Factors influencing compact-extended structure equilibrium in oligomers of aß1-40 peptide--an ion mobility mass spectrometry study.

Oligomers formed by amyloid ß (Aß) peptide are widely believed to be the main neurotoxic agent in Alzheimer's disease. Studies discovered a broad variety of oligomeric forms, which display different levels of toxicity. Some of these forms may further assemble into mature fibrils, while other might be off-pathway from conversion to fibrils and assemble into alternative forms. To better understand a relationship between the structure and toxicity of Aß oligomers, we require systematic characterization and classification of all possible forms, facilitating rational design of the beneficial modifiers of their activity. In previous ion mobility analysis of Aß1-40 oligomers, we have detected the coexistence of two alternative structural forms (compact and extended) in a pool of low-order Aß1-40 oligomers. These forms may represent two pathways of the oligomer evolution, leading either to fibrils or to off-pathway oligomers, which are potential candidates for the neurotoxic species. Here, we have analyzed the impact of incubation time, the presence of selected metal ions and the effect of a series of point mutations on mutual population of alternative forms. We have shown that a salt bridge D23K28 provides stabilization of the compact form whereas G25 is required for the existence of the extended form. We have found that binding of metal ions also stabilizes the compact form. These results improve our understanding of the possible molecular mechanism of the bifurcation of structural evolution of non-monomeric Aß species into an off-fibril pathway, ultimately leading to the formation of potentially neurotoxic species.

Ion mobility separation coupled with MS detects two structural states of Alzheimer's disease Aß1-40 peptide oligomers.

Mounting evidence points to the soluble oligomers of amyloid ß (Aß) peptide as important neurotoxic species in Alzheimer's disease, causing synaptic dysfunction and neuronal injury, and finally leading to neuronal death. The mechanism of the Aß peptide self-assembly is still under debate. Here, Aß1-40 peptide oligomers were studied using mass spectrometry combined with ion mobility spectrometry, which allowed separation of the signals of numerous oligomers and measurement of their collisional cross-section values (Ω). For several oligomers, at least two different species of different Ω values were detected, indicating the presence of at least two families of conformers: compact and extended. The obtained results are rationalized by a set of molecular models of Aß1-40 oligomer structure that provided a very good correlation between the experimental and theoretical Ω values, both for the compact and the extended forms. Our results indicate that mass spectrometry detects oligomeric species that are on-pathway in the process of fibril formation or decay, but also alternative structures which may represent off-pathway evolution of oligomers.

A direct determination of the dissociation constant for the Cu(II) complex of amyloid beta 1-40 peptide.

Interactions of amyloid beta (Abeta) peptides with Cu(II) are believed to play a crucial role in the molecular mechanisms of neurotoxicity of Alzheimer's disease. There is, however, a serious disagreement regarding the strength of Cu(II) binding to these peptides. We used recombinant amyloid beta peptide 1-40 (Abeta40) to determine the stoichiometry and dissociation constants of Cu(II)-Abeta40 complexes using fluorescence spectroscopy. A single Cu(Abeta40) complex, characterized with the conditional dissociation constant K(d)(cond) = 57 +/- 5 nM was identified. This complex does not bind Hepes buffer molecules, as indicated by the total lack of relationship between K(d)(cond) values and Hepes concentration. The differences between this and other determinations of this constant and its relevance for the understanding of Cu(II) interaction with Abeta peptides are discussed.

The binding constant for amyloid Abeta40 peptide interaction with human serum albumin.

Human serum albumin (HSA) is the major carrier of Abeta peptides in blood plasma. 1:1 interaction stoichiometries were established in previous indirect antibody-based studies for both Abeta40 and Abeta42, but corresponding binding constants were not provided. In this study we applied direct titrations of HSA with Abeta40 monitored using circular dichroism spectroscopy and obtained a dissociation constant (K(d)) of 5+/-1 microM for a HSA complex with Abeta40. The interaction resulted in an increase of the alpha-helical contents in the complex, compared to its components, which is quantitatively consistent with the known ability of Abeta40 to adopt a partially alpha-helical conformation in a hydrophobic environment. The relevance of these findings for the role of HSA in Abeta physiology is discussed.

[Expression of glutathione-S-transferase isoenzymes in larynx cancer]. / Ekspresja izoenzymów transferazy glutationowej w raku krtani.

Carcinoma planoepitheliale of larynx is the most common cancer present in the upper part of the respiratory system. The molecular mechanism of this cancer generation is unknown and there is no a specific marker for its early diagnosis. The properties, physiological function, as well as the results of studies on genetic polymorphism indicate that glutathione-S-transferase (GST) may play an important role in etiology of this cancer. In the present work the studies on expession of GST isoenzymes pi, alfa, mu4, and mu5 in larynx cancer (carcinoma planoepitheliale laryngis) were performed. Specimens of larynx cancer and their corresponding normal adjacent tissues were obtained from patients by surgery. Expression was studied on the level of mRNA using RT-PCR method. The results indicated raised expression of GST pi (the major isoform in larynx) and lowered expression of GSTs mu4 and alfa in cancer as compared with the control tissue. No expression of GST mu5 was observed in tumor and normal larynx tissues. Obtained results indicate various mechanism in regulation of GSTs expression in cancerous and normal larynx tissues. They also indicate a special role of GST pi in larynx carcinogenesis.