Leonid Breydo

Fibrillar Oligomers Nucleate the Oligomerization of Monomeric Amyloid β but Do Not Seed Fibril Formation

Soluble amyloid oligomers are potent neurotoxins that are involved in a wide range of human degenerative diseases, including Alzheimer disease. In Alzheimer disease, amyloid β (Aβ) oligomers bind to neuronal synapses, inhibit long term potentiation, and induce cell death. Recent evidence indicates that several immunologically distinct structural variants exist as follows: prefibrillar oligomers (PFOs), fibrillar oligomers (FOs), and annular protofibrils. Despite widespread interest, amyloid oligomers are poorly characterized in terms of structural differences and pathological significance. FOs are immunologically related to fibrils because they react with OC, a conformation-dependent, fibril-specific antibody and do not react with antibodies specific for other types of oligomers. However, fibrillar oligomers are much smaller than fibrils. FOs are soluble at 100,000 × g, rich in β-sheet structures, but yet bind weakly to thioflavin T. EPR spectroscopy indicates that FOs display significantly more spin-spin interaction at multiple labeled sites than PFOs and are more structurally similar to fibrils. Atomic force microscopy indicates that FOs are approximately one-half to one-third the height of mature fibrils. We found that Aβ FOs do not seed the formation of thioflavin T-positive fibrils from Aβ monomers but instead seed the formation of FOs from Aβ monomers that are positive for the OC anti-fibril antibody. These results indicate that the lattice of FOs is distinct from the fibril lattice even though the polypeptide chains are organized in an immunologically identical conformation. The FOs resulting from seeded reactions have the same dimensions and morphology as the initial seeds, suggesting that the seeds replicate by growing to a limiting size and then splitting, indicating that their lattice is less stable than fibrils. We suggest that FOs may represent small pieces of single fibril protofilament and that the addition of monomers to the ends of FOs is kinetically more favorable than the assembly of the oligomers into fibrils via sheet stacking interaction. These studies provide novel structural insight into the relationship between fibrils and FOs and suggest that the increased toxicity of FOs may be due to their ability to replicate and the exposure of hydrophobic sheet surfaces that are otherwise obscured by sheet-sheet interactions between protofilaments in a fibril.

Annealing Prion Protein Amyloid Fibrils at High Temperature Results in Extension of a Proteinase K-resistant Core

Amyloids are highly ordered, rigid β-sheet-rich structures that appear to have minimal dynamic flexibility in individual polypeptide chains. Here, we demonstrate that substantial conformational rearrangements occur within mature amyloid fibrils produced from full-length mammalian prion protein. The rearrangement results in a substantial extension of a proteinase K-resistant core and is accompanied by an increase in the β-sheet-rich conformation. The conformational rearrangement was induced in the presence of low concentrations of Triton X-100 either by brief exposure to 80 °C or, with less efficacy, by prolonged incubation at 37 °C at pH 7.5 and is referred to here as “annealing.” Upon annealing, amyloid fibrils acquired a proteinase K-resistant core identical to that found in bovine spongiform encephalopathy-specific scrapie-associated prion protein. Annealing was also observed when amyloid fibrils were exposed to high temperatures in the absence of detergent but in the presence of brain homogenate. These findings suggest that the amyloid fibrils exist in two conformationally distinct states that are separated by a high energy barrier and that yet unknown cellular cofactors may facilitate transition of the fibrils into thermodynamically more stable state. Our studies provide new insight into the complex behavior of prion polymerization and highlight the annealing process, a previously unknown step in the evolution of amyloid structures.

Site-specific Conformational Studies of Prion Protein (PrP) Amyloid Fibrils Revealed Two Cooperative Folding Domains within Amyloid Structure

Despite the ability of most proteins to form amyloid, very little is know about amyloid fibril structures and the factors that govern their stability. Using amyloid fibrils produced from full-length prion protein (PrP), we describe a reliable approach for determining both site-specific and global conformational stability of the fibrillar form. To measure site-specific stability, we produced six variants of PrP by replacing the residues at positions 88, 98, 127, 144, 196, and 230 with cysteine, labeled the new cysteines with the fluorescent dye acrylodan, and investigated their conformational status within the amyloid form in guanidine hydrochloride-induced denaturation experiments. We found that the fibrils labeled at positions 127, 144, 196, and 230 displayed cooperative unfolding and showed a very high C½ value similar to that observed for the global unfolding of the amyloid structure. The unfolding at residue 98 was also cooperative; however, it showed a C½ value substantially lower than that of global unfolding, whereas the unfolding of fibrils labeled at residue 88 was non-cooperative. These data illustrate that there are at least two independent cooperative folding domains within the amyloid structure of the full-length PrP. In addition, kinetic experiments revealed only a partial overlap between the region that constituted the fibrillar cross-β core and the regions that were involved in nucleation. This result illustrates that separate PrP regions accounted for the nucleation and for the formation of the conformationally most stable fibrillar core.

DNA alkylation by leinamycin can be triggered by cyanide and phosphines.

Previous work has shown that alkylation of DNA by the antitumor agent leinamycin (1) is potentiated by reaction of the antibiotic with thiols. Here, it is shown that other soft nucleophiles such as cyanide and phosphines can also trigger DNA alkylation by leinamycin. Overall, the results suggest that reactions of cyanide and phosphines with leinamycin produce the oxathiolanone intermediate (2), which is known to undergo rearrangement to the DNA-alkylating episulfonium ion 4.

Thiol-dependent DNA cleavage by 3H-1,2-benzodithiol-3-one 1,1-dioxide

Hydrodisulfides (RSSH) have previously been implicated as key intermediates in thiol-triggered oxidative DNA damage by the antitumor agent leinamycin. In an effort to better understand DNA damage by RSSH and to expand on the number and type of chemical systems that produce this reactive intermediate, the ability of 3H-1,2-benzodithiol-3-one 1,1-dioxide (11) to serve as a thiol-dependent DNA cleaving agent has been investigated. The findings reported here indicate that reaction of 11 with thiols results in release of RSSH and subsequent oxidative DNA strand cleavage.

Two (E,E)- and (Z,E)-thiazol-5-ylpenta-2,4-dienones.

In order to characterize the structural elements that might play a role in non-covalent DNA binding by the antitumor antibiotic leinamycin, we have solved the crystal structures of the two leinamycin analogs, methyl (R)-5-[2-[1-(tert-butoxycarbonylamino)ethyl]thiazol-4-yl]penta-(E,E)-2,4-dienoate, C(16)H(22)N(2)O(4)S, (II), and 2-methyl-8-oxa-16-thia-3,17-diazabicyclo[12.2.1]heptadeca-(Z,E)-1(17),10,12,14-tetraene-4,9-dione, C(14)H(16)N(2)O(3)S, (III). The penta-2,4-dienone moiety in both of these analogs adopts a conformation close to planarity, with the thiazole ring twisted out of the plane by 12.9 (2) degrees in (II) and by 21.4 (4) degrees in (III).

The macrocycle of leinamycin imparts hydrolytic stability to the thiol-sensing 1,2-dithiolan-3-one 1-oxide unit of the natural product

Reaction of cellular thiols with the 1,2-dithiolan-3-one 1-oxide moiety of leinamycin triggers the generation of DNA-damaging reactive intermediates. Studies with small, synthetic analogues of leinamycin reveal that the macrocyclic portion of the natural product imparts remarkable hydrolytic stability to the 1,2-dithiolan-3-one 1-oxide heterocycle without substantially compromising its thiol-sensing property.