Structural properties of Aβ protofibrils stabilized by a small molecule

Abstract

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of A␤ protofibrils. Library screening yielded several molecules that stimulate A␤ aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts A␤(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37°C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-A␤ aggregates exhibit a low thioflavin T response. Like A␤ fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-A␤ aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous A␤ fibril formation reaction: Ϸ12 of the 39 A␤(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the A␤ molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in A␤ conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester A␤ in a form and͞or location that cannot engage the toxic pathway.amyloid ͉ chemical biology ͉ hydrogen exchange ͉ proline scanning

Small-molecule conversion of toxic oligomers to nontoxic β-sheet–rich amyloid fibrils

“…However, it mostly remains unclear whether monomers, oligomers or protofibrillar structures are specifically targeted by modulators of amyloid aggregation 43,44 and whether compound effects on certain aggregate species (e.g. protofibrils) are toxic or beneficial for mammalian cells 13 . In this study, we have searched for small molecules that promote spontaneous A42 assembly.…”

“…However, more detailed timeresolved studies with well-defined A42 aggregate species are necessary to compare the results obtained with O4 and methylene blue. While O4 promotes the formation of long amyloid fibrils the compound calmidazolium chloride (CLC) was shown to stabilize protofibrillar structures 13 . A stabilization of -sheet-rich, nontoxic, spherical A conformations was observed with the compound scyllo-inositol 47 , while the polyphenol EGCG was shown to convert A42 peptides into non-toxic, unstructured oligomers 18 .…”

“…Biochemical and cell biological studies indicate that amyloid formation pathways can be manipulated with small molecules 9 Osmolytes such as trimethylamine N-oxide or glycerol as well as short peptides were shown to accelerate A fibrillogenesis 10,11 , while small molecules such as scyllo-inositol stabilize the formation of oligomers and protofibrils 12 . In contrast, compounds such as dopamine or calmidazolium chloride were found to block amyloid fibrillogenesis [13][14][15] . Substances selectively inhibiting A oligomerization 16 , redirecting amyloid formation pathways 17,18 or remodeling preformed amyloid structures 19 were also reported.…”

Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research

“…Whether some of these species represent off-or on-pathway intermediates to amyloid fibril formation remains unknown. Some of the key distinguishing features of protofibrillar aggregates are that they (i) contain a mixture of different secondary structure elements with a predominance of β-sheet 20 ; (ii) bind amyloid-specific dyes, such as Congo red and Thioflavin-T (ThT), although less strongly than mature fibrils [20][21][22] ; and (iii) are less stable than mature amyloid fibrils and exist in equilibrium with monomeric Aβ 20,21,23 . In the presence of monomeric Aβ, all these species convert to mature amyloid fibrils by monomer addition.…”

From Green Bacteria to Human Dementia: A Novel Model for Discovering Amyloid Assembly Inhibitors

“…In recent years, accumulating evidence has indicated that soluble amyloid assemblies rather than the mature amyloid plaques may actually be the pathological culprits that result in the degenerative phenomena ( Figure 1) (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13).…”