Structure and Dynamics of Human Chemokine CCL16-Implications for Biological Activity.

Human C-C motif ligand 16 (CCL16) is a chemokine that is distinguished by a large cleavable C-terminal extension of unknown significance. Conflicting data have been reported concerning its tissue distribution and modulation of expression, rendering the biological function of CCL16 enigmatic. Here, we report an integrated approach to the characterisation of this chemokine, including a re-assessment of its expression characteristics as well as a biophysical investigation with respect to its structure and dynamics. Our data indicate that CCL16 is chiefly synthesised by hepatocytes, without an appreciable response to mediators of inflammation, and circulates in the blood as a full-length protein. While the crystal structure of CCL16 confirms the presence of a canonical chemokine domain, molecular dynamics simulations support the view that the C-terminal extension impairs the accessibility of the glycosaminoglycan binding sites and may thus serve as an intrinsic modulator of biological activity.

Nanoparticle standards for immuno-based quantitation of α-synuclein oligomers in diagnostics of Parkinson's disease and other synucleinopathies.

Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by symptoms such as rigor, tremor and bradykinesia. A reliable and early diagnosis could improve the development of early therapeutic strategies before death of dopaminergic neurons leads to the first clinical symptoms. The sFIDA (surface-based fluorescence intensity distribution analysis) assay is a highly sensitive method to determine the concentration of α-synuclein (α-syn) oligomers which are presumably the major toxic isoform of α-syn and potentially the most direct biomarker for PD. Oligomer-based diagnostic tests require standard molecules that closely mimic the native oligomer. This is particularly important for calibration and assessment of inter-assay variation. In this study, we generated a standard in form of α-syn coated silica nanoparticles (α-syn-SiNaPs) that are in the size range of α-syn oligomers and provide a defined number of α-syn epitopes. The preparation of the sFIDA assay was realized on an automated platform to allow handling of high number of samples and reduce the effects of human error. The assay outcome was analyzed by determination of coefficient of variation and linearity for the applied α-syn-SiNaPs concentrations. Additionally, the limit of detection and lower limit of quantification were determined yielding concentrations in the lower femtomolar range.

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.