Biology subjects

Biernat, J.

Publications and source records attributed to Biernat, J..

3 recordsLinked to original sources

bioRxiv2020

Combinatorial native MS and LC-MS/MS approach reveals high intrinsic phosphorylation of human Tau but minimal levels of other key modifications

Abnormal changes in the neuronal microtubule-associated protein Tau, such as hyperphosphorylation and aggregation, are considered hallmarks of cognitive deficits in Alzheimer disease. Hyperphosphorylation is thought to take place before aggregation, and therefore it is often assumed that phosphorylation predisposes Tau towards aggregation. However, the nature and extent of phosphorylation has remained ill-defined. Tau protein contains up to 85 potential phosphorylation sites (80 Ser/Thr, and 5 Tyr P-sites), many of which can be phosphorylated by various kinases because the unfolded structure of Tau makes them accessible. However, limitations in methods (e.g. in mass spectrometry of phosphorylated peptides, or antibodies against phospho-epitopes) have led to conflicting results regarding the overall degree of phosphorylation of Tau in cells. Here we present results from a new approach, that is based on native mass spectrometry analysis of intact Tau expressed in a eukaryotic cell system (Sf9) which reveals Tau in different phosphorylation states. The extent of phosphorylation is remarkably heterogeneous with up to [~]20 phosphates (Pi) per molecule and distributed over 51 sites (including all P-sites published so far and additional 18 P-sites). The medium phosphorylated fraction Pm showed overall occupancies centered at 8 Pi ({+/-} 5 Pi) with a bell-shaped distribution, the highly phosphorylated fraction Ph had 14 Pi ({+/-} 6 Pi). The distribution of sites was remarkably asymmetric (with 71% of all P-sites located in the C-terminal half of Tau). All phosphorylation sites were on Ser or Thr residues, but none on Tyr. Other known posttranslational modifications of Tau were near or below our detection limit (e.g. acetylation, ubiquitination). None of the Tau fractions self-assemble readily, arguing that Tau aggregation is not promoted by phosphorylation per se but requires additional factors.

neuroscience↗

bioRxiv2020

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau fibers

Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism proposed for prion pathogenesis. In cell cultures, the process is usually monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD, with pro-aggregant mutation, e.g., {Delta}K280 or P301L, [~]13.5 kDa) fused to GFP-based FRET pairs (YFP or CFP, [~]28 kD). Since the diameter of the reporter GFP ([~]3 nm) is [~]6.5 times larger than the {beta}-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of GFP tagged (N- or C-terminally) TauRD and TauFL (full-length Tau) on their aggregation behavior in vitro. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD{Delta}K-GFP was severely inhibited, even in the presence of nucleation enhancers (heparin and/or pre-formed PHFs from TauRD{Delta}K). Some rare fiber-like particles had a very different subunit packing from proper PHFs, as judged by STEM. The mass per length (MPL) values of TauRD{Delta}K fibrils are equivalent to 4.45 molecules/nm, close to the expected value for a paired-helical fiber with 2 protofilaments and cross-{beta} structure. By contrast, the elongated particles formed by TauRD{Delta}K-GFP have MPL values around [~]2, less than half of the values expected for PHFs, indicating that the subunit packing is distinct. Thus, both kinetic and structural observations are incompatible with a model whereby external Tau can form a template for PHF assembly of Tau-GFP in recipient cells. As a consequence, the observed local increase of FRET in recipient cells must be caused by other signalling processes.

neuroscience↗

bioRxiv2020

Lipid membrane templated misfolding and self-assembly of intrinsically disordered tau protein

The aggregation of the intrinsically disordered tau protein into highly ordered {beta}-sheet fibrils is implicated in many neurodegenerative disorders. Fibrillation mechanism remains unresolved, particularly early events that trigger tau misfolding and assembly. We investigated the role membrane plays in modulating aggregation of three tau variants, the largest isoform hTau40, the truncated construct K18, and a hyperphosphorylation mutant hTau40/3Epi. Despite being charged and soluble, tau proteins were also highly surface active and favorably interacted with anionic, but not zwitterionic, lipid monolayer at the air/water interface. Membrane binding induced macroscopic tau phase separation and {beta}-sheet-rich tau oligomer formation. Concomitantly, membrane morphology and lipid packing became disrupted. Our findings support a general tau aggregation mechanism wherein taus inherent surface activity and favorable electrostatic interactions drive tau-membrane association, inducing tau phase separation that is accompanied by misfolding and self-assembly of disordered tau into {beta}-sheet-rich oligomers, which subsequently seed fibrillation and deposition into diseased tissues.

biophysics↗