Identification of a novel tetrameric structure for human apolipoprotein-D.

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded ß-barrel with a single adjacent α-helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimer's disease, where apolipoprotein-D overexpression reduces the amyloid-ß burden in Alzheimer's disease mouse models. Oligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids. Here, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a ∼95 to ∼100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

Proteomic analysis of cerebrospinal fluid in Alzheimer's disease: wanted dead or alive.

Clinical diagnosis of Alzheimer's disease (AD) relying on symptomatic features has a low specificity, emphasizing the importance of the pragmatic use of neurochemical biomarkers. The most advanced and reliable markers are amyloid-ß (Aß42), total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) with relatively high levels of sensitivity, specificity, and diagnostic accuracy. Recent advances within the field of proteomics offer the potential to search for novel biomarkers in CSF by using modern methods, such as microarrays. The purpose of this study was to identify pathognostic proteins in CSF obtained from patients whose clinical AD diagnosis was confirmed by the "core" biomarkers. CSF samples were obtained from 25 AD patients and 25 control individuals. The levels of Aß42, t-tau, and p-tau were measured by ELISA. In the microarray experiments, ultrasensitive slides representing of 653 antigens were used. Apolipoprotein E genotyping was also determined. A decrease of seven CSF proteins in AD were found, four of them (POLG, MGMT, parkin, and ApoD) have a protective function against neuronal death, while the remaining three proteins (PAR-4, granzyme B, Cdk5) trigger multiple pathways facilitating neuronal cell death. Since these proteins from CSF samples could not be identified by western blot, their decreased levels in AD patients were not verified. Our results provide new information of pathognostic importance of POLG and granzyme B in AD. Although the function of MGMT, parkin, ApoD, PAR-4, and Cdk5 was previously known in AD, the findings presented here provide novel evidence of the significance of CSF analysis in the mapping of the AD pathomechanism.