Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau

Abstract

Tau is an intrinsically disordered microtubule-associated protein that is implicated in several neurodegenerative disorders called tauopathies. In these diseases, Tau is found in the form of intracellular inclusions that consist of aggregated paired helical filaments (PHFs) in neurons. Given the importance of this irreversible PHF formation in neurodegenerative disease, Tau aggregation has been extensively studied. Several different factors, such as mutations or post translational modifications, have been shown to influence the formation of late-stage non-reversible Tau aggregates. It was recently shown that zinc ions accelerated heparin-induced oligomerization of Tau constructs. Indeed, in vitro studies of PHFs have usually been performed in the presence of additional co-factors, such as heparin, in order to accelerate their formation. Using turbidimetry, we investigated the impact of zinc ions on Tau in the absence of heparin and found that zinc is able to induce a temperature-dependent reversible oligomerization of Tau. The obtained oligomers were not amyloid-like and dissociated instantly following zinc chelation or a temperature decrease. Finally, a combination of isothermal titration calorimetry and dynamic light scattering experiments showed zinc binding to a high-affinity binding site and three low-affinity sites on Tau, accompanied by a change in Tau folding. Altogether, our findings stress the importance of zinc in Tau oligomerization. This newly identified Zn-induced oligomerization mechanism may be a part of a pathway different of and concurrent to Tau aggregation cascade leading to PHF formation.

Zinc promotes liquid–liquid phase separation of tau protein

“…Altogether, the present data clearly demonstrate that zinc strongly promotes LLPS of full-length tau and identify specific structural elements within the protein that are important for this effect. The finding that Cys residues are essential for Zn 2+ -induced LLPS is consistent with previous isothermal titration calorimetry studies that identified a single high affinity binding site (with reported Kd between 0.5 and 4 µM) in which zinc is coordinated by two Cys and two His residues (His330 and His362) within the R2-R3 tau region (22,24). However, this high affinity binding appears to be insufficient to promote LLPS, as zincinduced droplet formation was observed only at by guest on July 5, 2020 http://www.jbc.org/ Downloaded from Zn 2+ :tau441 molar ratios above 2:1.…”

“…1B). It should be noted that an increase in turbidity of tau solution (in 50 mM Tris buffer) upon zinc addition was also observed in a recent study by Roman et al (22). However, this effect was attributed to oligomerization of the protein, not to LLPS.…”

“…The finding that the extent of tau LLPS gradually increases at zinc concentrations corresponding to Zn 2+ to tau molar ratios above 4:1 (Fig. 1D) suggests that at least some of the low affinity binding sites become populated only at zinc concentration higher than expected for the reported average Kd value ~20 µM (22).…”

“…Thus, what is the mechanism by which zinc promotes this process? Certain clues in this regard are provided by the finding that zinc binding results in a more compact structure of tau monomers (22), suggesting that coordination through Cys and His residues induces local hairpin-like folding within the pseudo-repeat region (22,25). A similar local folding has also been suggested for the acidic Nterminal region that contains many potential zinc chelating amino acids (25).…”

“…However, this high affinity binding appears to be insufficient to promote LLPS, as zincinduced droplet formation was observed only at by guest on July 5, 2020 http://www.jbc.org/ Downloaded from Zn 2+ :tau441 molar ratios above 2:1. This conundrum may be explained by the most recent finding that, in addition to the high binding site, tau contains three low affinity zinc-binding sites, with an average Kd around 20 µM (22). These low affinity binding sites appear to be also essential for zinc's ability to promote LLPS of tau.…”