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...  Read more

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.

View all comments by Dominic Walsh

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...  Read more

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:
1. Lazo ND, Grant MA, Condron MC, Rigby AC, Teplow DB. On the nucleation of amyloid beta-protein monomer folding. Protein Sci. 2005 Jun;14(6):1581-96. Abstract

2. Borreguero JM, Urbanc B, Lazo ND, Buldyrev SV, Teplow DB, Stanley HE. 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. Epub 2005 Apr 18. Abstract

3. Cruz L, Urbanc B, Borreguero JM, Lazo ND, Teplow DB, Stanley HE. 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. Epub 2005 Dec 9. Abstract

4. Baumketner A, Bernstein SL, Wyttenbach T, Lazo ND, Teplow DB, Bowers MT, Shea JE. Structure of the 21-30 fragment of amyloid beta-protein. Protein Sci. 2006 Jun;15(6):1239-47. Abstract

5. Necula M, Kayed R, Milton S, Glabe CG. Small molecule inhibitors of aggregation indicate that amyloid beta oligomerization and fibrillization pathways are independent and distinct. J Biol Chem. 2007 Apr;282(14):10311-24. Epub 2007 Feb 6. Abstract

6. Lesne S, Koh MT, Kotilinek L, Kayed R, Glabe CG, Yang A, Gallagher M, Ashe KH. A specific amyloid-beta protein assembly in the brain impairs memory. Nature. 2006 Mar 16;440(7082):352-7. Abstract

7. Cheng IH, Scearce-Levie K, Legleiter J, Palop JJ, Gerstein H, Bien-Ly N, Puolivali J, Lesne S, Ashe KH, Muchowski PJ, Mucke L. 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. Epub 2007 Jun 4. Abstract

View all comments by Brigita Urbanc

On Computers, Flies, and Alzheimer Disease
Two recently published papers address the fundamental question of how amyloid proteins form neurotoxic assemblies (see Luheshi et al., 2007 and Cheon et al., 2007). Pat McCaffrey has written an informative and insightful news report that summarizes their key findings and implications. The work reported extends efforts by the ”Cambridge group” (broadly defined, and including those in Firenze, Italy; Busan, Korea; and Jülich, Germany) to explore ”generic” protein folding pathways and their biological consequences. In these latest publications, the group extends the idea of generic protein structures to generic toxicity, meaning that protein assemblies that share structural features also share toxic activity. Importantly, algorithms have been developed that allow prediction of assembly state and neurotoxicity from protein primary structure.

The technical rigor of the two studies is excellent. Thus, within the contexts of the...  Read more

On Computers, Flies, and Alzheimer Disease
Two recently published papers address the fundamental question of how amyloid proteins form neurotoxic assemblies (see Luheshi et al., 2007 and Cheon et al., 2007). Pat McCaffrey has written an informative and insightful news report that summarizes their key findings and implications. The work reported extends efforts by the ”Cambridge group” (broadly defined, and including those in Firenze, Italy; Busan, Korea; and Jülich, Germany) to explore ”generic” protein folding pathways and their biological consequences. In these latest publications, the group extends the idea of generic protein structures to generic toxicity, meaning that protein assemblies that share structural features also share toxic activity. Importantly, algorithms have been developed that allow prediction of assembly state and neurotoxicity from protein primary structure.

The technical rigor of the two studies is excellent. Thus, within the contexts of the experimental systems employed, namely in silico and in vivo (in Drosophila), one may have great confidence in the results. However, now comes the more philosophical and difficult question of meaning. Specifically, how do these results contribute to our understanding of diseases of protein folding?

In this brief discussion, I consider this question and raise a number of others for consideration by the reader. My goal in playing the metaphorical ”Devil's advocate” is to stimulate scientific discourse.

”Meaning” is a nebulous and malleable term for which a definition invariably depends upon the system of evaluation one employs. The goal of the studies of Luheshi et al. and Cheon et al. is to answer the questions of “... the molecular basis of amyloid formation and the nature of the toxic species.” In this context, it is reasonable to ask whether simulating the self-association of Aβ(16-22) or Aβ(25-35) has any relevance (meaning) to Alzheimer disease (AD) or any other disease. Why? Neither peptide is found in vivo. Historically, the former has been a favorite of theorists (including this writer), as its size makes it amenable to in silico study and it forms fibrils in vitro. The latter has been studied since 1990, when the suggestion was made that it was homologous to the tachykinin family of neuropeptides (Yankner et al., 1990). However, the homology relationship was tenuous (as are many when sequence length is so short), authentic tachykinin peptides had no trophic or toxic effects on neurons, and significant evidence supporting the tachykinin connection has not emerged in the subsequent 17 years. Thus, without compelling biological precedent, one must ask what study of these peptides can reveal. For example, are these peptides proxies for holo-Aβ? Clearly, the answer must be ”no,” as the critical determinant of peptide pathogenicity lies at the Aβ C-terminus in the form of the Ile-Ala dipeptide.

Why are people studying what may be irrelevant peptides, and why is such irrelevance not recognized? An answer may come from what, until recently, has been one of the most controversial and contentious fields of modern biology, i.e., prions. The prion theory postulates that the causative agent of a variety of neurodegenerative diseases in animals and humans is composed entirely of protein (no nucleic acid). In the last three decades, the status of this ”protein only” hypothesis in the scientific community has moved from heresy to orthodoxy. However, questions about the scientific appropriateness of this changing perspective have led some, including Laura Manuelidis, to suggest that a re-examination is warranted of ”the objectivity of science and whether it is a myth vanished.” Manuelidis opines that the acceptance of the theory reflects "the peculiarly American sport of betting on popular momentum” (Manuelidis, 2000). A more apropos metaphor, considering that one prion disease is bovine spongiform encephalopathy (“Mad Cow” disease), might have been that of “following the herd.”

Much research on AD could be subject to the same type of criticism. Consider the example of what may be called the "generic” herd. This herd believes that amyloid structure is "generic” because many (most? all?) proteins form amyloids with some common structural organization. Although amyloids, by definition, do share a number of biophysical and spectroscopic features, great structural diversity may be found in the assemblies formed by classical and non-classical amyloid proteins and peptides (e.g., see Sawaya et al., 2007). Importantly, no generic structure outside of the cross-β core of the amyloid fibril has been shown to exist, for obvious reasons. Regions outside of the core, which can be quite extensive in protein, as opposed to peptide amyloids, are likely to influence the biological behavior of the assemblies significantly.

Now, Cheon and colleagues suggest that amyloid formation involves a second generic process, a two-step mechanism of “collapse” of monomers and their subsequent rearrangement into amyloid fibrils. This idea appears to invoke known processes of globular protein folding in the context of amyloid formation, specifically the classical idea of hydrophobic collapse into a molten globule followed by proper arrangement of secondary structure elements to form the native tertiary structure. The idea that some peptides bypass this two-step pathway if they can immediately form hydrogen bonds in their eventual cross-β organization is quite interesting. However, although plausible for short, disordered peptides of the sort studied here, what happens in the common case of natively folded proteins forming amyloid? Here, and as the authors themselves suggest implicitly, factors other than the intrinsic properties of the protein monomer likely moderate amyloid assembly. This increased complexity requires me to question the value of this suggestion of generic mechanisms. Scientists, especially medicinal chemists, need targets. Does a “generic amyloid target” exist? Could a single compound directed at such a target be of value in the treatment of the greater than two score amyloid diseases defined thus far?

Maybe a generic target does exist. In Luheshi et al., studies of the effects of expression of human Aβ42 in Drosophila suggest that protofibril formation correlates with neuronal dysfunction and neurodegeneration. In addition, in a kind of Anfinsen redux (Anfinsen, 1973), an algorithm has been created to predict from primary structure alone the propensity of a protein to form toxic protofibrils. My question: Does the experimental assessment of Aβ-induced locomotor and longevity effects in flies, and its correlation with the toxicity metric, have any relevance to the consideration of Aβ-induced disease in humans? Granted, the same question is sometimes raised as gratuitous criticism of work in a variety of non-human animal models, and it is an easy concern to raise, but that does not diminish the significance of the question.

In closing, it may appear to some that the answers to the questions I have asked are implicit in the construction of the questions themselves. This certainly was not my intention. From a purely academic perspective, I found the publications rigorous, enjoyable to read, and quite thought-provoking. It is the provocation aspect of the experience that operates here, particularly with respect to establishing the meaning of the results and their impact on our shared efforts to understand and treat diseases of aberrant protein folding and assembly.

View all comments by David Teplow

Reply by Leila M. Luheshi, Giorgio Favrin, Damian C. Crowther, Michele Vendruscolo, and Christopher M. Dobson to Teplow Comment
We are pleased to have the opportunity of adding further observations to a recent commentary by David Teplow about the “generic hypothesis” of amyloid fibril formation (1). According to this hypothesis, the ability to form amyloid structures is an inherent property of polypeptide chains, although the propensity to form such structures can vary dramatically with their sequences (2).

This hypothesis is supported by a growing body of experimental evidence that has been summarized in a number of recent reviews (3). The generic nature of amyloid fibrils resides in their core cross-β structure, which is stabilized predominantly by backbone hydrogen bonding interactions (4). It has also been recently discovered that the range of proteins capable of forming toxic oligomers, that may well be precursors to mature amyloid fibrils, is very large and includes those with no known association with disease (5-7). Of course, there are many additional...  Read more

Reply by Leila M. Luheshi, Giorgio Favrin, Damian C. Crowther, Michele Vendruscolo, and Christopher M. Dobson to Teplow Comment
We are pleased to have the opportunity of adding further observations to a recent commentary by David Teplow about the “generic hypothesis” of amyloid fibril formation (1). According to this hypothesis, the ability to form amyloid structures is an inherent property of polypeptide chains, although the propensity to form such structures can vary dramatically with their sequences (2).

This hypothesis is supported by a growing body of experimental evidence that has been summarized in a number of recent reviews (3). The generic nature of amyloid fibrils resides in their core cross-β structure, which is stabilized predominantly by backbone hydrogen bonding interactions (4). It has also been recently discovered that the range of proteins capable of forming toxic oligomers, that may well be precursors to mature amyloid fibrils, is very large and includes those with no known association with disease (5-7). Of course, there are many additional complexities involved in misfolding diseases, and it is also clear that the aggregation of different proteins in vitro does result in fibrils of different morphologies, and the aggregation of different proteins in vivo causes diseases with very different characteristics.

So far the generic hypothesis has provided the inspiration for a number of innovative studies, including the two mentioned by David Teplow (8, 9). We have been astonished by the way that the principles observed in the test tube and in the computer are able to explain the behavior and lifespan of living organisms such as Drosophila. We are therefore optimistic that the insights such studies have given us will lead, in the next few years, to the development of effective therapeutic strategies to combat the debilitating and increasingly prevalent diseases associated with protein misfolding.

References:
1. Dobson CM. Protein misfolding, evolution and disease. Trends Biochem Sci. 1999 Sep;24(9):329-32. Review. No abstract available. Abstract

2. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM. Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature. 2003 Aug 14;424(6950):805-8. Abstract

3. Chiti F, Dobson CM. Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem. 2006;75:333-66. Review. Abstract

4. Knowles, T. P. J. et al. The Role of Inter-Molecular Forces in Defining the Material Properties of Fibrillar Protein Nanostructures. Science (2007) in press.

5. Bucciantini M, Giannoni E, Chiti F, Baroni F, Formigli L, Zurdo J, Taddei N, Ramponi G, Dobson CM, Stefani M. Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature. 2002 Apr 4;416(6880):507-11. Abstract

6. Baglioni S, Casamenti F, Bucciantini M, Luheshi LM, Taddei N, Chiti F, Dobson CM, Stefani M. Prefibrillar amyloid aggregates could be generic toxins in higher organisms. J Neurosci. 2006 Aug 2;26(31):8160-7. Abstract

7. Kayed R, Glabe CG. Conformation-dependent anti-amyloid oligomer antibodies. Methods Enzymol. 2006;413:326-44. Abstract

8. Luheshi LM, Tartaglia GG, Brorsson AC, Pawar AP, Watson IE, Chiti F, Vendruscolo M, Lomas DA, Dobson CM, Crowther DC. Systematic in vivo analysis of the intrinsic determinants of amyloid Beta pathogenicity. PLoS Biol. 2007 Oct 30;5(11):e290. Abstract

9. Cheon M, Chang I, Mohanty S, Luheshi LM, Dobson CM, Vendruscolo M, Favrin G. Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils. PLoS Comput Biol. 2007 Sep 14;3(9):1727-38. Abstract

View all comments by Leila Luheshi