In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to alzheimer's disease β-protein

Rempel

Proc. Natl. Acad. Sci. U.S.A.

et al. 2013

112

179

Probing the conformational changes of amyloid beta (Aβ) peptide aggregation is challenging owing to the vast heterogeneity of the resulting soluble aggregates. To investigate the formation of these aggregates in solution, we designed an MS-based biophysical approach and applied it to the formation of soluble aggregates of the Aβ 42 peptide, the proposed causative agent in Alzheimer's disease. The approach incorporates pulsed hydrogen-deuterium exchange coupled with MS analysis. The combined approach provides evidence for a self-catalyzed aggregation with a lag phase, as observed previously by fluorescence methods. Unlike those approaches, pulsed hydrogen-deuterium exchange does not require modified Aβ 42 (e.g., labeling with a fluorophore). Furthermore, the approach reveals that the center region of Aβ 42 is first to aggregate, followed by the C and N termini. We also found that the lag phase in the aggregation of soluble species is affected by temperature and Cu 2+ ions. This MS approach has sufficient structural resolution to allow interrogation of Aβ aggregation in physiologically relevant environments. This platform should be generally useful for investigating the aggregation of other amyloid-forming proteins and neurotoxic soluble peptide aggregates.soluble Aβ oligomers | amyloid beta peptide | copper | electrospray ionization | Finke-Watsky mode P rotein aggregation is one of the immediate causes of Alzheimer's, Parkinson, and Huntington diseases, motivating biophysical studies of the responsible proteins. More than 20 small proteins undergo amyloidosis in humans. In Alzheimer's disease (AD), the aggregation of the 40-or 42-aa-long amyloid beta (Aβ) peptide, generally called Aβ 40 or Aβ 42 , respectively, is proposed to be involved in the onset of the disease (1, 2). Aβ 42 is more amyloidogenic and more neurotoxic than Aβ 40 . Although the amyloid-cascade hypothesis suggests that the Aβ-containing amyloid plaques are responsible for neurodegeneration (3-7), other studies suggest that soluble aggregates of Aβ 42 are more neurotoxic than the amyloid plaques (8-13).The amyloid plaques in AD-affected brains contain high levels of copper, zinc, and iron (14-20). Among these, Cu has drawn the most attention because the Aβ precursor protein is likely a Cuchaperone protein (21). Several studies of Cu 2+ -Aβ 40 interactions show that Cu 2+ can promote Aβ 40 aggregation (14,18,19).The structure of Aβ 42 and its aggregates, although studied extensively, remains of high interest. Studies of amyloid fibrils invoke X-ray crystallography (22-24), EM (19,25,26), and thioflavin T fluorescence (19, 27), revealing the polypeptide's global behavior, whereas NMR studies provide residue-level information for the fibrils (28-30). Nevertheless, we know little about soluble Aβ aggregates owing to their intrinsically high heterogeneity.MS should offer an opportunity for investigating soluble aggregates of Aβ 42 . Thus far, there are no MS-based, timedependent studies of the formation of soluble aggregates. Moreover, there are no ot...

Section: Resultsmentioning

confidence: 99%

“…Studies of amyloid fibrils invoke X-ray crystallography (22)(23)(24), EM (19,25,26), and thioflavin T fluorescence (19,27), revealing the polypeptide's global behavior, whereas NMR studies provide residue-level information for the fibrils (28)(29)(30). Nevertheless, we know little about soluble Aβ aggregates owing to their intrinsically high heterogeneity.…”

mentioning

confidence: 99%

Pulsed hydrogen–deuterium exchange mass spectrometry probes conformational changes in amyloid beta (Aβ) peptide aggregation

Rempel

Proc. Natl. Acad. Sci. U.S.A.

et al. 2013

112

179

“…The fl/A4 amyloid protein in amyloid fibrils is organized into a cross-fl conformation in which the peptide backbone is perpendicular to the fiber axis [8]. Studies using synthetic peptides containing partial-and full-length sequences of fl/A4 amyloid protein have suggested that the physical properties of fl/A4 amyloid protein themselves are the causes of amyloid fibril assembly and the formation of insoluble aggregates [9][10][11][12][13][14].…”

Section: Introductionmentioning

confidence: 99%

Cross‐linking of a synthetic partial‐length (1–28) peptide of the Alzheimer β/A4 amyloid protein by transglutaminase

1993

Cerebral deposits of iliA4 amyloid protein is a pathologic sign of Alzheimer's disease. A synthetic partial-length (I 28) peptide of this protein contains one glutamine and two lysine residues. Here we show that this peptide can be a substrate oftransglutaminase, which catalyzes cross-linking between glutamine and lysine residues in peptides, by demonstrating the formation of multimeric peptides due to the action of this enzyme. A modified (Lys 2s to L-norleucine) version of the synthetic peptide was also cross-linked, but another modified version (Lys ~6 to L-norleucine) was very poorly cross-linked, indicating that Lys 16 is involved exclusively in the cross-linking of the partial-length peptide catalyzed by transglutaminase.Transglutaminase; Amyloid beta-protein; Alzheimer's disease.

European Journal of Biochemistry

“…The partially membrane-bound protein precluded the possibility of studying the whole PA4 sequence since it would be a difficult task to mimic NMR solvent conditions whereby part of the peptidelprotein under study is in an 'extracellular' environment and the other part in a 'membrane' environment. Due to these limitations, we are studying synthetic extracellular fragments of PA4 which have demonstrated in vitro formation of aniyloid fibrils under physiological conditions (Castano et al, 1986;Gorevic et al, 1987;., 1987). The NMR data is then combined with computational techniques to generate possible three-dimensional structures and to assist in the understanding of local or long-range interactions.…”

mentioning

confidence: 99%

Structure determination of extracellular fragments of amyloid proteins involved in Alzheimer's disease and Dutch‐type hereditary cerebral haemorrhage with amyloidosis

Sorimachi

Craik

1994

Amyloid deposition is a biochemical and histopathologic hallmark of various clinical forms of amyloidoses including Alzheimer's disease and the Dutch‐type hereditary cerebral haemorrhage with amyloidosis. The self‐aggregating peptides responsible for these irreversible deposits have been sequenced but the mechanisms involved in the aggregation processes are not well understood. In order to gain an understanding of the possible structures prior to self‐association, the extracellular fragment of the Alzheimer amyloid protein (βA4) responsible for the deposits (the ‘native’ fragment) and a mutant of this with a single residue substitution (which is responsible for deposits in the Dutch‐type amyloidosis) were examined by 1H‐NMR spectroscopy. Interproton distance constraints were derived from NMR experimental data and incorporated into tertiary structure calculations using a simulated annealing protocol. Solution conformations of the fragment peptides associated with the two forms of amyloidoses are presented and compared. Although in both peptides the existence of a mixture of conformations in equilibrium is likely, one such population of structures possesses a flexible N‐terminus and a well defined C‐terminal region. The latter region includes a helical segment and a terminal turn‐like structure. These structural features may be important for the basis of amyloid formation. Comparison of the calculated structures of the two peptides revealed a conformationally different region arising from the conservative substitution of Gln22 for Glu22. This region may be responsible for altered binding in the mutant peptide, giving rise to the clinically different form of amyloidosis.