3 September 2010. Oligomers of amyloid-β (Aβ) have created quite a stir since they were discovered to be perhaps the most toxic forms of the peptide. But oligomers are notoriously difficult to study. In fact, without much stirring, they spontaneously morph into larger and larger molecules that may be less germane to Alzheimer disease than their smaller counterparts. How is the researcher to keep tabs on what’s in the test tube? A little protein engineering may help. In the August 13 PNAS online, researchers led by Torleif Hard at the Swedish University of Agricultural Sciences, Uppsala, report that covalently constraining Aβ monomers into a specific β-hairpin structure accelerates formation of toxic oligomers. “These oligomers are completely stable and never convert to amyloid,” Hard told ARF. With about 50 times the toxic potency of natural Aβ monomers or fibrils, the stable, engineered peptides, Hard believes, could prove useful for studying oligomer dynamics and toxicity, and may even provide better epitopes for vaccination or raising antibodies for therapeutics.
Previously, Hard and colleagues found that certain proteins with antibody-like properties, called affibodies, stabilized Aβ peptides with a specific β-hairpin structure between amino acids 17 and 36 (see ARF related news story). The β structure resembled, but was not quite the same as, the β-sheet structures seen in amyloid fibrils. In the latter, the bonds between the β-sheets are intermolecular, whereas in affibody-stabilized peptides, they are intramolecular. Nevertheless, that work led to a proposed model of Aβ fibrillization where peptides with intramolecular β-sheets stacked upon one another before undergoing a conformation flip that resulted in intermolecular bonding. “We postulated that that β-sheet hairpin is a preferred conformation, and may be present in toxic oligomers,” said Hard.
To test that idea, first author Anders Sandberg and colleagues introduced two cysteine residues into the Aβ peptide, replacing alanine 21 and alanine 30, which are juxtaposed in the β-sheet hairpin. The theory was that a disulphide bond between the cysteines would stabilize that conformation. The researchers expressed the engineered peptide, dubbed Aβcc, in Escherichia coli. Aβcc readily formed oligomers with a range of conformations as judged by size exclusion chromatography, and formed protofibrils visible in the transmission electron microscope, but it failed to form fibrils unless it was first treated with a reducing agent to break the disulphide bond. The findings are in keeping with the need for a conformational change and rearrangement of intramolecular bonding for Aβ to form amyloid fibrils.
The covalently stabilized Aβcc may form oligomers, but how do those oligomers relate to the toxic ones that are released by cells (see ARF related news story), synthesized (see Lambert et al., 1998), and isolated from human tissue (see ARF related news story)? Sandberg found that various Aβcc species were highly toxic to SH-SY5Y neuroblastoma cells. In this assay, the potency of mid-molecular-weight oligomers and protofibrils of Aβcc was on par with that of wild-type Aβ42 oligomers and about 50 times higher than that of wild-type monomers or Aβ42 fibrils. “We think that the Aβcc oligomers, or the protofibrils formed by Aβcc oligomers, are very close to the naturally toxic form of Aβ,” said Hard.
“This was an ambitious project, the results of which support prior suggestions that oligomeric Aβ assemblies are particularly neurotoxic relative to monomeric and fibrillar forms of the peptide, and, therefore, that oligomers are attractive therapeutic targets,” suggested David Teplow, University of California, Los Angeles (see full comment below). Hard told ARF that some of those therapeutics could include monoclonal antibodies or vaccines. MIVAC Development AB, a biotechnology company in Gothenburg, Sweden, has begun looking into these possibilities. Hard is a consultant for, and holds stock in, this company.—Tom Fagan.
Sandberg A, Luheshi LM, Sollvander S, de Barros TP, Macao B, Knowles TPJ, Biverstal H, Lendl C, Ekholm-Petterson F, Dubnovitsky A, Lannfelt L, Dobson CM, Hard T. Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering. Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15595-600. Abstract