X-raying Aβ Oligomers, Unraveling Mysteries of Amyloid Aggregation
Amyloid sure likes to keep its secrets. Despite decades of research, scientists still do not know precisely what makes amyloidogenic molecules such as Aβ gang up and go bad. Two new papers yield some clues to the puzzle. In the January 26 Journal of Neuroscience, researchers led by Stewart Nuttall at Commonwealth Scientific and Industrial Research Organization in Parkville, Victoria, Australia, describe the first x-ray crystallographic structure of an oligomeric Aβ fragment—though not yet oligomers of the most pathogenic forms of Aβ. Intriguingly, the structure looks quite different from fibrillar Aβ. Its features jibe with computational models, although only further research will reveal whether the form occurs in vivo. Researchers led by Sheena Radford at the University of Leeds, U.K., focused on the question of what causes an amyloidogenic protein to begin sticking together, using β2-microglobulin as their test subject. In the January 21 Molecular Cell, the scientists detail how subtle changes in shape can tilt the molecule toward aggregation, changing a formerly well-behaved protein into a bad seed. The authors demonstrate a mechanism that allows a handful of these twisted molecules to corrupt a much larger mass of correctly folded proteins.
Much of scientists’ current understanding of Aβ structure rests on studies of fibrillar amyloid. One seminal nuclear magnetic resonance study led by Robert Tycko at the National Institutes of Health showed that Aβ fibrils have an ordered β-turn-β structure (see ARF related news story on Petkova et al., 2002). In recent years, however, the scientific spotlight has zoomed in on oligomers, now widely believed to be the most toxic form of Aβ. Oligomers have frustrated scientists’ efforts to crystallize them, because they appear never to hold still in solution, forming larger aggregates that spontaneously fall apart.
First author Victor Streltsov in Nuttall’s group got around these problems by making use of a single chain shark immunoglobulin to box in the Aβ oligomers as they formed. Streltsov and colleagues created chimeric proteins in which the immunoglobulin was bound to the p3 fragment of Aβ (residues 17 to 42, created by successive α and γ cleavage). As Aβ peptides began to stick together in solution, the attached antibody formed a cage around the amyloid fragment, trapping the oligomer and preventing further aggregation. The authors then crystallized this immobilized complex and analyzed its structure by x-ray. To their surprise, they saw not the classic β-hairpin shape known from fibrils, but instead a tetrameric, globular structure consisting of two connected loops.
“This paper is important because it finally shows that the structure of oligomers is not like a piece of a fibril,” said Brigita Urbanc of Drexel University in Philadelphia, Pennsylvania.
The general features of this oligomer match some recent predictions, Urbanc said. Computer modeling from her lab suggests that oligomers form compact shapes, with hydrophobic residues buried in the center and hydrophilic residues exposed, and predicted the key roles of residues identified by Streltsov and colleagues as central to tetramer formation (see Urbanc et al., 2004 and Urbanc et al., 2010). The observed structure also fits with spectroscopic data from Charles Glabe’s lab at the University of California in Irvine (see ARF related news story).
But does this structure occur naturally in brains? “That’s the million dollar question,” Urbanc said, pointing out that because of the presence of the shark proteins, the oligomer may form differently than in vivo.
Urbanc notes, however, that several details of the structure do fall in line with other studies. For example, Streltsov and colleagues show that the lysine 28 side chain can take on two different configurations, a finding detailed in several previous experimental and computational studies by Dave Teplow and colleagues at the University of California in Los Angeles (see Lazo et al., 2005; Borreguero et al., 2005; ARF related news story on Cruz et al., 2005; Baumketner et al., 2006; Grant et al., 2007; and Baumketner et al., 2008).
The choice of the p3 Aβ fragment raises additional questions about the pathogenic significance of this structure. The p3 fragment is found in amyloid plaques of Alzheimer’s disease and in the pre-amyloid lesions of Down's syndrome, but has typically been considered both harmless and non-amyloidogenic. A recent study led by Ratnesh Lal at the University of California in San Diego reported that p3 peptides can form channels in cell membranes, leading to calcium influx and toxicity (see ARF related news story on Jang et al., 2010). The fragment is also amyloidogenic, Lal contends, although it aggregates more slowly than full-length Aβ (see Pike et al., 1995). The data from the new paper are not conclusive on these points: The complex of Aβ and shark proteins was not toxic to cells, Streltsov and colleagues found, but the surrounding shark proteins may have blocked Aβ’s effects. Streltsov said that they are working on removing the oligomer from its protein cage to directly test its toxicity. One potential complication is that without the surrounding shark immunoglobulins, Aβ will be free to aggregate further, leading to a more heterogeneous mixture. The authors are also making oligomers of longer Aβ fragments inside scaffolding proteins, Streltsov said, and will analyze those structures by x-ray as well.
In the second paper, Radford and colleagues focused on what prompts amyloidogenic proteins to assume their toxic forms. The authors used β2-microglobulin, which is part of the Class I major histocompatibility complex (MHC-I) and forms amyloid deposits if not properly cleared by the kidney, as when people are on dialysis. These deposits contain a truncated form of β2-microglobulin called ΔN6 that lacks six N-terminal amino acids and is highly amyloidogenic. First author Timo Eichner began by analyzing the structure of ΔN6, finding that in the amyloid form, the peptide bond at proline32 was changed from a cis to a trans configuration. This small change led to widespread repacking of the hydrophobic core of the protein, altering surface charges and making the protein more sticky and aggregation-prone. The missing N-terminal region normally serves to lock the protein into its native structure and preserve the cis bond, the authors conclude. This is the first description of the atomic-resolution structure of ΔN6, points out Lila Gierasch of the University of Massachusetts, Amherst, in an accompanying commentary.
Eichner and colleagues next wondered how native β2-microglobulin might change into an aggregation-prone state. They found that adding even 1 percent ΔN6 to a β2-microglobulin solution catalyzed the assembly of all the native protein into amyloid fibrils over a period of several weeks. Amyloid conversion occurred by collision; when a misfolded protein bumped into a native protein in just the right orientation, Radford said, the native protein reorganized into the toxic conformation. NMR measurements revealed that the contact caused the AB-loop (residues 13-22) of β2-microglobulin to relax. This in turn displaced a strand from the protein’s β sandwich structure, allowing the proline32 bond to flip into the trans conformation. In ongoing studies, Radford said, they hope to further elucidate the exact mechanism by which ΔN6 promotes this change. The reaction occurred more quickly under mildly acidic conditions, which the authors traced to the effect of a histidine residue picking up a proton. This positive charge destabilized the protein and made the alternate, amyloidogenic shape more favorable.
“We learned that proteins can change their conformation on a collision, and that very subtle changes such as picking up a proton or isomerizing a single bond is enough to cause the conversion,” Radford said. Next, they would like to use the methods they have developed to look at later steps in the aggregation pathway, Radford said. Knowing the mechanisms behind aggregation might provide clues as to how to prevent it, Radford suggested, for example, by designing small molecules to inhibit the initial conformational change.
It is not clear if a similar mechanism occurs in Aβ aggregation, Radford said, as unlike β2-microglobulin, Aβ does not start as a folded protein. Nonetheless, the basic finding that subtle changes in shape can have dramatic effects on aggregation might well hold for Aβ, Radford speculated. In addition, the research suggests that, “Potentially any protein in a dangerous conformation could convert an innocuous protein,” Radford said. “Catalytic conversion could be a general phenomenon in amyloidosis.” If true, this might provide a mechanism for the emerging data that Aβ can spread in a prion-like way through the brain (see ARF related news story on Eisele et al., 2009). In other words, just as 1 percent ΔN6 sets off aggregation in a β2-microglobulin solution, a minor increase in the concentration of Aβ42 might trigger fibril formation in the brain.—Madolyn Bowman Rogers
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University College Dublin
This elegant study by Eichner et al. considerably advances atomic-level understanding of the molecular features that underlie human β2-microglobulin (β2m) amyloidogenecity, and should facilitate the development of novel therapeutics for dialysis-related amyloidosis (DRA). Using NMR, they solved a solution structure of an amyloidogenic intermediate of N-terminal truncated β2m (ΔN6), a protein variant that is present in renal amyloid deposits of DRA patients. Furthermore, using NMR, Eichner et al. established specific conformational changes in full-length β2m that arose from bimolecular interactions with ΔN6 that presumably reflected the truncated protein’s prion-like seeding of β2m fibrils. Their findings reinforce that invaluable insight on the molecular basis of amyloidogenic polypeptides can be obtained by probing, at the atomic level, the native and non-native structures and aggregation propensities of pathogenically relevant variants (unmodified and post-translationally modified forms).
In contrast with the central pathogenic role that soluble Aβ oligomers are believed to have in Alzheimer’s disease, the pathogenic process in DRA is likely to be the abundant renal deposition of amyloid that contains β2m fibrils and various accessory molecules. Similar with other amyloidogenic polypeptides, β2m aggregates to form amyloid fibrils via distinct pathways that generate heterogeneous assemblies in dynamic equilibria (1). Nevertheless, this study, and others, suggests that discrete assemblies of ΔN6 are relevant to the disease process. First, greater than 20 percent of β2m in renal amyloid deposits of DRA patients is proteolyzed and contains the truncated ΔN6 form (2). Second, solid-state NMR studies indicate that the non-native trans-prolyl peptide bond at position 32, which is present in the ΔN6 intermediate, is also present in in-vitro generated β2m fibrils (3). Third, ΔN6 forms amyloid fibrils more readily than the full-length protein (4). Fourth, ΔN6 interacts more strongly with collagen than the full-length protein, and β2m amyloid deposits more readily in collagen-rich joints (5). Lastly, Eichner et al. showed that the trans-prolyl ΔN6 intermediate acted in a prion-like manner by specifically seeding fibril formation by the less amyloidogenic full-length β2m. If, indeed, ΔN6 assemblies are a primary culprit for DRA, a novel therapeutic strategy may be reagents that specifically inhibit proteolysis of the N-terminal of β2m.
As mentioned above, more than one assembly of β2m is likely to contribute to DRA. Thus, it would be interesting to establish if the ΔN6 intermediate studied by Eichner et al., or an equivalent full-length β2m assembly, is a pre-nucleus for fibrils or a direct precursor for oligomers that are formed by domain-swapped β2m variants (6,7). Alternatively, the latter conformers may form amyloid fibrils by distinct pathways that reflect the heterogeneous nature of β2m fibrils (8). Lastly, the finding by Eichner et al. that non-native structure in the ΔN6 intermediate is anchored by a propyl-peptide rearrangement is similar to the central role that proline has in stabilizing non-native conformations of several other amyloidogenic proteins, for example, the variable light chain from the κ4 Bence Jones protein, Len (9).
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O'Nuallain B, Allen A, Ataman D, Weiss DT, Solomon A, Wall JS. Phage display and peptide mapping of an immunoglobulin light chain fibril-related conformational epitope. Biochemistry. 2007 Nov 13;46(45):13049-58. PubMed.View all comments by Brian O’Nuallain
Massachusetts General Hospital & Harvard Medical School
The study describes an elegant tool for what has been a frustrating problem. Of particular interest to us, this study shows that Aβ dimers have striking structural similarities to the dimeric forms of the antimicrobial peptides human NP2, horseshoe crab tachystatin B, and mouse α-defensin. We recently reported on the antimicrobial activity of Aβ, and our newest data suggest dimerization is a key event in turning relatively inactive Aβ monomers into forms that can attack bacteria.
This is not a phenomenon restricted to Aβ. It has been known for some time that oligomerization has an important role in the action and targeting of a number of antimicrobial proteins (AMPs), including the archetypal antimicrobial peptide LL-37 which can form oligomers, fibrils, and birefringent amyloid (albeit, LL-37 amyloid has only been observed in vitro to date). Oligomerization is also a key mechanism for antimicrobial protein-mediated agglutination and inactivation of viral particles.
Despite this central role in AMP mechanisms, relatively few studies have characterized the oligomerization of antimicrobial peptides. Potentially, studies like this one could help us understand the cytotoxic action of not only Aβ, but AMPs as well. Notably, using conditions developed to promote Aβ oligomerization, we have generated LL-37 dimers with potentiated activity that are also resistant to bacterial defense proteases targeted at AMPs.View all comments by Robert Moir
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