Oligomer Formation by Amyloid-ß42 in a Membrane-Mimicking Environment in Alzheimer's Disease.

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 Aß40 and Aß42, which are derived by the processing of the amyloid pre-cursor protein (APP) by two proteases called ß-secretase and γ-secretase. Aß42 differs from Aß40 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, Aß 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 Aß aggregates called oligomers. The low levels and heterogeneity of Aß 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.

VEGF receptor-1 modulates amyloid ß 1-42 oligomer-induced senescence in brain endothelial cells.

Aggregated amyloid ß (Aß) peptides in the Alzheimer's disease (AD) brain are hypothesized to trigger several downstream pathologies, including cerebrovascular dysfunction. Previous studies have shown that Aß 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 Aß1-42 oligomers can readily enter a senescence phenotype. To determine the effect of Aß oligomers on brain ECs, we treated early passaged human brain microvascular ECs and HUVECs with high MW Aß1-42 oligomers (5 µM, for 72 h). For controls, we used no peptide treatment, 5 µM Aß1-42 monomers, and 5 µM Aß1-42 fibrils, respectively. Brain ECs treated with Aß1-42 oligomers showed increased senescence-associated ß-galactosidase staining and increased senescence-associated p21/p53 expression. Treatment with either Aß1-42 monomer or Aß1-42 fibrils did not induce senescence in this assay. We then measured vascular endothelial growth factor receptor (VEGFR) expression in the Aß1-42 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 Aß1-42 oligomer treatment. Our studies show that exposure to Aß1-42 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.-Angom, R. S., Wang, Y., Wang, E., Pal, K., Bhattacharya, S., Watzlawik, J. O., Rosenberry, T. L., Das, P., Mukhopadhyay, D. VEGF receptor-1 modulates amyloid ß 1-42 oligomer-induced senescence in brain endothelial cells.

Solvent Deuterium Oxide Isotope Effects on the Reactions of Organophosphorylated Acetylcholinesterase.

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.

A Second Look at the Crystal Structures of Drosophila melanogaster Acetylcholinesterase in Complex with Tacrine Derivatives Provides Insights Concerning Catalytic Intermediates and the Design of Specific Insecticides.

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 Cß 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.

Decarbamoylation of acetylcholinesterases is markedly slowed as carbamoyl groups increase in size.

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.

Distinct spatiotemporal accumulation of N-truncated and full-length amyloid-ß42 in Alzheimer's disease.

Accumulation of amyloid-ß peptides is a dominant feature in the pathogenesis of Alzheimer's disease; however, it is not clear how individual amyloid-ß species accumulate and affect other neuropathological and clinical features in the disease. Thus, we compared the accumulation of N-terminally truncated amyloid-ß and full-length amyloid-ß, depending on disease stage as well as brain area, and determined how these amyloid-ß species respectively correlate with clinicopathological features of Alzheimer's disease. To this end, the amounts of amyloid-ß species and other proteins related to amyloid-ß 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-ß accumulation (normal ageing, n = 13), and neurologically normal elderly with cortical amyloid-ß accumulation (pathological ageing, n = 15). We observed that N-terminally truncated amyloid-ß42 and full-length amyloid-ß42 accumulations distributed differently across disease stages and brain areas, while N-terminally truncated amyloid-ß40 and full-length amyloid-ß40 accumulation showed an almost identical distribution pattern. Cortical N-terminally truncated amyloid-ß42 accumulation was increased in Alzheimer's disease compared to pathological ageing, whereas cortical full-length amyloid-ß42 accumulation was comparable between Alzheimer's disease and pathological ageing. Moreover, N-terminally truncated amyloid-ß42 were more likely to accumulate more in specific brain areas, especially some limbic areas, while full-length amyloid-ß42 tended to accumulate more in several neocortical areas, including frontal cortices. Immunoprecipitation followed by mass spectrometry analysis showed that several N-terminally truncated amyloid-ß42 species, represented by pyroglutamylated amyloid-ß11-42, were enriched in these areas, consistent with ELISA results. N-terminally truncated amyloid-ß42 accumulation showed significant regional association with BACE1 and neprilysin, but not PSD95 that regionally associated with full-length amyloid-ß42 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-ß42 accumulation than those of other amyloid-ß 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-ß11-x fibrils than full-length amyloid-ß fibrils. Retrospective review of clinical records showed that accumulation of N-terminally truncated amyloid-ß42 in cortical areas was associated with disease onset, duration and cognitive scores. Collectively, N-terminally truncated amyloid-ß42 species have spatiotemporal accumulation patterns distinct from full-length amyloid-ß42, likely due to different mechanisms governing their accumulations in the brain. These truncated amyloid-ß species could play critical roles in the disease by linking other clinicopathological features of Alzheimer's disease.

Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study.

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 binding mode of various ligand families and will be of importance in designing specific reversible ligands of BChE that behave as inhibitors or reactivators.

Development of acetophenone ligands as potential neuroimaging agents for cholinesterases.

Association of cholinesterase with ß-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 (18F) 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 18F for PET imaging. In vitro kinetic analysis indicates tight binding of the trifluoro derivatives to cholinesterases. Compound 1 has a Ki value of 7nM for acetylcholinesterase and 1300nM for butyrylcholinesterase while for compound 2 these values are 0.4nM and 26nM, respectively. Tight binding of these compounds to cholinesterase encourages their development for PET imaging detection of cholinesterase associated with Alzheimer's disease pathology.

Substrate-targeting gamma-secretase modulators.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.

The natural product dihydrotanshinone I provides a prototype for uncharged inhibitors that bind specifically to the acetylcholinesterase peripheral site with nanomolar affinity.

Cholinergic synaptic transmission often requires extremely rapid hydrolysis of acetylcholine by acetylcholinesterase (AChE). AChE is inactivated by organophosphates (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 B1, 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 (KI) of 750 nM, provides a good lead compound for the development of high-affinity, uncharged inhibitors with specificity for the P-site.

A common pathway for detergent-assisted oligomerization of Aß42.

Amyloid beta (Aß) aggregation is a slow process without seeding or assisted nucleation. Sodium dodecyl sulfate (SDS) micelles stabilize Aß42 small oligomers (in the dimer to tetramer range); subsequent SDS removal leads to a 150-kD Aß42 oligomer. Dodecylphosphorylcholine (DPC) micelles also stabilize an Aß42 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 ß-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 Aß aggregation at cellular interfaces.

Probing the peripheral site of human butyrylcholinesterase.

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 near 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 triad 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.

A mass spectrometric approach for characterization of amyloid-ß aggregates and identification of their post-translational modifications.

Endogenous amyloid-ß (Aß) 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 Aß 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 Aß(1-40) monomer sample that had been cross-linked with transglutaminase (TGase) and digested with pepsin. From PIM analysis of an Aß(4-13) MS/MS fragment, precursor ions were identified that corresponded to peptic fragments of three TGase cross-linked species: Aß(4-19)--(4-19), Aß(4-19)--(20-34), and Aß(1-19)--(20-34). Next, we demonstrate the applicability of the PIM technique to an endogenous Aß sample that had been purified and concentrated by immunoaffinity chromatography. Without pepsin digestion, we successfully identified the full length and C-terminally truncated monomeric Aß 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 Aß oligomers.

Out-of-Register Parallel ß-Sheets and Antiparallel ß-Sheets Coexist in 150-kDa Oligomers Formed by Amyloid-ß(1-42).

We present solid-state NMR measurements of ß-strand secondary structure and inter-strand organization within a 150-kDa oligomeric aggregate of the 42-residue variant of the Alzheimer's amyloid-ß peptide (Aß(1-42)). We build upon our previous report of a ß-strand spanned by residues 30-42, which arranges into an antiparallel ß-sheet. New results presented here indicate that there is a second ß-strand formed by residues 11-24. Contrary to expectations, NMR data indicate that this second ß-strand is organized into a parallel ß-sheet despite the co-existence of an antiparallel ß-sheet in the same structure. In addition, the in-register parallel ß-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 ß-sheet. We corroborated this unexpected scheme for ß-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.

Cathepsin D regulates cerebral Aß42/40 ratios via differential degradation of Aß42 and Aß40.

BACKGROUND: Cathepsin D (CatD) is a lysosomal protease that degrades both the amyloid ß-protein (Aß) 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 Aß proteostasis in vivo and to more completely characterize the degradation of Aß42 and Aß40 by CatD. METHODS: We quantified Aß degradation rates and levels of endogenous Aß42 and Aß40 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 Aß42 vs. Aß40, and the degradation of aggregated Aß42 species was also characterized. Competitive inhibition assays were used to interrogate the relative inhibition of full-length human and mouse Aß42 and Aß40, as well as corresponding p3 fragments. RESULTS: Genetic deletion of CatD resulted in 3- to 4-fold increases in insoluble, endogenous cerebral Aß42 and Aß40, exceeding the increases produced by deletion of an insulin-degrading enzyme, neprilysin or both, together with readily detectable intralysosomal deposits of endogenous Aß42-all by 3 weeks of age. Quite significantly, CatD-KO mice exhibited ~ 30% increases in Aß42/40 ratios, comparable to those induced by presenilin mutations. Mechanistically, the perturbed Aß42/40 ratios were attributable to pronounced differences in the kinetics of degradation of Aß42 vis-à-vis Aß40. Specifically, Aß42 shows a low-nanomolar affinity for CatD, along with an exceptionally slow turnover rate that, together, renders Aß42 a highly potent competitive inhibitor of CatD. Notably, the marked differences in the processing of Aß42 vs. Aß40 also extend to p3 fragments ending at positions 42 vs. 40. CONCLUSIONS: Our findings identify CatD as the principal intracellular Aß-degrading protease identified to date, one that regulates Aß42/40 ratios via differential degradation of Aß42 vs. Aß40. The finding that Aß42 is a potent competitive inhibitor of CatD suggests a possible mechanistic link between elevations in Aß42 and downstream pathological sequelae in AD.

Biophysical analyses of synthetic amyloid-beta(1-42) aggregates before and after covalent cross-linking. Implications for deducing the structure of endogenous amyloid-beta oligomers.

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-beta (Abeta) peptides. Several lines of evidence indicate that soluble Abeta aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Abeta 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 Abeta oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Abeta have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Abeta aggregates in vitro that resemble the endogenous soluble Abeta aggregates, we previously found that monomeric Abeta(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 Abeta(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 Abeta cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Abeta(1-42) with three cross-linking agents: transglutaminase, 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.

Rate-limiting step in the decarbamoylation of acetylcholinesterases with large carbamoyl groups.

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.

Accumulation of pathological tau species and memory loss in a conditional model of tauopathy.

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.

Analysis of the reaction of carbachol with acetylcholinesterase using thioflavin T as a coupled fluorescence reporter.

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 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.

Crystal structure of thioflavin T bound to the peripheral site of Torpedo californica acetylcholinesterase reveals how thioflavin T acts as a sensitive fluorescent reporter of ligand binding to the acylation site.

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 approximately 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 acetylcholinesterase 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.