Examination of the N-terminal sequence of non-Aβ component of Alzheimer's Disease amyloid (NAC) revealed a degree of similarity to regions crucial for aggregation and toxicity of three other amyloidogenic proteins, namely amyloid β peptide (Aβ), prion protein (PrP) and islet amyloid polypeptide (IAPP), leading us to believe that this might be the part of the molecule responsible for causing aggregation. Secondary structure prediction analysis of NAC indicated that the N-terminal half was likely to form a β-structure whereas the C-terminal half was likely to form an A-helix. NAC in solution altered from random coil to β-sheet structure upon ageing, a process that has previously been shown to lead to fibril formation. To delineate the region of NAC responsible for aggregation we synthesised two fragments, NAC-(1Ϫ18)-peptide and NAC-(19Ϫ35)-peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC-(1Ϫ18)-peptide became congophilic and aggregated to form fibrils of diameter 5Ϫ10 nm, whereas NAC-(19Ϫ35)-peptide did not bind Congo Red and remained in solution. Circular dichroism spectroscopy was used to study the secondary structure of NAC and the two fragments. In trifluoroethanol/water mixtures, NAC and NAC-(19Ϫ35)-peptide adopted A-helical structure but NAC-(1Ϫ18)-peptide did not. NAC-(1Ϫ18)-peptide and NAC formed β-sheet in acetonitrile/water mixtures more readily than did NAC-(19Ϫ35)-peptide. CD spectra of NAC or NAC-(1Ϫ18)-peptide in aqueous solution indicate the formation of β-sheet on ageing. We propose that the N-terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles Aβ, and other amyloidogenic proteins, in that aggregation is dependent upon β-sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.
The non-beta-amyloid (Ab) component of Alzheimer's disease amyloid (NAC) and its precursor a-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and a-synuclein both form b-sheet structures upon ageing, aggregate to form ®brils, and are neurotoxic. We recently established that a peptide comprising residues 3±18 of NAC retains these properties. To pinpoint the exact region responsible we have carried out assays of toxicity and physicochemical properties on smaller fragments of NAC. Toxicity was measured by the ability of fresh and aged peptides to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by rat pheochromocytoma PC12 cells and human neuroblastoma SHSY-5Y cells. On immediate dissolution, or after ageing, the fragments NAC(8±18) and NAC(8±16) are toxic, whereas NAC(12±18), NAC(9±16) and NAC(8±15) are not. Circular dichroism indicates that none of the peptides displays b-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce b sheet, fragments NAC(8±18) and NAC(8±16) both form b-sheet structure. Only NAC(8±18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms ®brils, as determined by electron microscopy. These ®ndings indicate that residues 8±16 of NAC, equivalent to residues 68±76 in a-synuclein, comprise the region crucial for toxicity.
The non-Ab component of Alzheimer's disease amyloid (NAC) and its precursor a-synuclein have been linked to amyloidogenesis in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Previously we have shown that NAC forms b-sheet structures and fibrils [El-Agnaf, O.M.A., Bodles, A.M., Guthrie, D.J.S., Harriott, P. & Irvine, G.B. (1998) Eur. J. Biochem. 258, 157±163]. As a measure of their neurotoxic potential we have examined the ability of fresh and aged NAC and fragments thereof to inhibit the reduction of the redox dye 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide by rat pheochromocytoma PC12 cells. Micromolar concentrations of NAC and fragments thereof display varying degrees of toxicity. On immediate dissolution and after an incubation period for 3 days at 37 8C the full-length peptide and fragments NAC(3±18) and NAC(1±18) scrambled sequence [NAC(1±18 s)] were toxic, whereas fragments NAC(1±13) and NAC(6±14) were not. CD indicates that NAC(3±18) and NAC(1±18 s) exhibit b-sheet secondary structure in aqueous solution, whereas NAC(1±13) and NAC(6±14) do not. NAC(3±18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days measured by an HPLC assay, and forms fibrils, as determined by electron microscopy. However, although some fibrils were detected for NAC(1±18 s) it does not come out of solution to a significant degree. Fragments NAC(1±13) and NAC(6±14) form few fibrils and remain in solution. These findings indicate that the ability of the central region of NAC to form b-sheet secondary structures is important for determining the toxicity of the peptide. This contrasts with what has been reported previously for most Ab peptides as their toxicity appears to require the peptide to have formed fibrillary aggregates as well as displaying b-sheet. These results suggest that an intermediate, which exhibits b-sheet structure, may be responsible for the toxic properties of NAC and provides further evidence for the role of NAC in the pathogenesis of AD, PD and DLB.
Alzheimer's β-amyloid peptide (Aβ) is a 39-to 43-amino-acid peptide that is the major component of neuritic plaques found in Alzheimer's disease (AD). The central region of Aβ plays a crucial role in many of its properties, including aggregation, neurotoxicity, proteolytic processing and interactions with other proteins, such as apolipoprotein E. Two mutations in this region, Ala21→Gly and Glu22→Gln, give rise to early onset forms of disease. We have studied several peptides based on the central region of Aβ in order to clarify the influence of specific amino acid residues on physicochemical behaviour. To avoid difficulties due to oxidation of Met35, the latter was replaced by the amino acid isostere, norleucine (Ahx), giving [Ahx35]Aβ-(25Ϫ35)-amide as a prototype structure. To this prototype, addition of pairs of amino acid residues from the sequence of Aβ, forming the corresponding 23-, 21-and 19Ϫ35 derivatives, resulted in peptides that aggregated to form fibrils of diameter 6Ϫ10 nm. The rate of aggregation was more rapid as peptide length increased. Circular dichroism spectra of aged solutions of peptides revealed that aggregation was accompanied by a transition from random structure to β sheet for some, but not all, peptides. The mutation from Ala to Gly at position 21 increased the rate of aggregation and altered the tendency to adopt secondary structure in the direction away from A helix and towards β sheet. In individuals with the Ala21→Gly mutation, these results would suggest that truncated species with N-termini in the region containing residues 17Ϫ20 would be more amyloidogenic than the wild type homologues.
Thionoleucine S-anilide (Leut-anilide), Leut-Gly-OEt and Leut-Phe-OMe were synthesized and shown to be competitive inhibitors of leucine aminopeptidase from pig kidney. The kinetics of inhibition were determined in the presence of leucine 4-methylcoumarin-7-amide as substrate. Although the compounds showed only moderate inhibitory potency, it was found that all were resistant to hydrolysis by the enzyme, in contrast with the reported behaviour of some thionopeptide analogues of substrates for other Zn2+-peptidases such as carboxypeptidase A and angiotensin-converting enzyme.
1. A 1H-n.m.r. method was used to measure concentrations of valine, alanine, lactate, acetate, hyaluronan and lipids in synovial fluid obtained, during the normal course of examination from the knee joints of patients attending rheumatology and orthopaedic clinics. Fluid was available from 16 patients with osteoarthritis, 18 patients with rheumatoid arthritis, four patients with meniscal tear and one patient each with systemic lupus erythematosis, mono-arthritis, synovitis and loose bodies. Four normal specimens were obtained for comparison. 2. Valine, alanine and acetate levels all showed a normal Gaussian distribution, reflecting the distributions within the serum of the sample population. 3. Lactate concentrations divided into two distinct patterns. At concentrations below 2.5 mmol/l the lactate levels showed a Gaussian distribution, reflecting the distribution in normal serum. The normal synovial fluid specimens belong to this distribution. Above 2.5-3.0 mmol/l, lactate levels were asymmetric in distribution with a long tail at higher concentrations. These high levels of lactate can be explained by the generation of lactate through anaerobic metabolism within the synovial cavity. This metabolic process is triggered by a general inflammatory condition such as in rheumatoid arthritis. 4. The distribution of n.m.r.-observable lipid concentrations in rheumatoid arthritis and osteoarthritis each shows a normal distribution and the mean concentration is significantly higher in rheumatoid arthritis. 5. An increased n.m.r.-observable hyaluronan concentration is associated with an inflammatory situation. 6. It is concluded that raised levels of lactate and n.m.r.-observable hyaluronan and lipids are useful markers to aid the clinical distinction between rheumatoid arthritis and osteoarthritis.(ABSTRACT TRUNCATED AT 250 WORDS)
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