. Familial Alzheimer's disease mutations alter the stability of the amyloid beta-protein monomer folding nucleus. Proc Natl Acad Sci U S A. 2007 Oct 16;104(42):16522-7. PubMed.

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  1. Aβ Takes a Turn for the Better?
    Using a powerful combination of biochemistry, biophysics, and mathematics, Grant and colleagues build on their earlier observation that wild-type Aβ1-40 and 1-42 contain a stretch of 10 amino acids (spanning residues 21-30) that has sufficient structure to render it relatively insensitive to proteolysis (Lazo et al., 2005) and which in nature is not cleaved by any of the known Aβ-degrading enzymes (Carson and Turner, 2002). Surprisingly, Aβ21-30 exhibited a similar protease resistance to that seen with the full-length peptide, and initial solution-state NMR analysis of the fragment indicated the presence of a relatively stable turn.

    But from the Lazo study, it is not clear if the putative turn represents a structure that is on path to oligomer formation. To address this point, the authors undertook a study of the 5 intra-Aβ mutations associated with Alzheimer’s or Alzheimer’s-like pathologies, the basic premise being that if, as burgeoning data suggest, oligomers play a pivotal role in disease, then mutations associated with disease may function to promote oligomer formation. Consequently, if the turn detected in wild-type Aβ represents a structure on path to oligomerization, one might expect that disease-associated mutations would stabilize turn formation. However, if the turn is not pro-oligomerization, then the mutations may act to destabilize the turn, thus facilitating alternative folding.

    Limited proteolysis and solution-state NMR studies of wild-type Aβ21-30 and of Aβ21-30 peptides bearing disease-associated or design substitutions were conducted to ascertain the effects of these mutations on turn stability. Limited proteolysis with trypsin, which specifically cleaves after lysine residues, was used to determine the accessibility of Lys28, a residue implicated in stabilizing the turn structure. It was found that three (E22G D23N, E22K) of the five disease-associated mutations caused a dramatic increase in proteolysis, one caused a modest (E22Q) increase, and the other (A21G) was not significantly different from wild-type. The three disease-associated mutations (E22G D23N, E22K) and the two design substitutions (D23G and D23Orn) that rendered Aβ21-30 more prone to proteolysis also yielded NMR spectra indicative of destabilization of the turn. As the differential sensitivity to trypsin is linked with structural stability, the authors cleverly used the data in their trypsin proteolysis progress curves to determine the free energies of folding (delta,deltaGf). Comparison of the folding stability calculated in this study with prior estimates of oligomer formation for Aβ1-40 and Aβ1-42 peptides bearing the same mutations (Bitan et al., 2003) revealed that the magnitude of turn destabilization correlated with the propensity for oligomer formation. Taken together, these data indicate that the turn is not pro-oligomer forming, and that oligomerization is enhanced by destabilization of this structure.

    These results suggest that small molecules that stabilize this turn structure should inhibit oligomerization and therefore may offer a target for therapeutic development. However, before such therapies are contemplated it will be important to test the effects of design substitutions that further stabilize the turn structure. Specifically, it will be crucial to test the toxic potential of “stabilized Aβ.” Moreover, if stabilizing small molecules can be developed, it will be important to determine if such molecules affect APP processing.

    Disclosure: DMW admits to being a long-standing friend and admirer of DBT.

    References:

    . On the nucleation of amyloid beta-protein monomer folding. Protein Sci. 2005 Jun;14(6):1581-96. PubMed.

    . Beta-amyloid catabolism: roles for neprilysin (NEP) and other metallopeptidases?. J Neurochem. 2002 Apr;81(1):1-8. PubMed.

    . Elucidation of primary structure elements controlling early amyloid beta-protein oligomerization. J Biol Chem. 2003 Sep 12;278(37):34882-9. PubMed.

  2. Implications of Aβ Folding Stability
    Alzheimer disease (AD) belongs to a class of diseases associated with protein misfolding and aberrant aggregation. Compelling evidence indicates that initial assembly stages of amyloid-β protein (Aβ), which exists in two main alloforms, Aβ40 and Aβ42, are critically involved in AD neurodegeneration. The present study by Grant et al. builds on the prior work done in the Teplow lab: in the search for the earliest events of Aβ misfolding, Lazo et al. used limited proteolysis and mass spectroscopy to identify a 10-residue segment within folded Aβ40 and Aβ42 conformations with a stable structure protected from proteolysis [1]. The homologous decapeptide, Aβ(21-30), displayed an identical protease resistance, and thus the region A21-A30 was hypothesized to represent the folding nucleus of both Aβ40 and Aβ42. The accompanied NMR study showed a turn in the V24-K28 region, stabilized by an effective hydrophobic interaction between V24 and the butyl side chain of K28, and long-range electrostatic interactions between E22/D23 and K28 [1]. Several computational studies using different molecular dynamics methods, two of which were conducted in our group, showed that the effective hydrophobic interaction between V24 and K28 was key to the turn structure, while the occasional formation of salt bridges E22-K28 and D23-K28 contributed to the stability of the turn [2-4].

    The present study of Grant et al. examines effects of seven clinically relevant amino acid substitutions at positions 21, 22, and 23 on the stability of the folded structure of a decapeptide Ala21-Ala30 in comparison to the wild-type (WT). These mutations were Ala21Gly (Flemish), Glu22Gly (Arctic), Glu22Gln (Dutch), Glu22Lys (Italian), Asp23Asn (Iowa), and in addition Asp23Gly and Asp23Orn. Using a combination of limited proteolysis, mass spectroscopy, and solution-state NMR spectroscopy, Grant et al. found three groups with different degrees of folding stability: (1) Asp23Orn, Glu22Gly, Asp23Gly; (2) Asp23Asn, Glu22Lys; and (3) Glu22Gln, WT, Ala21Gly. Here group (1) was the least and group (3) was the most stable. Data analysis showed that the decreased stability of the decapeptide fold due to various mutations as determined by limited proteolysis using Lys-specific protease, trypsin, is closely correlated with the mobility of the Lys28 side chain as measured by the side chain resonances of Lys28 using solution-state NMR TOCSY data. Based on external condition parameters of the limited proteolysis experiment and using kinetic equations for the concentrations of both Aβ and trypsin, Grant et al. derived peptide digestion progress curves for all eight different homologues, which enabled an estimation of probabilities for individual peptides to be in an unfolded and thus digestible state. Finally, Grant et al. drew a correlation between destabilization of the decapeptide folded structure and the propensity of full-length Aβ to assemble into oligomers. This is an impressive result providing evidence for existence of a major folding event involving a relatively small number of residues of the full-length Aβ that may have a major impact on oligomerization pathways.

    From a computer simulations perspective, a strong inverse correlation between stability of the folded structure and aggregation propensity is not surprising. A strongly folded structure is synonymous with an optimum conformation in which hydrophobic side chain groups are the least exposed to the solvent. Such strongly folded proteins need to partially unfold so as to expose the hydrophobic side chains of different molecules to each other and thus increase the propensity of effective hydrophobic intermolecular interactions leading to their assembly. Consequently, the more the decapeptide fold is destabilized due to an amino acid substitution, the higher will be its propensity to assemble. In the present work of Grant et al., this tendency is observed to a different degree in six of seven mutations under study. On the other hand, the effect of an amino acid substitution may result in a differently folded structure, which may be more stable than the original fold. In such a case, the propensity to assemble would not increase but rather decrease or remain unchanged. This present study provides compelling evidence for the hypothesis that the WT structure of Aβ is the most stable of all studied homologues and thus most protective against formation of toxic assemblies.

    This study opens up new questions asking for more detailed structural considerations that can also be addressed using computer simulations. In the earlier study, Lazo et al. found two families of WT decapeptide folded structures. The present study of Grant et al. addressed an average stability of folded structures for each of the eight homologues. The question remains whether different mutations destabilize both WT families of folded structure, only one of them, or perhaps even induce new stable families of folded structures. Addressing such detailed structural questions is important because an existence of several different folded structures may imply different independent pathways of assembly [5], some leading to protofibrils and fibrils and others leading to structurally distinct and stable oligomers, such as, for example, the dodecamer Aβ*56 observed in the transgenic mouse model [6,7]. In conclusion, a combined work of Lazo et al. and Grant et al. is unique as it zooms in to the earliest Aβ folding events, shows a profound impact of these initial events on the subsequent Aβ assembly, and suggests new future research directions.

    Disclosure: BU is a close collaborator of DBT and has great respect for his work.

    References:

    . On the nucleation of amyloid beta-protein monomer folding. Protein Sci. 2005 Jun;14(6):1581-96. PubMed.

    . Folding events in the 21-30 region of amyloid beta-protein (Abeta) studied in silico. Proc Natl Acad Sci U S A. 2005 Apr 26;102(17):6015-20. PubMed.

    . Solvent and mutation effects on the nucleation of amyloid beta-protein folding. Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18258-63. PubMed.

    . Structure of the 21-30 fragment of amyloid beta-protein. Protein Sci. 2006 Jun;15(6):1239-47. PubMed.

    . Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem. 2007 Apr 6;282(14):10311-24. PubMed.

    . A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006 Mar 16;440(7082):352-7. PubMed.

    . Accelerating amyloid-beta fibrillization reduces oligomer levels and functional deficits in Alzheimer disease mouse models. J Biol Chem. 2007 Aug 17;282(33):23818-28. PubMed.

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