Protein aggregation into amyloid fibrils is associated with several widespread neurodegenerative disorders. Alzheimer's disease is one of the most prominent cases, with an ever-increasing number of afflicted patients. The disorder is characterized by memory loss and is considered to be caused by amyloid-β extracellular plaques and neurofibrillary tangles made from hyperphosphorylated Tau. The main physiological function of the Tau protein is to maintain microtubule dynamics and promote polymerization. In vitro Tau protein filament formation is induced by the presence of polyanions such as heparin. A truncated form of Tau protein (dGAE), which can assemble into filaments without polyanions, forms a proteolytically stable core region in full-length Tau paired helical filament (PHF) [1]. The dGAE fragment could serve as a model system for the search for inhibitors of Tau protein aggregation [2].
Considering the rise of Alzheimer's disease incidences worldwide, understanding the process of amyloid aggregation is a crucial step in developing drugs. Research of dementia is heavily focused on the process of how pathogenic Tau can spread throughout the brain, as it may provide a key therapeutic target for slowing the development of tauopathies. There are a lot of disputed questions concerning the physiological and pathological consequences of PHF-core Tau self-assembly, therefore we aim to elucidate the influence of dGAE fragment on Tau protein aggregation.
Tau 2N4R gene was inserted into pET Champion His-SUMO vector by TA cloning method to create the ULP1-cleavable N-terminally His-SUMO-tagged Tau. The recombinant His-SUMO-tagged Tau (2N4R isoform) protein was purified by immobilized metal affinity chromatography and consequently cleaved by the ULP1 protease to remove His-SUMO-tag. A truncated form of Tau protein (297-391) (dGAE) was purified by cation exchange chromatography. Both proteins were purified further by size-exclusion chromatography. We used purified Tau 2N4R protein and dGAE fragment for monitoring the amyloid aggregation process. All performed aggregation kinetics were followed using a thioflavin-T fluorescence assay. Atomic force microscopy was performed to analyze the morphology of the formed aggregates. We used FTIR spectroscopy to observe the differences in secondary structures of the dGAE fragment and dGAE+Tau protein aggregates.
Looking at the results we can see the aggregation halftime and lag time decrease when dGAE fragment concentration increases. Thus, the results obtained confirm the fact that is stated in the articles that at higher concentrations the dGAE fragment aggregates faster. The apparent rate constant varies within error at different concentrations. In vitro FTIR analysis indicates that the soluble Tau protein is a natively unfolded protein dominated by random coil structure, whereas Alzheimer PHFs show an increase in the level of β-structure. The results support a model in which the repeat domain of Tau (which lies within the core of PHFs) adopts an increasing level of β-structure during aggregation. FTIR spectra show that when dGAE aggregates, the resulting dGAE fibrils associate with monomeric Tau protein.