Familial Alzheimer's disease mutations alter the stability of the amyloid β-protein monomer folding nucleus

Abstract

Amyloid ␤-protein (A␤) oligomers may be the proximate neurotoxins in Alzheimer's disease (AD). Recently, to elucidate the oligomerization pathway, we studied A␤ monomer folding and identified a decapeptide segment of A␤, 21 Ala-22 Glu-23 Asp-24 Val-25 Gly-26 Ser-27 Asn-28 Lys-29 Gly-30 Ala, within which turn formation appears to nucleate monomer folding. The turn is stabilized by hydrophobic interactions between Val-24 and Lys-28 and by longrange electrostatic interactions between Lys-28 and either Glu-22 or Asp-23. We hypothesized that turn destabilization might explain the effects of amino acid substitutions at Glu-22 and Asp-23 that cause familial forms of AD and cerebral amyloid angiopathy. To test this hypothesis, limited proteolysis, mass spectrometry, and solution-state NMR spectroscopy were used here to determine and compare the structure and stability of the A␤(21-30) turn within wild-type A␤ and seven clinically relevant homologues. In addition, we determined the relative differences in folding free energies (⌬⌬G f) among the mutant peptides. We observed that all of the disease-associated amino acid substitutions at Glu-22 or Asp-23 destabilized the turn and that the magnitude of the destabilization correlated with oligomerization propensity. The Ala21Gly (Flemish) substitution, outside the turn proper (Glu-22-Lys-28), displayed a stability similar to that of the wild-type peptide. The implications of these findings for understanding A␤ monomer folding and disease causation are discussed.A bundant evidence links the amyloid ␤-protein (A␤) with the neuropathogenesis of Alzheimer's disease (AD) (for recent reviews, see refs. 1 and 2). A␤ is a normal metabolite of the A␤ precursor (A␤PP), from which A␤ is produced by endoproteolysis (3). Two predominant forms of A␤ exist in vivo, A␤40 and A␤42, which are 40-and 42-aa in length, respectively (2, 4, 5). Recent experimental and clinical evidence suggests that the primary neurotoxins in AD are A␤ oligomers or protofibrils (1, 6-10). Understanding the folding and oligomerization of nascent A␤ monomers thus has become an especially important aspect of current strategies for understanding AD etiology and developing therapeutic agents.We have applied a multidisciplinary approach to the A␤ assembly problem. Initial studies used limited proteolysis coupled with mass spectrometry to determine whether monomeric A␤ possessed any stable or quasistable structure that could protect the peptide from proteolysis. Surprisingly, a 10-residue segment within both A␤40 and A␤42, Ala-21-Ala-30, was identified (11). The homologous decapeptide, A␤(21-30), displayed protease resistance identical to that of full-length A␤, suggesting that this region could organize monomer folding and thus be a folding nucleus. This suggestion was consistent with the observation that many folding nuclei studied in isolation are structurally stable (12-16). In fact, NMR studies of the A␤(21-30) peptide revealed a turn in the Val-24-Lys-28 region that was stabilized by hydrophobic interactions between...

β-Amyloid Precursor Protein Mutants Respond to γ-Secretase Modulators

“…Surprisingly, A␤ could not be detected for the K28E mutant in cultured cells and the cell-free assay system by SDS-PAGE (data not shown). This may reflect a potential alteration of the folding of Lys-28 mutant A␤ peptides (45), which could possibly have affected their detectability in some of the previous reports (22,46,47 FIGURE 4. Validation of cell-free assay system concerning the cleavage specificity of purified ␥-secretase on mutant APP C-terminal fragment based C100-His 6 substrates.…”

Structure and Dynamics of the Aβ21–30 Peptide from the Interplay of NMR Experiments and Molecular Simulations

“…Lazo et al report a longrange i,i + 8 Glu22 H α to Ala30 HN crosspeak, 22 and more recent work from the same group assigns the peak to an overlap of the original Glu22 H α to Ala30 HN interaction and i,i + 2 Lys28 H α to Ala30 HN in spectra collected at 500 MHz. 35 The observation of the long-range ROE is critical to their proposed NMR model, which is significantly collapsed. However, because of the higher resolution of the spectrum we have collected at 900 MHz, we interpret the crosspeak to be solely attributed to the i,i + 2 contact between Lys28 H α and Ala30 HN, as shown in Figure 4, upper panel.…”

“…In fact, they arise from separate structural populations of local turn structure in regions 23-27 and 27-30 that together comprise onlỹ 40% of the total equilibrium ensemble and thus provide a qualitatively different result from the single structural model reported by Lazo and co-workers. 22,35 The very good quality of results of this validation study on Aβ [21][22][23][24][25][26][27][28][29][30] paves the way for simulating the structural ensemble of the Aβ 1-40 and Aβ 1-42 systems, because solution NMR experiments on these peptides are inherently more difficult because of peptide aggregation. Furthermore, we believe that this study shows that the interplay of modern molecular simulation and highquality NMR experiments has reached a fruitful stage for characterizing structural ensembles of disordered peptides and proteins in general.…”

“…They proposed that WT Aβ [21][22][23][24][25][26][27][28][29][30] folds into a single conformer corresponding to a unique bend structure, identified through long-range (i,i+8) crosspeaks for Glu22 H α to Ala30 HN, and (i,i+6) side chain-side chain crosspeaks for Glu22 to Lys28 in the ROESY spectrum of WT Aβ [21][22][23][24][25][26][27][28][29][30] ; these crosspeaks were found to be absent in many of the FAD mutants. 35 Replica exchange molecular dynamics simulations by Baumketner et al by using the OPLS and TIP3P all-atom models for peptide and water, 36 showed that 40% of the peptide ensemble is folded into two distinct bend structures stabilized primarily by Asp23 side-chain interactions with the Ser26 side chain and backbone. Borreguero and co-workers studied the peptide by a coarse-grained model in which they find collapsed structures stabilized by hydrophobic interactions between Val24 and Lys28, as well as electrostatic interactions between Asp23 and Lys28.…”

“…24,[26][27][28][29][30][31][32][33][34] A number of experimental and computational studies have attempted to determine what stable structure in the Aβ [21][22][23][24][25][26][27][28][29][30] monomer accounts for its protease resistance. Teplow and coworkers studied the wild-type 22 as well as five FAD mutants 35 of the Aβ [21][22][23][24][25][26][27][28][29][30] peptide by using rotating-frame nuclear Overhauser effect spectroscopy (ROESY) NMR experiments. They proposed that WT Aβ [21][22][23][24][25][26][27][28][29][30] folds into a single conformer corresponding to a unique bend structure, identified through long-range (i,i+8) crosspeaks for Glu22 H α to Ala30 HN, and (i,i+6) side chain-side chain crosspeaks for Glu22 to Lys28 in the ROESY spectrum of WT Aβ [21][22][23][2...…”