. Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils. PLoS Comput Biol. 2007 Sep;3(9):1727-38. PubMed.


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  1. Hydrophobic Residues Exposed to Solvent: A Cause of Oligomer Toxicity?
    Assembly of proteins into toxic soluble oligomers and highly ordered fibrils is believed to be critical to amyloidogenic diseases associated with protein misfolding and aberrant aggregation. Thus, understanding these processes at atomic resolution has become the center of many computational studies. Computational approaches need to be simplified to enable studies of processes starting from separated protein molecules into ordered aggregates. Cheon et al. employ constant temperature, Monte Carlo simulations and an implicit water protein model which incorporates all atoms but reduces the degrees of freedom to Ramachandran and side-chain torsional angles only. Using a thus simplified computational approach, Cheon et al. study early stages of oligomer and fibril formation of two amyloid-β protein (Aβ) fragments, Aβ(16-22) and Aβ(25-35).

    Cheon et al. demonstrate that the process of aggregation into amorphous versus ordered species is determined by a competition between the hydrophobicity of the primary structure and the tendency of amino acids to form arrays of hydrogen bonds. The two fragments, Aβ(16-22) and Aβ(25-35), differ by the degree of their overall hydrophobicity, with Aβ(16-22) being significantly more hydrophobic than Aβ(25-35). Consequently, Cheon et al. make an important observation that while formation of disordered oligomers, primarily driven by hydrophobic collapse, is significantly stronger in Aβ(16-22), Aβ(25-35) proceeds to form ordered fibril-like aggregates with no significant amount of hydrophobically collapsed oligomers. This conclusion nicely complements a more general work on assembly of polyalanine molecules done in the Hall group [1-4] as well as more particular studies of full-length Aβ assembly done in our group [5-8].

    During the formation of oligomers, hydrophobic residues are buried on the inside, away from the solvent. Later on, when hydrogen bonds start forming, eventually yielding an ordered fibrillar structure, the hydrophobic residues are forced to get more exposed to the solvent. When the fibrillar structure grows from the initial seed, the ratio of surface to volume steadily decreases, decreasing the total solvent exposure of hydrophobic residues. Thus, the intermediate-size oligomers with some β-strand structure are the ones with a maximal solvent exposure of hydrophobic residues. Based on this observation and on the fact that oligomers of intermediate sizes are typically associated with the highest degree of toxic function, Cheon et al. suggest a relationship between the degree of solvent exposure of hydrophobic residues in an assembly and the cytotoxicity of the assembly. This is a very appealing general hypothesis that needs to be tested in future in-vitro and in-vivo studies.


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  2. 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.


    . Systematic in vivo analysis of the intrinsic determinants of amyloid Beta pathogenicity. PLoS Biol. 2007 Oct 30;5(11):e290. PubMed.

    . Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils. PLoS Comput Biol. 2007 Sep;3(9):1727-38. PubMed.

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    . Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature. 2007 May 24;447(7143):453-7. PubMed.

    . Principles that govern the folding of protein chains. Science. 1973 Jul 20;181(4096):223-30. PubMed.

    View all comments by David Teplow
  3. 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.


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    . Protein misfolding, functional amyloid, and human disease. Annu Rev Biochem. 2006;75:333-66. PubMed.

    . Role of intermolecular forces in defining material properties of protein nanofibrils. Science. 2007 Dec 21;318(5858):1900-3. PubMed.

    . Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases. Nature. 2002 Apr 4;416(6880):507-11. PubMed.

    . Prefibrillar amyloid aggregates could be generic toxins in higher organisms. J Neurosci. 2006 Aug 2;26(31):8160-7. PubMed.

    . Conformation-dependent anti-amyloid oligomer antibodies. Methods Enzymol. 2006;413:326-44. PubMed.

    . Systematic in vivo analysis of the intrinsic determinants of amyloid Beta pathogenicity. PLoS Biol. 2007 Oct 30;5(11):e290. PubMed.

    . Structural reorganisation and potential toxicity of oligomeric species formed during the assembly of amyloid fibrils. PLoS Comput Biol. 2007 Sep;3(9):1727-38. PubMed.

    View all comments by Leila Luheshi

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