Field of Science

Probing amyloid, one oligomer at a time
One of the more important paradigm shifts in our understanding of the Alzheimer’s disease-causing amyloid protein in the last few years has been the recognition of differences between the well known polymer aggregates of amyloid and their smaller, soluble oligomer counterparts. For a long time it was believed that the fully formed 40-42 amino acid protein aggregate found in autopsies was the causative agent in AD, or at least the most toxic one. This understanding has radically changed in the last few years, partly through elegant work done in identifying oligomers and partly through the unfortunate results of clinical trials targeting amyloid. The new understanding is that it’s not the fully formed aggregates but the smaller oligomers that are the real toxic species.

Identifying these different monomers, dimers, trimers and tetramers is a valuable goal. But until now their recognition has mainly depended on raising specific antibodies against them, a tedious and expensive process. Small molecule probes that specifically identify each oligomer have been missing. In a recent JACS communication, a team from the University of Michigan uses a simple but clever technique to develop such probes and makes a promising step in this direction.

The probes are based on the idea that the best antidote against a poison is another poison. In this case the poison is the specific sequence of amino acids that makes up amyloid. In particular, a sequence of five amino acids- KLVFF- has been found to be sufficient for aggregation and toxicity. The aggregates form by the stacking of beta sheets principally driven by hydrophobic interaction between the FF residues; each pair thus serves as a growth site for addition of further such residues. The insight then is that if one could construct a mimic of the sequence, this mimic would basically act as a competitive inhibitor and bind to the normal sequence, inhibiting further growth. In this case the strategy was to use KLVFF segments themselves which would sort of wrap around newly formed oligomers of different constitution and sequester them from further self-assembly. So the team essentially constructed two KLVFF segments joined by a linker. The linker would also serve the purpose of providing an entropic advantage to the two segments so that they would not be at an energetic disadvantage during binding. The important question was how long the linker should be.

To decide on the length of the linker the team made some clever use of molecular dynamics simulations. Since you can estimate the approximate thickness of every oligomer, you can estimate the linker length that would be required to keep two KLVFF segments at the same distance as the thickness of the oligomer. For instance, the distances between the segments needed to wrap around the oligomers were 14-15 A for the dimer, 19-20 A for the trimer and 24-25 A for the tetramer.

But the linker should also keep the segments stable at that distance. To probe this the team used MD simulations. The MD simulations revealed the length of the linker required to keep the two segments separated at the specific distances by indicating how much time the assembly spent at those distances.

To test these results, the team then generated mixtures of different kinds of KLVFF oligomers and then added each probe to the solution. A streptavidin moiety was attached to every probe. Silver staining revealed that each probe was specifically binding to an oligomer of a certain type dictated by the compatibility of the intraprobe distance and oligomer thickness. Trimers and tetramers could be clearly identified but there was more ambiguity in case of dimers, presumably because of their less ordered structure.

Most interestingly, the team then added the probes to cerebrospinal fluid (CSF). Since amyloid is part of normal physiology, it is present in CSF. Gratifyingly they found that the probes could very clearly label trimers and tetramers against a background of several other proteins and intermediates in CSF. This experiment notably demonstrates that the method can selectively detect amyloid oligomers in complex mixtures.

I think that this work is valuable and paves the way toward the development of similar small-molecule based probes for identifying the key intermediates in amyloid formation. It could also be very useful in exploring amyloid formation in normal physiology and in exploring the stages of protein self-assembly in diverse amyloid-based diseases.

Reinke, A., Ung, P., Quintero, J., Carlson, H., & Gestwicki, J. (2010). Chemical Probes That Selectively Recognize the Earliest Aβ Oligomers in Complex Mixtures Journal of the American Chemical Society DOI: 10.1021/ja106291e

1 comment:

  1. Wanna just say something about amyloid. Oxidizing the methionine residue near the c-terminus of a-beta has an effect on the aggregation properties (to the point where it's used as synthetic strategy). This is such a minor change, how do you expect a five-mer to be a useful model system, especially in light of the fact that any aggregates, even of the wild type protein, are polymorphic.

    That's why all solid-phase structural studies of the peptide are done by making a sample, and using it to seed and multiply a single structural example. We don't even know if some structure are more or less relatively toxic than others.

    "This experiment notably demonstrates that the method can selectively detect amyloid oligomers in complex mixtures."

    It could also be selectively trapping monomers as the oligomers that you'd like to see.


Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="">FoS</a> = FoS