NMR spectroscopy of Tau, a naturally unfolded protein


The neuronal tau protein is associated with tauopathies, most notably Alzheimer’s disease (AD). tauopathies are characterised by intraneuronal aggregation of tau protein into filaments causing neurofibrillary degeneration and synaptic dysfunctions, which lead to dementia. The urgent need for better diagnostics for AD and related tauopathies, as well as disease-modifying treatments, is immense due to the expected growing burden link to demographic changes. Our aim is to understand the relationship between phosphorylation and cellular dysfunctions induced by tau pathogenesis, based on collaborations with experts in the AD field in Lille, combining our complementary approaches to decipher key mechanisms of tau dysregulation. The team was the first to publish on NMR signal assignments of tau (Lippens et al., 2004; Smet et al., 2004). In addition, in a milestone paper, we showed the potential of nuclear magnetic resonance spectroscopy to characterize protein phosphorylation, a methodology that has been widely adopted nowadays (Landrieu et al., 2006). Our focus of interest is the impact of phosphorylation on tau structure and function. We have investigated the impact of tau phosphorylation on its interaction with physiological partners, such as the AD genetic determinant Bin1 (Sartori et al., 2019, Lasorsa et al. 2018, Malki et al., 2017, Sottejeau et al., 2015), the link between phosphorylation and conformational perturbation of tau (Ahuja et al., 2016, Gandhi et al., 2015) and finally the relation between specific phosphorylation of tau and its aggregation susceptibility ( Despres et al., 2019, Despres et al., 2017; Qi et al., 2016). We have also investigated modulation of protein-protein interaction, involving phospho-Tau epitopes, using peptide-based compounds (Andrei et al., 2018, Milroy et al., 2015; Smet et al., 2005). Another strategy to slow down Tau aggregation is the use of single domain antibodies (Dupré et al., 2019) and monoclonals (Albert et al., 2019) targeting the central region of tau.

Our project is supported by the ANR ToNIC, ANR PykAlz, ISite ULNE Tunable and the Labex Distalz

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Tau_1
A. Superimposed 1H, 15N, HSQC spectra of Tau (red) with tau mixed with non-labelled single domain antibody spectra (overlaid in blue). Each resonance in this spectrum can be linked to a specific amino acid residue in Tau sequence (assignment).
B. The graph shows the normalized intensities I/I0 of corresponding resonances in the two-dimensional spectra of Tau with equimolar quantity of the single domain antibody (I, blue spectrum) or free in solution (I0, red spectrum) for residues along the Tau sequence. The interaction region is boxed in red and corresponds to peaks that are broadened beyond detection (peaks that disappear in the blue spectrum). The identified interaction region is shown along the Tau sequence as a green bar. PRD corresponds to the proline riche region of Tau. R1 to R4 are the (partially) repeated sequences in the microtubule binding domain of Tau.
C. 3D crystal structure of a single domain antibody (green ribbon) in interaction with Fyn SH3 domain (cyan ribbon). Interaction is mediated by the complementarity determining regions CDR 1 to 3 (red, pink and blue loops). The crystals visualized in a drop correspond to complexes between a phospho-Tau single domain antibody and a Tau phospho-peptide.
D. Sensorgram (in red) of Tau titration by a single domain antibody, monitored by surface plasmon resonance spectroscopy or SPR: Single cycle kinetics analysis was performed on surface-immobilized biotinylated Tau, with five injections of increasing concentration of the antibody. Black line corresponds to the fit of the data to a one-to-one binding model.
E. Thioflavin T fluorescence in vitro Tau aggregation assay, in the presence of heparin: increasing concentration of a single domain antibody directed against Tau (from red curve to blue one, stoichiometries Tau: antibody, 1:0 red curve, 1:0.2 green curve, 1:0.5 purple curve, 1.1 blue curve, control without heparin orange curve) decreases Tau aggregation in this assay, as monitored by the fluorescence intensity emitted at 490nm.