High-Affinity Binding of Monomeric but Not Oligomeric Amyloid-ß to Ganglioside GM1 Containing Nanodiscs.

The interaction of the amyloid-ß protein (Aß) with neuronal cell membranes plays a crucial role in Alzheimer's disease. Aß undergoes structural changes upon binding to ganglioside GM1 containing membranes leading to altered molecular characteristics of the protein. The physiological role of the Aß interaction with the ganglioside GM1 is still unclear. In order to further elucidate the molecular requirements of Aß membrane binding, we tested different nanodiscs varying in their lipid composition, regarding the charge of the headgroups as well as ganglioside GM1 concentration. Nanodiscs are excellent model membrane systems for studying protein membrane interactions, and we show here their suitability to investigate the membrane interaction of Aß. In particular, we set out to investigate whether the binding activity of GM1 to Aß is specific for the assembly state of Aß and compared the binding affinities of monomeric with oligomeric Aß. Using fluorescence titration experiments, we demonstrate high-affinity binding of Aß(1-40) to GM1 containing nanodiscs, with dissociation constants, KD, in the range from 25 to 41 nM, in a GM1 concentration-dependent manner. Biolayer interferometry experiments confirmed the high-affinity binding of monomeric Aß(1-40) (KD of 24 nM to 49 nM) as well as of Aß(1-42) (KD of 30 nM) to GM1 containing nanodiscs, and no binding to phospholipid containing nanodiscs. Interestingly, and in contrast to monomeric Aß, neither oligomeric Aß(1-40) nor oligomeric Aß(1-42) binds to GM1 nanodiscs. To the best of our knowledge, this is the first report of a loss of function for monomeric Aß upon aggregation.

Optimization of d-Peptides for Aß Monomer Binding Specificity Enhances Their Potential to Eliminate Toxic Aß Oligomers.

Amyloid-beta (Aß) oligomers are thought to be causative for the development and progression of Alzheimer's disease (AD). Starting from the Aß oligomer eliminating d-enantiomeric peptide D3, we developed and applied a two-step procedure based on peptide microarrays to identify D3 derivatives with increased binding affinity and specificity for monomeric Aß(1-42) to further enhance the Aß oligomer elimination efficacy. Out of more than 1000 D3 derivatives, we selected seven novel d-peptides, named ANK1 to ANK7, and characterized them in more detail in vitro. All ANK peptides bound to monomeric Aß(1-42), eliminated Aß(1-42) oligomers, inhibited Aß(1-42) fibril formation, and reduced Aß(1-42)-induced cytotoxicity more efficiently than D3. Additionally, ANK6 completely inhibited the prion-like propagation of preformed Aß(1-42) seeds and showed a nonsignificant tendency for improving memory performance of tg-APPSwDI mice after i.p. application for 4 weeks. This supports the hypothesis that stabilization of Aß monomers and thereby induced elimination of Aß oligomers is a suitable therapeutic strategy.

Arabidopsis DEAD-box RNA helicase UAP56 interacts with both RNA and DNA as well as with mRNA export factors.

The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.