Biomedical subjects

D J Guthrie

Publications and source records attributed to D J Guthrie.

At least 19 recordsLinked to original sources

PubMed2001

Identification of the region of non-Abeta component (NAC) of Alzheimer's disease amyloid responsible for its aggregation and toxicity.

The non-beta-amyloid (Abeta) component of Alzheimer's disease amyloid (NAC) and its precursor alpha-synuclein have been linked to amyloidogenesis in several neurodegenerative diseases. NAC and alpha-synuclein both form beta-sheet structures upon ageing, aggregate to form fibrils, 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 beta-sheet structure; rather all remain random coil throughout 24 h. However, in acetonitrile, an organic solvent known to induce beta sheet, fragments NAC(8-18) and NAC(8-16) both form beta-sheet structure. Only NAC(8-18) aggregates, as indicated by concentration of peptide remaining in solution after 3 days, and forms fibrils, as determined by electron microscopy. These findings indicate that residues 8-16 of NAC, equivalent to residues 68-76 in alpha-synuclein, comprise the region crucial for toxicity.

Acetonitriles↗

PubMed2000

Toxicity of non-abeta component of Alzheimer's disease amyloid, and N-terminal fragments thereof, correlates to formation of beta-sheet structure and fibrils.

The non-Abeta component of Alzheimer's disease amyloid (NAC) and its precursor alpha-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 beta-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 degrees C 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 beta-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 beta-sheet secondary structures is important for determining the toxicity of the peptide. This contrasts with what has been reported previously for most Abeta peptides as their toxicity appears to require the peptide to have formed fibrillary aggregates as well as displaying beta-sheet. These results suggest that an intermediate, which exhibits beta-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 Disease↗

PubMed1998

Comparative studies on peptides representing the so-called tachykinin-like region of the Alzheimer Abeta peptide [Abeta(25-35)].

In an attempt to answer the question of whether or not the so-called tachykinin-like region of the Alzheimer beta-amyloid protein [Abeta(25-35)] can act as a tachykinin, the sequences Abeta(25-35), Abeta(25-35)amide and their norleucine-35 and phenylalanine-31 analogues were synthesized. These peptides were examined with ligand binding studies, electron microscopy, CD and NMR. In all cases some differences were found between the Abeta(25-35) analogue and the corresponding Phe31 peptide. In addition, in ligand displacement studies on tachykinin NK1 receptors, only the Phe31 analogue showed activity comparable to that of genuine tachykinins. We conclude that peptides based on Abeta(25-35) but with a Phe residue at position 31 do display properties typical of a tachykinin, but that peptides with Ile at this position do not.

Alzheimer Disease↗

PubMed1998

The N-terminal region of non-A beta component of Alzheimer's disease amyloid is responsible for its tendency to assume beta-sheet and aggregate to form fibrils.

Examination of the N-terminal sequence of non-A beta 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 beta peptide (A beta), 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 beta-structure whereas the C-terminal half was likely to form an alpha-helix. NAC in solution altered from random coil to beta-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-(1-18)-peptide and NAC-(19-35)-peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC-(1-18)-peptide became congophilic and aggregated to form fibrils of diameter 5-10 nm, whereas NAC-(19-35)-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-(19-35)-peptide adopted alpha-helical structure but NAC-(1-18)-peptide did not. NAC-(1-18)-peptide and NAC formed beta-sheet in acetonitrile/water mixtures more readily than did NAC-(19-35)-peptide. CD spectra of NAC or NAC-(1-18)-peptide in aqueous solution indicate the formation of beta-sheet on ageing. We propose that the N-terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles A beta, and other amyloidogenic proteins, in that aggregation is dependent upon beta-sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.

Aging↗

PubMed1998

The influence of the central region containing residues 19-25 on the aggregation properties and secondary structure of Alzheimer's beta-amyloid peptide.

Alzheimer's beta-amyloid peptide (Abeta) 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 Abeta 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 Abeta 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]Abeta-(25-35)-amide as a prototype structure. To this prototype, addition of pairs of amino acid residues from the sequence of Abeta, forming the corresponding 23-, 21- and 19-35 derivatives, resulted in peptides that aggregated to form fibrils of diameter 6-10 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 beta 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 alpha helix and towards beta sheet. In individuals with the Ala21-->Gly mutation, these results would suggest that truncated species with N-termini in the region containing residues 17-20 would be more amyloidogenic than the wild type homologues.

Alzheimer Disease↗

PubMed1998