Amyloidoses are a varied and complex set of syndromes caused by aggregation of specific proteins such as the amyloid-β, found in the brains of Alzheimer's patients, and transthyretin, which is found in a variety tissues damaged by systemic amyloidosis. In this week's early edition of PNAS, two papers explore why these amyloidogenic proteins form such stable and deadly aggregates.
Michel Steinmetz and Chris Dobson, from the Paul Scherrer Institute, Switzerland, and the University of Cambridge, U.K., respectively, together with colleagues elsewhere, report the synthesis of a novel peptide that may be extremely useful for testing the whys and wherefores of amyloid formation. Dubbed ccβ, this 17-residue peptide exists as a coiled-coil under ambient conditions, but converts into fibrils when the temperature is raised.
One of the beauties of ccβ is that it readily forms crystals which can be studied by x-ray diffraction. Such crystal structures have been difficult to obtain using natural amyloid peptides. Analysis of the crystals by Richard Kammerer and colleagues shows that the molecule exists as a coil of three parallel α-helices. When Kammerer and colleagues elevated the temperature of these peptides, the helical structures broke down and circular dichroism spectra revealed the emergence of β-sheet formations. By playing with the parent compound (S I R E L E X R I R E L E X R I G, where X-7 and X-14 are alanine and leucine, respectively), the authors were able to find substitutions that dramatically accelerate the formation of β fibrils; methionines at the 7 and 14 positions, for example, enhanced fibril formation by about 60-fold.
Further biophysical measurements showed that the β fibrils form as laminated cross-β sheets, a structure that has been proposed for a variety of amyloid fibrils, including the amyloid-β of Alzheimer's disease. The authors state that the "ccβ system provides an exceptionally favorable platform from which to develop a more detailed understanding of the origin and progression of the increasing number of human disorders associated with amyloid formation."
In the second paper, Joel Buxbaum and colleagues at The Scripps Research Institute, La Jolla, California, used tissue cultures to test the toxicity of various oligomeric forms of the transthyretin protein (TTR). Amyloidogenic peptides generally pass through several intermediary forms on their way from free soluble protein to amyloid aggregates, and evidence has mounted that these various protofibrillary forms are actually the most toxic entities (see ARF related news story).
First author Natalia Reixach found that native TTR, which exists as a homotetramer, has little effect on neuroblastoma cells, whereas the amyloidogenic mutant V30M TTR (in which valine at position 30 is substituted by methionine) induced apoptosis. To determine if the monomers play any role in the toxicity of the methionine mutant, Reixach carried out the same experiment in the presence of either resveratrol or chlorinated biaryl amine, two chemicals which have been shown to stabilize the tetrameric form of the protein. Both chemicals protected the cells against the V30M mutant. To confirm that the monomers are the toxic entities, Reixach tested variants of TTR that cannot form tetramers. All of these monomeric TTRs (M-TTRs) proved toxic, even those without the V30M mutation. The results suggest that it is the propensity to form monomers that confers toxicity on TTR mutants.
The monomeric TTRs also had a propensity to aggregate. Could the aggregates, therefore, be causing the toxicity? To test this, Reixach incubated M-TTR (without the V30M mutation) for five days, recovered the aggregates by gel filtration, and tested them in the neuroblastoma cultures. Aggregates over 100 KDa in size did not cause cytotoxicity. The authors conclude that monomers, or possibly aggregates of two or six monomers (as judged by their size on gel filtration columns) may be the toxic species. In the case of the multimers, they must be very active because Reixach found that the numbers of these being formed were very low.—Tom Fagan
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