvent deuterium oxide isotope effects for k21 decreased with larger carbamoyls. • X-ray crystal structures of AChE phosphorylated by paraoxon suggest mechanism. • The k21 rate-limiting step may shift from proton transfer to conformation change. [ABSTRACT FROM AUTHOR]

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. Although closely related, these enzymes display very different substrate specificities that only partially overlap. This disparity is largely due to differences in the number of aromatic residues lining the active site gorge, which leads to large differences in the shape of the gorge and potentially to distinct interactions with an individual ligand. Considerable structural information is available for the binding of a wide diversity of ligands to AChE. In contrast, structural data on the binding of reversible ligands to BChE are lacking. In a recent effort, an inhibitor competition approach was used to probe the overlap of ligand binding sites in BChE. Here, we extend this study by solving the crystal structures of human BChE in complex with five reversible ligands, namely, decamethonium, thioflavin T, propidium, huprine, and ethopropazine. We compare these structures to equivalent AChE complexes when available in the protein data bank and supplement this comparison with kinetic data and observations from isothermal titration calorimetry. This new information now allows us to define the bindingmode of various ligand families and will be of importance in designing specific reversible ligands of BChE that behave as inhibitors or reactivators. [ABSTRACT FROM AUTHOR]

Natural product inhibitors of AChE are of interest both because they offer promise as inexpensive drugs for symptomatic relief in Alzheimer’s disease and because they may provide insights into the structural features of the AChE catalytic site. Hopeahainol A is an uncharged polyphenol AChE inhibitor from the stem bark of Hopea hainanensis with a constrained, partially dearomatized bicyclic core. Molecular modeling indicates that hopeahainol A binds at the entrance of the long but narrow AChE active site gorge because it is too bulky to be accommodated within the gorge without severe distortion of the gorge as depicted in AChE crystal structures. We conducted inhibitor competition experiments in which AChE inhibition was measured with hopeahainol A together with either edrophonium (which binds at the base of the gorge) or thioflavin T (which binds to the peripheral or P-site near the gorge mouth). The results agreed with the molecular modeling and indicated that hopeahainol A at lower concentrations (<200 μM) bound only to the P-site, as hopeahainol A and thioflavin T were unable to form a ternary complex with AChE while hopeahainol A and edrophonium did form a ternary complex with essentially no competition between them. Inhibition increased to a striking extent at higher concentrations of hopeahainol A, with plots analogous to classic Dixon plots showing a dependence on hopeahainol A concentrations to the third- or fourth order. The inhibition at higher hopeahainol A concentrations was completely reversed on dilution and blocked by bound edrophonium. We hypothesize that bound hopeahainol A induces conformational changes in the AChE active site that allow binding of additional hopeahainol A molecules, a phenomenon that would be unprecedented for a reversible inhibitor that apparently forms no covalent bonds with AChE. [ABSTRACT FROM AUTHOR]

ABSTRACT: Acetyicholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acylated enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex with free AChE (E) and proceed to an acylated enzyme intermediate (EC), which is then hydrolyzed. However, the hydrolysis of EC is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here, we focus on the reaction of carbachol (carbamoylcholine) with AChE. The kinetics and thermodynamics of this reaction are of special interest because carbachol is an isosteric analogue of the physiological substrate acetylcholine. We show that the reaction can be monitored with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. Analysis of the fluorescence reaction profiles was challenging because four thermodynamic parameters and two fluorescence coefficients were fitted from the combined data both for E and for EC. Respective equilibrium dissociation constants of 6 and 26 mM were obtained for carbachol binding to the A- and P-sites in E and of 2 and 32 mM for carbachol binding to the A- and P-sites in EC. These constants for the binding of carbachol to the P-site are about an order of magnitude larger (i.e., indicating lower affinity) than previous estimates for the binding of acetylthiocholine to the P-site. [ABSTRACT FROM AUTHOR]

Abstract: Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme–substrate complex and proceed to an acylated enzyme intermediate which is then hydrolyzed. However, the hydrolysis of the carbamoylated enzyme is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we show that the reaction of carbachol (carbamoylcholine) with AChE can be monitored both with acetylthiocholine as a reporter substrate and with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. These fluorescence changes allow not only the monitoring of the course of the carbamoylation reaction but also the determination of carbachol affinities for the A- and P-sites. [Copyright &y& Elsevier]

Abstract: Acetylcholinesterase (AChE) hydrolyzes its physiological substrate acetylcholine at one of the highest known catalytic rates. Two sites of ligand interaction have been identified: an acylation site or A-site at the base of the active site gorge, and a peripheral site or P-site at its mouth. Despite a wealth of information about the AChE structure and the role of specific residues in catalysis, an understanding of the catalytic mechanism and the role of the P-site has lagged far behind. In recent years we have clarified how the P- and A-sites interact to promote catalysis. Our studies have revealed that the P-site mediates substrate trapping and that ligand binding to the P-site can result in steric blockade of the A-site as well as allosteric activation. We have demonstrated this activation only for the acylation step of the catalytic reaction, but others have proposed that it involves the deacylation step. To investigate this point, we have measured the reaction of carbamoyl esters (carbamates) with AChE. With these slowly hydrolyzed substrates, the carbamoylation (acylation) and decarbamoylation (deacylation) steps can be resolved and analyzed separately. Carbamoylcholine is one of the closest structural analogs of acetylcholine, and we monitored these steps in continuous mixed assays with acetylthiocholine as a reporter substrate. At high concentrations of carbamoylcholine, decarbamoylation was inhibited but no activation of carbamoylation was observed. However, high concentrations of acetylthiocholine had no effect on the decarbamoylation rate constants. We concluded that the binding of acetylthiocholine to the P-site does not activate deacylation reactions. [Copyright &y& Elsevier]

Presents a study on the evidence of the reduction of the association and dissociation rate constants for N-methylacridinium binding to the acetylcholinesterase active site. Binding of the neurotoxin fasciculin 2 to the acetylcholinesterase peripheral site; Fluorescence temperature jump relaxation kinetics; Consequences of fasciculin binding.

Organophosphates (OPs) are esters of substituted phosphates, phosphonates or phosphoramidates that react with acetylcholinesterase (AChE) by initially transferring the organophosphityl group to a serine residue in the enzyme active site, concomitant with loss of an alcohol or halide leaving group. With substituted phosphates, this transfer is followed by relatively slow hydrolysis of the organophosphoryl AChE, or dephosphorylation, that is often accompanied by an aging reaction that renders the enzyme irreversibly inactivated. Aging is a dealkylation that converts the phosphate triester to a diester. OPs are very effective AChE inhibitors and have been developed as insecticides and chemical warfare agents. We examined three reactions of two organophosphoryl AChEs, dimethyl- and diethylphosphorylated AChE, by comparing rate constants and solvent deuterium oxide isotope effects for hydrolysis, aging and oxime reactivation with pralidoxime (2-PAM). Our study was motivated (1) by a published x-ray crystal structure of diethylphosphorylated AChE, which showed severe distortion of the active site that was restored by the binding of pralidoxime, and (2) by published isotope effects for decarbamoylation that decreased from 2.8 for N-monomethylcarbamoyl AChE to 1.1 for N,N-diethylcarbamoyl AChE. We previously reconciled these results by proposing a shift in the rate-limiting step from proton transfer for the small carbamoyl group to a likely conformational change in the distorted active site of the large carbamoyl enzyme. This proposal was tested but was not supported in this report. The smaller dimethylphosphoryl AChE and the larger diethylphosphoryl AChE gave similar isotope effects for both oxime reactivation and hydrolysis, and the isotope effect values of about two indicated that proton transfer was rate limiting for both reactions. [ABSTRACT FROM AUTHOR]

Over recent decades, crystallographic software for data processing and structure refinement has improved dramatically, resulting in more accurate and detailed crystal structures. It is, therefore, sometimes valuable to have a second look at "old" diffraction data, especially when earlier interpretation of the electron density maps was rather difficult. Here, we present updated crystal structures of Drosophila melanogaster acetylcholinesterase (DmAChE) originally published in [Harel et al., Prot Sci (2000) 9:1063-1072], which reveal features previously unnoticed. Thus, previously unmodeled density in the native active site can be interpreted as stable acetylation of the catalytic serine. Similarly, a strong density in the DmAChE/ZA complex originally attributed to a sulfate ion is better interpreted as a small molecule that is covalently bound. This small molecule can be modeled as either a propionate or a glycinate. The complex is reminiscent of the carboxylate butyrylcholinesterase complexes observed in crystal structures of human butyrylcholinesterases from various sources, and demonstrates the remarkable ability of cholinesterases to stabilize covalent complexes with carboxylates. A very strong peak of density (10 σ) at covalent distance from the Cβ of the catalytic serine is present in the DmAChE/ZAI complex. This can be undoubtedly attributed to an iodine atom, suggesting an unanticipated iodo/hydroxyl exchange between Ser238 and the inhibitor, possibly driven by the intense X-ray irradiation. Finally, the binding of tacrine-derived inhibitors, such as ZA (1DX4) or the iodinated analog, ZAI (1QON) results in the appearance of an open channel that connects the base of the active-site gorge to the solvent. This channel, which arises due to the absence of the conserved tyrosine present in vertebrate cholinesterases, could be exploited to design inhibitors specific to insect cholinesterases. The present study demonstrates that updated processing of older diffraction images, and the re-refinement of older diffraction data, can produce valuable information that could not be detected in the original analysis, and strongly supports the preservation of the diffraction images in public data banks. [ABSTRACT FROM AUTHOR]

Amyloid beta (Aβ) aggregation is a slow process without seeding or assisted nucleation. Sodium dodecyl sulfate (SDS) micelles stabilize Aβ42 small oligomers (in the dimer to tetramer range); subsequent SDS removal leads to a 150-kD Aβ42 oligomer. Dodecylphosphorylcholine (DPC) micelles also stabilize an Aβ42 tetramer. Here we investigate the detergent-assisted oligomerization pathway by solid-state NMR spectroscopy and molecular dynamics simulations. SDS- and DPC-induced oligomers have the same structure, implying a common oligomerization pathway. An antiparallel β-sheet formed by the C-terminal region, the only stable structure in SDS and DPC micelles, is directly incorporated into the 150-kD oligomer. Three Gly residues (at positions 33, 37, and 38) create holes that are filled by the SDS and DPC hydrocarbon tails, thereby turning a potentially destabilizing feature into a stabilizing factor. These observations have implications for endogenous Aβ aggregation at cellular interfaces. A study jointly using solid-state NMR spectroscopy and molecular dynamics simulations reveals a common pathway for the detergent-assisted oligomerization of Aβ42. [ABSTRACT FROM AUTHOR]

Oligomeric forms of amyloid-β (Aβ), tau, and TDP-43 play important roles in Alzheimer's disease (AD), and therefore are promising biomarkers. We previously generated single chain antibody fragments (scFvs) that selectively bind disease-related variants of these proteins including A4, C6T, and E1, which bind different oligomeric Aβ variants; D11C, which binds oligomeric tau; and AD-TDP1 and AD-TDP2, which bind disease related TDP-43 variants. To determine the utility of these disease-related variants as early biomarkers, we first analyzed 11 human sera samples obtained ∼2 years prior to an initial mild cognitive impairment (MCI) diagnosis. While the subsequent diagnoses for the cases covered several different conditions, all samples had elevated protein variant levels relative to the plasma controls although with different individual biomarker profiles. We then analyzed a set of longitudinal human plasma samples from four AD (encompassing time points prior to MCI diagnosis and continuing until after conversion to AD) and two control cases. Pre-MCI samples were characterized by high TDP-43 variant levels, MCI samples by high Aβ variant levels, and AD samples by high Aβ and tau variant levels. Sample time points ranged from ∼7 years pre-MCI to ∼9 years after AD conversion. Bivariate correlations showed a negative correlation with TDP-43 levels and positive correlations with cumulative Aβ and oligomeric tau levels indicating an increase in neurodegenerative processes with time in AD. Detection of disease related protein variants not only readily selects AD cases from controls, but also stages progression of AD and holds promise for a pre-symptomatic blood-based biomarker profile for AD. [ABSTRACT FROM AUTHOR]

Abstract: Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) initiate the neurotoxicity that eventually leads to dementia in Alzheimer's disease. Knowledge on soluble aggregate structures will enhance our understanding of the relationship between structures and toxicities. Our group has reported a stable and homogeneous preparation of Aβ(1–42) oligomers that has been characterized by various biophysical techniques. Here, we have further analyzed this species by solid state nuclear magnetic resonance (NMR) spectroscopy and compared NMR results to similar observations on amyloid fibrils. NMR experiments on Aβ(1–42) oligomers reveal chemical shifts of labeled residues that are indicative of β-strand secondary structure. Results from two-dimensional dipolar-assisted rotational resonance experiments indicate proximities between I31 aliphatic and F19 aromatic carbons. An isotope dilution experiment further indicates that these contacts between F19 and I31 are intermolecular, contrary to models of Aβ oligomers proposed previously by others. For Aβ(1–42) fibrils, we observed similar NMR lineshapes and inter-side-chain contacts, indicating similar secondary and quaternary structures. The most prominent structural differences between Aβ(1–42) oligomers and fibrils were observed through measurements of intermolecular 13C–13C dipolar couplings observed in PITHIRDS-CT experiments. PITHIRDS-CT data indicate that, unlike fibrils, oligomers are not characterized by in-register parallel β-sheets. Structural similarities and differences between Aβ(1–42) oligomers and fibrils suggest that folded β-strand peptide conformations form early in the course of self-assembly and that oligomers and fibrils differ primarily in schemes of intermolecular organization. Distinct intermolecular arrangements between Aβ(1–42) oligomers and fibrils may explain why this oligomeric state appears off-pathway for monomer self-assembly to fibrils. [Copyright &y& Elsevier]

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) catalyze the hydrolysis of the neurotransmitter acetylcholine and, thereby, function as coregulators of cholinergic neurotransmission. For both enzymes, hydrolysis takes place neae the bottom of a 20 Å deep active site gorge. A number of amino acid residues within the gorge have been identified as important in facilitating efficient catalysis and inhibitor binding. Of particular interest is the catalytic triad, consisting of serine, histidine, and glutamate residues, that mediates hydrolysis. Another site influencing the catalytic process is located above the catalytic tnad toward the periphery of the active site gorge. This peripheral site (P-site) contains a number of aromatic amino acid residues as well as an aspartate residue that is able to interact with cationic substrates and guide them down the gorge to the catalytic triad. In human AChE, certain aryl residues in the vicinity of the anionic aspartate residue (D74), such as W286, have been implicated in ligand binding and have therefore been considered part of the P-site of the enzyme. The present study was undertaken to explore the P-site of human BuChE and determine whether, like AChE, aromatic side chains near the peripheral aspartate (D70) of this enzyme contribute to ligand binding. Results obtained, utilizing inhibitor competition studies and BuChE mutant species, indicate the participation of aryl residues (F329 and Y332) in the E-helix component of the BuChE active site gorge, along with the anionic aspartate residue (D70), in binding ligands to the P-site of the enzyme. [ABSTRACT FROM AUTHOR]

Abstract: Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to the catalytic efficiency of substrate hydrolysis by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Ligands that bind to the A-site invariably inhibit the hydrolysis of all AChE substrates, but ligands that bind to the P-site inhibit the hydrolysis of some substrates but not others. To clarify the basis of this difference, we focus here on second-order rate constants for substrate hydrolysis (k E), a parameter that reflects the binding of ligands only to the free form of the enzyme and not to enzyme–substrate intermediates. We first describe an inhibitor competition assay that distinguishes whether a ligand is inhibiting AChE by binding to the A-site or the P-site. We then show that the P-site-specific ligand thioflavin T inhibits the hydrolysis of the rapidly hydrolyzed substrate acetylthiocholine but fails to show any inhibition of the slowly hydrolyzed substrates ATMA (3-(acetamido)-N,N,N-trimethylanilinium) and carbachol. We derive an expression for k E that accounts for these observations by recognizing that the rate-limiting steps for these substrates differ. The rate-limiting step for the slow substrates is the general base-catalyzed acylation reaction k 2, a step that is unaffected by bound thioflavin T. In contrast, the rate-limiting step for acetylthiocholine is either substrate association or substrate migration to the A-site, and these steps are blocked by bound thioflavin T. [ABSTRACT FROM AUTHOR]

Mechanisms of altered vagal control in heart failure: influence of muscarinic receptors and acetylcholinesterase activity. Am J Physiol Heart Circ Physiol 285: H1632-H1640, 2003. First published June 26, 2003; 10.1152/ajpheart.01051.2002.—Parasympathetic control of the heart is attenuated in heart failure (HF). We investigated possible mechanisms and sites of altered vagal control in dogs with HF induced by rapid pacing. Muscarinic blockade reduced the R-R interval by 308 ms in controls but only by 32 ms in HF, indicating low levels of resting vagal tone. Vagomimetic doses of atropine sulfate prolonged the R-R interval by 109 ms in controls and increased standard deviation of the R-R interval by 66 ms but only by 46 and 16 ms, respectively, in HF. Bradycardia elicited by electrical stimulation of the vagus nerve was also attenuated in the HF group. Conversely, muscarinic receptor activation by bethanechol, and indirectly by neostigmine, elicited exaggerated R-R interval responses in HF. To investigate possible mechanisms, we measured muscarinic receptor density (B[sub max]) and acetylcholinesterase activity in different areas of the heart. In sinoatrial nodes, B[sub max] was increased (230 ± 75% of control) and acetylcholinesterase decreased (80 ± 6% of control) in HF. We conclude that muscarinic receptors are upregulated and acetylcholinesterase is reduced in the sinus node in HF. Therefore, reduced vagal control in HF is most likely due to changes of presynaptic function (ganglionic), because postsynaptic mechanisms augment vagal control in HF. [ABSTRACT FROM AUTHOR]

Provides information on an experimental study addressing the question of how ligand binding to the peripheral sites alters acetylcholinesterase (AChe) catalytic activity. Methodology used to conduct the experiment; Results of the experiment; Information on simulations of kinetic equations; Discussion on the results.

GlcN(acyl)PtdIns, a derivative of phosphatidylinositol (PtdIns) in which glucosamine and a fatty acid are linked to inositol hydroxyl groups, has been proposed to be an intermediate in the mammalian biosynthetic pathway for glycosylphosphatidylinositol (glycosyl-PtdIns) anchors of membrane proteins. In this report, GlcN(anyl) PtdIns metabolically labeled with [³H]inositol is shown to accumulate in a HeLa S3 cell subline. The amount of GlcN(acyl) PtdIns in these HeLa S3 cells is about 107 molecules/cell, a level comparable to those of the most abundant glycosyl-PtdIns-containing molecules reported to date. GlcN(acyl) PtdIns was purified by a two-step procedure involving octyl-Sepharose and thin-layer chromatography. Octyl-Sepharose separated phospholipids according to their number of hydrocarbon chains; one in 2-lysoPtdIns, two in PtdIns, and three in GlcN(acyl)PtdIns. Purification also was aided by prior treatment of lipid extracts with bee venom phospholipase A2, an enzyme that did ot cleave GleN(acyl) PtdIns. The GlcN-inositol head group in purified GlcN(acyl) PtdIns was confirmed by a number of procedures, including cation-exchange chromatography and mass spectrometry: after radiomethylation, and equal molar ratio of GlcN(Me)2/inositol was measured. Fatty acid analysis indicated an overall stoichiometry of 2.3 mol fatty acid/mol inositol with palmitic (16:0), stearic (18:0) and oleic (18:1) acids being predominant. Analysis of GlcN(anyl) inositol produced by HF fragmentation showed that palmitate was the acyl group attached to inositol and indicated that stearic and olieic acids were in the glycerolipid. Base methanolysis revealed that about 15% of the purified GleN(acyl) PtdIns contained alkylglycerol. A substantial conversion of GlcN(acyl) PtdIns to a slightly more polar lipid occurred after overnight incubation in even mildly alkaline buffers. Although the current data do not allow proposal of a structure of for this lipid, its formation from GlcN(acyl)PtdIns may be important because the conversion appeared to occur in vivo. [ABSTRACT FROM AUTHOR]

We present solid-state NMR measurements of β-strand secondary structure and inter-strand organization within a 150-kDa oligomeric aggregate of the 42-residue variant of the Alzheimer's amyloid-β peptide (Aβ(1–42)). We build upon our previous report of a β-strand spanned by residues 30–42, which arranges into an antiparallel β-sheet. New results presented here indicate that there is a second β-strand formed by residues 11–24. Contrary to expectations, NMR data indicate that this second β-strand is organized into a parallel β-sheet despite the co-existence of an antiparallel β-sheet in the same structure. In addition, the in-register parallel β-sheet commonly observed for amyloid fibril structure does not apply to residues 11–24 in the 150-kDa oligomer. Rather, we present evidence for an inter-strand registry shift of three residues that likely alternate in direction between adjacent molecules along the β-sheet. We corroborated this unexpected scheme for β-strand organization using multiple two-dimensional NMR and 13C–13C dipolar recoupling experiments. Our findings indicate a previously unknown assembly pathway and inspire a suggestion as to why this aggregate does not grow to larger sizes. Unlabelled Image • The assembly pathways and structural details of Aβ oligomers are not understood. • Two β-strand regions were located on the Aβ(1–42) sequence in 150-kDa oligomers. • The N-strand (E11–V24) was characterized to arrange into out-of-register parallel β-sheets. • The out-of-register parallel β-sheets and the antiparallel β-sheets coexist in the oligomer structure. • The co-existence of β-sheets with distinct organizations may differentiate oligomer and fibril assembly pathways. [ABSTRACT FROM AUTHOR]

Background: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid β-protein (Aβ) and the microtubule-associated protein, tau, and has been genetically linked to late-onset Alzheimer disease (AD). Here, we sought to examine the consequences of genetic deletion of CatD on Aβ proteostasis in vivo and to more completely characterize the degradation of Aβ42 and Aβ40 by CatD. Methods: We quantified Aβ degradation rates and levels of endogenous Aβ42 and Aβ40 in the brains of CatD-null (CatD-KO), heterozygous null (CatD-HET), and wild-type (WT) control mice. CatD-KO mice die by ~ 4 weeks of age, so tissues from younger mice, as well as embryonic neuronal cultures, were investigated. Enzymological assays and surface plasmon resonance were employed to quantify the kinetic parameters (KM, kcat) of CatD-mediated degradation of monomeric human Aβ42 vs. Aβ40, and the degradation of aggregated Aβ42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aβ42 and Aβ40, as well as corresponding p3 fragments. Results: Genetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aβ42 and Aβ40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aβ42—all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aβ42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aβ42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aβ42 vis-à-vis Aβ40. Specifically, Aβ42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aβ42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aβ42 vs. Aβ40 also extend to p3 fragments ending at positions 42 vs. 40. Conclusions: Our findings identify CatD as the principal intracellular Aβ-degrading protease identified to date, one that regulates Aβ42/40 ratios via differential degradation of Aβ42 vs. Aβ40. The finding that Aβ42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aβ42 and downstream pathological sequelae in AD. [ABSTRACT FROM AUTHOR]

Aggregated amyloid β (Aβ) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aβ peptides can have antiangiogenic properties, which may contribute to vascular dysfunction in the early stages of the disease process. We have generated data showing that brain endothelial cells (ECs) exposed to toxic Aβ1-42 oligomers can readily enter a senescence phenotype. To determine the effect of Aβ oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aβ1-42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aβ1-42 monomers, and 5 µM Aβ1-42 fibrils, respectively. Brain ECs treated with Aβ1-42 oligomers showed increased senescence-associated β-galactosidase staining and increased senescence-associated p21/p53 expression. Treatment with either Aβ1-42 monomer or Aβ1-42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aβ1-42 oligomer-treated ECs, and these cells showed significantly increased VEGFR-1 expression and decreased VEGFR-2 levels. Overexpression of VEGFR-1 in brain ECs readily induced senescence, suggesting a direct role of VEGFR-1 signaling events in this paradigm. More importantly, small interfering RNA-mediated knockdown of VEGFR-1 expression in brain ECs was able to prevent up-regulation of p21 protein expression and significantly reduced induction of senescence following Aβ1-42 oligomer treatment. Our studies show that exposure to Aβ1-42 oligomers may impair vascular functions by altering VEGFR-1 expression and causing ECs to enter a senescent phenotype. Altered VEGFR expression has been documented in brains of AD patients and suggests that this pathway may play a role in AD disease pathogenesis. These studies suggest that modulating VEGFR-1 expression and signaling events could potentially prevent senescence and rejuvenate EC functions, and provides us with a novel target to pursue for prevention and treatment of cerebrovascular dysfunction in AD. [ABSTRACT FROM AUTHOR]

Abstract Carbamates are esters of substituted carbamic acids that react with acetylcholinesterase (AChE) by initially transferring the carbamoyl group to a serine residue in the enzyme active site accompanied by loss of the carbamate leaving group followed by hydrolysis of the carbamoyl enzyme. This hydrolysis, or decarbamoylation, is relatively slow, and half-lives of carbamoylated AChEs range from 4 min to more than 30 days. Therefore, carbamates are effective AChE inhibitors that have been developed as insecticides and as therapeutic agents. We show here, in contrast to a previous report, that decarbamoylation rate constants are independent of the leaving group for a series of carbamates with the same carbamoyl group. When the alkyl substituents on the carbamoyl group increased in size from N -monomethyl- to N , N -dimethyl-, N -ethyl- N -methyl-, or N , N -diethyl-, the decarbamoylation rate constants decreased by 4-, 70-, and 800-fold, respectively. We suggest that this relationship arises as a result of active site distortion, particularly in the acyl pocket of the active site. Furthermore, solvent deuterium oxide isotope effects for decarbamoylation decreased from 2.8 for N -monomethylcarbamoyl AChE to 1.1 for N , N -diethylcarbamoyl AChE, indicating a shift in the rate-limiting step from general acid-base catalysis to a likely conformational change in the distorted active site. [ABSTRACT FROM AUTHOR]

Accumulation of amyloid-β peptides is a dominant feature in the pathogenesis of Alzheimer's disease; however, it is not clear how individual amyloid-β species accumulate and affect other neuropathological and clinical features in the disease. Thus, we compared the accumulation of N-terminally truncated amyloid-β and full-length amyloid-β, depending on disease stage as well as brain area, and determined how these amyloid-β species respectively correlate with clinicopathological features of Alzheimer's disease. To this end, the amounts of amyloid-β species and other proteins related to amyloid-β metabolism or Alzheimer's disease were quantified by enzyme-linked immunosorbent assays (ELISA) or theoretically calculated in 12 brain regions, including neocortical, limbic and subcortical areas from Alzheimer's disease cases (n = 19), neurologically normal elderly without amyloid-β accumulation (normal ageing, n = 13), and neurologically normal elderly with cortical amyloid-β accumulation (pathological ageing, n = 15). We observed that N-terminally truncated amyloid-β42 and full-length amyloid-β42 accumulations distributed differently across disease stages and brain areas, while N-terminally truncated amyloid-β40 and full-length amyloid-β40 accumulation showed an almost identical distribution pattern. Cortical N-terminally truncated amyloid-β42 accumulation was increased in Alzheimer's disease compared to pathological ageing, whereas cortical full-length amyloid-β42 accumulation was comparable between Alzheimer's disease and pathological ageing. Moreover, N-terminally truncated amyloid-β42 were more likely to accumulate more in specific brain areas, especially some limbic areas, while full-length amyloid-β42 tended to accumulate more in several neocortical areas, including frontal cortices. Immunoprecipitation followed by mass spectrometry analysis showed that several N-terminally truncated amyloid-β42 species, represented by pyroglutamylated amyloid-β11-42, were enriched in these areas, consistent with ELISA results. N-terminally truncated amyloid-β42 accumulation showed significant regional association with BACE1 and neprilysin, but not PSD95 that regionally associated with full-length amyloid-β42 accumulation. Interestingly, accumulations of tau and to a greater extent apolipoprotein E (apoE, encoded by APOE) were more strongly correlated with N-terminally truncated amyloid-β42 accumulation than those of other amyloid-β species across brain areas and disease stages. Consistently, immunohistochemical staining and in vitro binding assays showed that apoE co-localized and bound more strongly with pyroglutamylated amyloid-β11-x fibrils than full-length amyloid-β fibrils. Retrospective review of clinical records showed that accumulation of N-terminally truncated amyloid-β42 in cortical areas was associated with disease onset, duration and cognitive scores. Collectively, N-terminally truncated amyloid-β42 species have spatiotemporal accumulation patterns distinct from full-length amyloid-β42, likely due to different mechanisms governing their accumulations in the brain. These truncated amyloid-β species could play critical roles in the disease by linking other clinicopathological features of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Association of cholinesterase with β-amyloid plaques and tau neurofibrillary tangles in Alzheimer’s disease offers an opportunity to detect disease pathology during life. Achieving this requires development of radiolabelled cholinesterase ligands with high enzyme affinity. Various fluorinated acetophenone derivatives bind to acetylcholinesterase with high affinity, including 2,2,2-trifluoro-1-(3-dimethylaminophenyl)ethanone ( 1 ) and 1-(3- tert -butylphenyl)-2,2,2-trifluoroethanone ( 2 ). Such compounds also offer potential for incorporation of radioactive fluorine ( 18 F) for Positron Emission Tomography (PET) imaging of cholinesterases in association with Alzheimer’s disease pathology in the living brain. Here we describe the synthesis of two meta -substituted chlorodifluoroacetophenones using a Weinreb amide strategy and their rapid conversion to the corresponding trifluoro derivatives through nucleophilic substitution by fluoride ion, in a reaction amenable to incorporating 18 F for PET imaging. In vitro kinetic analysis indicates tight binding of the trifluoro derivatives to cholinesterases. Compound 1 has a K i value of 7 nM for acetylcholinesterase and 1300 nM for butyrylcholinesterase while for compound 2 these values are 0.4 nM and 26 nM, respectively. Tight binding of these compounds to cholinesterase encourages their development for PET imaging detection of cholinesterase associated with Alzheimer’s disease pathology. [ABSTRACT FROM AUTHOR]

Mutations in triggering receptor expressed on myeloid cells 2 (TREM2), which has been proposed to regulate the inflammatory responses and the clearance of apoptotic neurons and/or amyloid-β, are genetically linked to increased risk for late-onset Alzheimer's disease (AD). Interestingly, a missense variant in TREM-like transcript 2 (TREML2), a structurally similar protein encoded by the same gene cluster with TREM2 on chromosome 6, has been shown to protect against AD. However, the molecular mechanisms by which TREM2 and TREML2 regulate the pathogenesis of AD, and their functional relationship, if any, remain unclear. Here, we show that lipopolysaccharide (LPS) stimulation significantly suppressed TREM2 but increased TREML2 expression in mouse brain. Consistent with this in vivo result, LPS or oligomeric amyloid-β treatment down regulated TREM2 but up-regulated TREML2 expression in primary microglia. Most important, modulation of TREM2 or TREML2 levels had opposing effects on inflammatory responses with enhancement or suppression of LPS-induced proinflammatory cytokine gene expression observed on TREM2 or TREML2 down regulation, respectively. In addition, the proliferation of primary microglia was significantly decreased when TREM2 was down regulated, whereas it was increased on TREML2 knockdown. Together, our results suggest that several microglial functions are strictly regulated by TREM2 and TREML2, whose dysfunctions likely contribute to AD pathogenesis by impairing brain innate immunity. Our findings provide novel mechanistic insights into the functions of TREM2 and TREML2 in microglia and have implications on designing new therapeutic strategies to treat AD. [ABSTRACT FROM AUTHOR]

Understanding the molecular structures of amyloid-β (Aβ) oligomers and underlying assembly pathways will advance our understanding of Alzheimer's disease (AD) at the molecular level. This understanding could contribute to disease prevention, diagnosis, and treatment strategies, as oligomers play a central role in AD pathology. We have recently presented a procedure for production of 150-kDa oligomeric samples of Aβ(1-42) (the 42-residue variant of the Aβ peptide) that are compatible with solid-state nuclear magnetic resonance (NMR) analysis, and we have shown that these oligomers and amyloid fibrils differ in intermolecular arrangement of β-strands. Here we report new solid-state NMR constraints that indicate antiparallel intermolecular alignment of β-strands within the oligomers. Specifically, 150-kDa Aβ(1-42) oligomers with uniform 13 C and 15 N isotopic labels at I32, M35, G37, and V40 exhibit β-strand secondary chemical shifts in 2-dimensional (2D) finite-pulse radiofrequency-driven recoupling NMR spectra, spatial proximities between I32 and V40 as well as between M35 and G37 in 2D dipolar-assisted rotational resonance spectra, and close proximity between M35 H α and G37 H α in 2D CHHC spectra. Furthermore, 2D dipolar-assisted rotational resonance analysis of an oligomer sample prepared with 30% labeled peptide indicates that the I32-V40 and M35-G37 contacts are between residues on different molecules. We employ molecular modeling to compare the newly derived experimental constraints with previously proposed geometries for arrangement of Aβ molecules into oligomers. [ABSTRACT FROM AUTHOR]

Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organo-phosphates (OPs) in chemical warfare nerve agents. The resulting accumulation of acetylcholine disrupts cholinergic synaptic transmission and can lead to death. A potential long-term strategy for preventing ACHE inactivation by OPs is based on evidence that OPs must pass through a peripheral site or P-site near the mouth of the AChE active site gorge before reacting with a catalytic serine in an acylation site or A-site at the base of the gorge. An ultimate goal of this strategy is to design compounds that bind tightly at or near the P-site and exclude OPs from the active site while interfering minimally with the passage of acetylcholine. However, to target the AChE P-site with ligands and potential drugs that selectively restrict access, much more information must be gathered about the structure-activity relationships of ligands that bind specifically to the P-site. We apply here an inhibitor competition assay that can correctly determine whether an AChE inhibitor binds to the P-site, the A-site, or both sites. We have used this assay to examine three uncharged, natural product inhibitors of AChE, including aflatoxin Bl, dihydrotanshinone I, and territrem B. The first two of these inhibitors are predicted by the competition assay to bind selectively to the P-site, while territrem B is predicted to span both the P- and A-sites. These predictions have recently been confirmed by X-ray crystallography. Dihydrotanshinone I, with an observed binding constant (K1) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site. [ABSTRACT FROM AUTHOR]

Abstract: Hydrolysis of acetylcholine by acetylcholinesterase (AChE) is extremely rapid, with a second-order hydrolysis rate constant k E (often denoted k cat/K M) that approaches 108 M−1 s−1. AChE contains a deep active site gorge with two sites of ligand binding, an acylation site (or A-site) containing the catalytic triad at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site is known to contribute to catalytic efficiency with acetylthiocholine (AcSCh) by transiently trapping the substrate in a low affinity complex on its way to the A-site, where a short-lived acyl enzyme intermediate is produced. Here we ask whether the P-site does more than simply trap the substrate but in fact selectively gates entry to the A-site to provide specificity for AcSCh (and acetylcholine) relative to the close structural analogs acetyl(homo)thiocholine (Ac-hSCh, which adds one additional methylene group to thiocholine) and acetyl(nor)thiocholine (Ac-nSCh, which deletes one methylene group from thiocholine). We synthesized Ac-hSCh and Ac-nSCh and overcame technical difficulties associated with instability of the northiocholine hydrolysis product. We then compared the catalytic parameters of these substrates with AChE to those of AcSCh. Values of k E for Ac-hSCh and Ac-nSCh were about 2% of that for AcSCh. The k E for AcSCh is close to the theoretical diffusion-controlled limit for the substrate association rate constant, but k E values for Ac-hSCh or Ac-nSCh are too low to be limited by diffusion control. However, analyses of kinetic solvent isotope effects and inhibition patterns for P-site inhibitors indicate that these two analogs also do not equilibrate with the A-site prior to the initial acylation step of catalysis. We propose that k E for these substrates is partially rate-limited by a gating step that involves the movement of bound substrate from the P-site to the A-site. [Copyright &y& Elsevier]

Endogenous amyloid-β (Aβ) oligomeric aggregates have been proposed as toxic agents in Alzheimer's disease (AD). Knowledge of their structures not only may provide insight into the basis of their neurotoxicities but also may reveal new targets for therapeutic drugs and diagnostic tools. However, the low levels of these Aβ oligomers have impeded structural characterization. Evidence suggests that the endogenous oligomers are covalently modified in vivo. In this report, we demonstrate an established mass spectrometry (MS) methodology called precursor ion mapping (PIM) that potentially may be applied to endogenous oligomer characterization. First, we illustrate the use of this PIM technique with a synthetic Aβ(l-40) monomer sample that had been cross-linked with transglutaminase (TGase) and digested with pepsin. From PIM analysis of an Aβ(4-13) MS/MS fragment, precursor ions were identified that corresponded to peptic fragments of three TGase cross-linked species: Aβ(4-19)-(4-19), Aβ(4-19)-(20-34), and Aβ(l-19)-(20-34). Next, we demonstrate the applicability of the PIM technique to an endogenous Aβ sample that had been purified and concentrated by immunoaffinity chromatography. Without pepsin digestion, we successfully identified the full length and C-terminally truncated monomeric Aβ species 1-35 to 1-42, along with select methionine-oxidized counterparts. Because PIM focuses only on a subpopulation of ions, namely the related precursor ions, the resulting spectra are of increased specificity and sensitivity. Therefore, this methodology shows great promise for structural analysis and identification of post-translational modification(s) in endogenous Aβ oligomers. [ABSTRACT FROM AUTHOR]

Abstract: By rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase terminates neurotransmission at cholinergic synapses. Acetylcholinesterase is a very fast enzyme, functioning at a rate approaching that of a diffusion-controlled reaction. The powerful toxicity of organophosphate poisons is attributed primarily to their potent inhibition of acetylcholinesterase. Acetylcholinesterase inhibitors are utilized in the treatment of various neurological disorders, and are the principal drugs approved thus far by the FDA for management of Alzheimer''s disease. Many organophosphates and carbamates serve as potent insecticides, by selectively inhibiting insect acetylcholinesterase. The determination of the crystal structure of Torpedo californica acetylcholinesterase permitted visualization, for the first time, at atomic resolution, of a binding pocket for acetylcholine. It also allowed identification of the active site of acetylcholinesterase, which, unexpectedly, is located at the bottom of a deep gorge lined largely by aromatic residues. The crystal structure of recombinant human acetylcholinesterase in its apo-state is similar in its overall features to that of the Torpedo enzyme; however, the unique crystal packing reveals a novel peptide sequence which blocks access to the active-site gorge. [Copyright &y& Elsevier]

A neuropathological hallmark of Alzheimer's disease (AD) is the presence of large numbers of senile plaques in the brain. These deposits are rich in fibrils that are composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that soluble Aβ aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Aβ aggregates make them difficult to characterize, but several species in extracts of AD brains have been detected by gel electrophoresis in sodium dodecyl sulfate (SDS) and immunoblotting. Individual Aβ oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Aβ have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Aβ aggregates in vitro that resemble the endogenous soluble Aβ aggregates, we previously found that monomeric Aβ(1-42) rapidly forms soluble oligomers in the presence of dilute SDS micelles. Here we extend this work in two directions. First, we contrast the size and secondary structure of these oligomers with those of synthetic Aβ(1-42) fibrils. SEC and multiangle light scattering were used to obtain a molecular mass of 150 kDa for the isolated oligomers. The oligomers partially dissociated to monomers through nonamers when incubated with SDS, but in contrast to endogenous oligomers, we saw no evidence of these discrete species prior to SDS treatment. One hypothesis to explain this difference is that endogenous oligomers are stabilized by covalent cross-linking induced by unknown cellular agents. To explore this hypothesis, optimal mass spectrometry (MS) analysis procedures need to be developed for Aβ cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Aβ(1-42) with three cross-linking agents: transglutamitiase, glutaraldehyde, and Cu(II) with peroxide. We compared the efficiency of covalent cross-linking with these agents, the effect of cross-linking on peptide secondary structure, the stability of the cross-linked structures to thermal unfolding, and the sites of peptide cross-linking obtained from proteolysis and MS. [ABSTRACT FROM AUTHOR]

Acetylcholinesterase plays a key role in cholinergic synaptic transmission by hydrolyzing the neurotransmitter acetylcholine with one of the highest known catalytic rate constants. Hydrolysis occurs in a narrow and deep gorge that contains two sites of ligand binding: A peripheral site, or P-site, near the gorge entrance that contributes to catalytic efficiency both by transiently trapping substrate molecules as they enter the gorge and by allosterically accelerating the transfer of the substrate acyl group to a serine hydroxyl in an acylation site or A-site at the base of the gorge. Thioflavin T is a useful reporter of ligand interactions with the A-site. It binds specifically to the P-site with fluorescence that is enhanced ~1000-fold over that of unbound thioflavin T, and the enhanced fluorescence is quenched 1.5- to 4-fold when another ligand binds to the A-site in a ternary complex. To clarify the structural basis of this advantageous signal change, we here report the X-ray structure of the complex of thioflavin T with Torpedo californica acetylcholinesterase. The two aromatic rings in thioflavin T are coplanar and are packed snugly parallel to the aromatic side chains of Trp279, Tyr334, and Phe330. Overlays of this structure with the crystal structures of Torpedo californica acetyicholinesterase complexes with either edrophonium or m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone, two small aromatic ligands that bind specifically to the A-site, indicate that the phenyl side chain of Phe330 must rotate to sterically accommodate both thioflavin T and the A-site ligand in the ternary complex. This rotation may allow some relaxation of the strict coplanarity of the aromatic rings in the bound thioflavin T and result in partial quenching of its fluorescence. [ABSTRACT FROM AUTHOR]

Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid β precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimer's disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Aβ1- 40 transgenes in APP mouse models. Expression of BRI2-Aβ1- 40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild-type human BRI2 reduces cerebral Aβ deposition in an AD mouse model. Additional data indicate that the 23 aa peptide, Bri23, released from BRI2 by normal processing, is present in human CSF, inhibits Aβ aggregation in vitro and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Aβ deposition in vivo. [ABSTRACT FROM AUTHOR]

Alzheimer' s disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-β (Aβ) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Aβ species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Aβ are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Aβ, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Aβ aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Aβ aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Aβ(1—40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Aβ(1—42) undergoes an immediate conversion to a predominant β-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8—14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30—60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Aβ aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Aβ aggregation procedures. [ABSTRACT FROM AUTHOR]

Neurofibrillary tangles (NFTs) are a pathological hallmark of Alzheimer's disease and other tauopathies, but recent studies in a conditional mouse model of tauopathy (rTg4510) have suggested that NFT formation can be dissociated from memory loss and neurodegeneration. This suggests that NFTs are not the major neurotoxic tau species, at least during the early stages of pathogenesis. To identify other neurotoxic tau protein species, we performed biochemical analyses on brain tissues from the rTg4510 mouse model and then correlated the levels of these tau proteins with memory loss. We describe the identification and characterization of two forms of tau multimers (140 and 170 kDa), whose molecular weight suggests an oligomeric aggregate, that accumulate early in the pathogenic cascade in this mouse model. Similar tau multimers were detected in a second mouse model of tauopathy (JNPL3) and in tissue from patients with Alzheimer's disease and FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17). Moreover, levels of the tau multimers correlated consistently with memory loss at various ages in the rTg4510 mouse model. Our findings suggest that accumulation of early-stage aggregated tau species, before the formation of NFT, is associated with the development of functional deficits during the pathogenic progression of tauopathy. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that fibrillar Aβ and especially soluble Aβ aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Aβ aggregates generated from monomeric Aβ in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily β-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Aβ aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Aβ aggregation have found that amphiphilic interactions and micellar-like Aβ structures may play a role. We previously described the formation of Aβ(1–40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Aβ(1–40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Aβ(1–40) was introduced. With HFIP, the change involved an increase in predominant/3-structure content and in fluorescence with thioflavin T, while with SDS, a partial α-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in β-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Aβ(1–40) monomers effectively. [ABSTRACT FROM AUTHOR]

The brains of Alzheimer's disease (AD) patients contain large numbers of amyloid plaques that are rich in fibrils composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that fibrillar Aβ and especially soluble Aβ aggregates are important in the etiology of AD. Recent reports also stress that amyloid aggregates are polymorphic and that a single polypeptide can fold into multiple amyloid conformations. Here we demonstrate that Aβ-(1-40) can form soluble aggregates with predominant β-structures that differ in stability and morphology. One class of aggregates involved soluble Aβ protofibrils, prepared by vigorous overnight agitation of monomeric Aβ-(1-40) at low ionic strength. Dilution of these aggregation reactions induced disaggregation to monomers as measured by size exclusion chromatography. Protofibril concentrations monitored by thioflavin T fluorescence decreased in at least two kinetic phases, with initial disaggregation (rate constant ∼1 h-1) followed by a much slower secondary phase. Incubation of the reactions without agitation resulted in less disaggregation at slower rates, indicating that the protofibrils became progressively more stable over time. In fact, protofibrils isolated by size exclusion chromatography were completely stable and gave no disaggregation. A second class of soluble Aβ aggregates was generated rapidly (<10 min) in buffered 2% hexafluoroisopropanol (HFIP). These aggregates showed increased thioflavin T fluorescence and were rich in β-structure by circular dichroism. Electron microscopy and atomic force microscopy revealed initial globular clusters that progressed over several days to soluble fibrous aggregates. When diluted out of HFIP, these aggregates initially were very unstable and disaggregated completely within 2 min. However, their stability increased as they progressed to fibers. Relative to Aβ protofibrils, the HFIP-induced aggregates seeded elongation by Aβ monomer deposition very poorly. The techniques used to distinguish these two classes of soluble Aβ aggregates may be useful in characterizing Aβ aggregates formed in vivo. [ABSTRACT FROM AUTHOR]

Accumulation of aggregated amyloid-β peptide (Aβ) in the brain is a pathological hallmark of Alzheimer' s disease (AD). In vitro studies indicate that the 40- to 42-residue Aβ peptide in solution will undergo self-assembly leading to the transient appearance of soluble protofibrils and ultimately to insoluble fibrils. The Aβ peptide is amphiphilic and accumulates preferentially at a hydrophilic/hydrophobic interface. Solid surfaces and air-water interfaces have been shown previously to promote Aβ aggregation, but detailed characterization of these aggregates has not been presented. In this study Aβ(1-40) introduced to aqueous buffer in a two-phase system with chloroform aggregated 1-2 orders of magnitude more rapidly than Aβ in the buffer alone. The interface-induced aggregates were released into the aqueous phase and persisted for 24-72 h before settling as a visible precipitate at the interface. Thioflavin T fluorescence and circular dichroism analyses confirmed that the Aβ aggregates had a β-sheet secondary structure. However, these aggregates were far less stable than Aβ(1 -40) protofibrils prepared in buffer alone and disaggregated completely within 3 min on dilution. Atomic force microscopy revealed that the aggregates consisted of small globules 4-5 nm in height and long flexible fibers composed of these globules aligned roughly along a longitudinal axis, a morphology distinct from that of Aβ protofibrils prepared in buffer alone. The relative instability of the fibers was supported by fiber interruptions apparently introduced by brief washing of the AFM grids. To our knowledge, unstable aggregates of Aβ with β-sheet structure and fibrous morphology have not been reported previously. Our results provide the clearest evidence yet that the intrinsic β-sheet structure of an in vitro Aβ aggregate depends on the aggregation conditions and is reflected in the stability of the aggregate and the morphology observed by atomic force microscopy. Resolution of these structural differences at the molecular level may provide important clues to the further understanding of amyloid formation in vivo. [ABSTRACT FROM AUTHOR]

The acetylcholinesterase (ACHE) active site consists of a narrow gorge with two separate ligand binding sites: an acylation site (or A-site) at the bottom of the gorge where substrate hydrolysis occurs and a peripheral site (or P-site) at the gorge mouth. AChE is inactivated by organophosphates as they pass through the P-site and phosphorylate the catalytic serine in the A-site. One strategy to protect against organophosphate inactivation is to design cyclic ligands that will bind specifically to the P-site and block the passage of organophosphates but not acetylcholine. To accelerate the process of identifying cyclic compounds with high affinity for the AChE P-site, we introduced a cysteine residue near the rim of the P-site by site-specific mutagenesis to generate recombinant human H287C ACHE. Compounds were synthesized with a highly reactive methanethiosulfonyl substituent and linked to this cysteine through a disulfide bond. The advantages of this tethering were demonstrated with H287C AChE modified with six compounds, consisting of cationic trialkylammonium, acridinium, and tacrine ligands with tethers of varying length. Modification by ligands with short tethers had little effect on catalytic properties, but longer tethering resulted in shifts in substrate hydrolysis profiles and reduced affinity for acridinium affinity resin. Molecular modeling calculations indicated that cationic ligands with tethers of intermediate length bound to the P-site, whereas those with long tethers reached the A-site. These binding locations were confirmed experimentally by measuring competitive inhibition constants K[sub I2] for propidium and tactine, inhibitors specific for the P- and A-sites, respectively. Values of K[sub I2] for propidium increased 30- to 100-fold when ligands had either intermediate or long tethers. In contrast, the value of K[sub I2] for tacrine increased substantially only when ligands had long tethers. These relative changes in propidium and tactine affinities thus provided a sensitive molecular ruler for assigning the binding locations of the tethered cations. [ABSTRACT FROM AUTHOR]

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a > 1 give rise to a hydrolysis profile called substrate activation, and the AChE sitespecific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a[sub 2] of 1.3, while propidium failed to accelerate. Second,... [ABSTRACT FROM AUTHOR]

Examines the growth of amyloid plaques in brain tissue. Impact of sodium chloride on protofibril elongation; Determination of the binding of thioflavine T to amyloid; Protofibril growth of monomer.

Studies the hydrogen bonds at the active site of human acetylcholinesterase. Interaction of trifluoroacetophenone (TMTFA) with AChE; Interaction of paraoxon with AChE; Effect of pH on the deshielded resonances of AChE; Interaction of AChE with NPMP.

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G2a form. In Triton X-100 extracts of the subline K562–243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562–243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562–48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562–48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766–18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503–508]. In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. The sialidase-resistant G1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented. [ABSTRACT FROM AUTHOR]

Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18 766-18 775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors. [ABSTRACT FROM AUTHOR]

The article provides information on the findings of a study on the mechanisms of action of anti-Aβ antibodies in Alzheimer's disease mouse model. A stable monoclonal antibodies (mAbs):Aβ complex is created by peripheral administration of anti-mAb in the plasma. The half-life of plasma Aβ is prolonged from minutes to days by binding mAbs to Aβ.

Abstract: The active site gorge of acetylcholinesterase (AChE) contains two distinct sites of ligand interaction: an acylation site (A-site) at the base of the gorge and a peripheral site (P-site) near the mouth of the gorge at the enzyme surface. While the role of the P-site in catalysis has been elusive, our recent studies have shown that it serves as an intermediate binding site for cationic ligands as they proceed to the A-site. P-site binding also can lead to allosteric activation of the acylation step and/or to steric blockade, defined as an equal reduction in the association and dissociation rate constants for the binding of a second ligand to the A-site when the P-site is occupied. Compelling evidence that allosteric activation occurs at the acylation step came from examination of the hydrolysis of a close substrate analog of acetylcholine, the acetanilide ATMA [J.L. Johnson, B. Cusack, M.P. Davies, A. Fauq, T.L. Rosenberry, Biochemistry 42 (2003) 5438–5452]. The acylation step in the AChE hydrolysis of ATMA can be isolated because of the slow cleavage of the anilide bond, revealing the positive deviation from Michealis-Menten kinetics that defines substrate activation. However, not all substrates that bind to the P-site show allosteric activation of acylation. In fact, the only substrates that we have identified thus far are ATMA and, to a lesser extent, acetylthiocholine. Carbamates are also poor substrates of AChE, but they differ from ATMA in that both the formation and hydrolysis of the intermediate acyl enzyme are slow. Thus, the hydrolysis of carbamates by AChE can be resolved into two distinct catalytic steps, carbamoylation and decarbamoylation. The ability to kinetically isolate these two catalytic steps also allows us to investigate which step, if any, is subject to allosteric activation. [Copyright &y& Elsevier]

Protection of the enzyme acetylcholinesterase (AChE) from the toxic effects of organophosphate insecticides and chemical warfare agents (OPs) may be provided by inhibitors that bind at the peripheral binding site (P-site) near the mouth of the active-site gorge. Compounds that bind to this site may selectively block access to the acylation site (A-site) catalytic serine for OPs, but not acetylcholine itself. To identify such compounds, we employed a virtual screening approach using A utoD ock 4.2 and AutoDock Vina, confirmed using compounds experimentally known to bind specifically to either the A-site or P-site. Both programs demonstrated the ability to correctly predict the binding site. Virtual screening of the NCI Diversity Set II was conducted using the apo form of the enzyme, and with acetylcholine bound at the crystallographic locations in the A-site only and in both and A- and P-sites. The docking identified 32 compounds that were obtained for testing, and one was demonstrated to bind specifically to the P-site in an inhibitor competition assay. [ABSTRACT FROM AUTHOR]