Inhibition of tau aggregation by the CCT3 and CCT7 apical domains

Abstract

The eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) is a molecular chaperone that assists protein folding in an ATP-driven manner. It consists of two stacked identical rings that are each made up of eight distinct subunits. Here, we show that the apical domains of subunits CCT3 and CCT7 from humans are strong inhibitors of tau aggregation, which is associated with several neurological disorders such as Alzheimer's and Parkinson's diseases. Kinetic analyses and negative-stain electron microscopy indicate that the mechanism of inhibition of tau aggregation by the apical domains of subunits CCT3 and CCT7 differ. Aggregation of tau alone, or in the presence of the apical domain of subunit CCT7, can be described by a fragmentation model whereas in the presence of the apical domain of subunit CCT3, it fits a saturating elongation and fragmentation mechanism. Coarse-grained molecular dynamics simulations show that tau interacts with different regions in the apical domains of subunits CCT3 and CCT7, in agreement with their different inhibition mechanisms.

Keywords: Alzheimer's disease; CCT/TRiC; chaperonins; protein aggregation; tau.

FIGURE 1

Inhibition of tau^4R fibril formation by the CCT3 and CCT7 apical domains is dose dependent. The kinetics of tau^4R fibril formation were monitored in the presence of different concentrations of the apical domains of CCT3 (a) and CCT7 (b) by measuring ThT fluorescence as a function of time as described in section 3. At least 12 curves were collected for each condition in two or three independent experiments and averaged. The continuous red lines through the data points are fits to Equation (1).

FIGURE 2

Negative stain electron microscopy of tau^4R fibrils. Representative images show tau^4R fibrils alone and the effect of adding 15 μM of apiCCT3, apiCCT7, and apiCCT8. Samples were prepared and imaged as described in section 3. The scale bars correspond to 200 nm.

FIGURE 3

Double‐logarithmic plots of aggregation half‐time as a function of tau^4R concentration in the absence or in the presence of apiCCT3 or apiCCT7. Aggregation half‐times (t_0.5, hours) were estimated from fits to Equation (1) of plots of ThT fluorescence as a function of time at varying tau^4R concentrations (in μM) alone (a) or in the presence of 1.75 μM apiCCT3 (b) or 2.5 μM apiCCT7 (c), as described in section 3. The dashed lines represent the linear fits to the data. The concentration of apiCCT3 was selected to be lower than that of apiCCT7 as a smaller amount of the former was sufficient to induce a significant change in t_0.5. The errors in the values of log₁₀(t_0.5) are under 3%.

FIGURE 4

Structural analysis of the interaction between tau^4R and CCT apical domains. Heat maps show interaction probabilities between tau residues and residues in apiCCT3 (a), apiCCT7 (b), and apiCCT8 (c), which range from 0 (white, no interaction) to 0.5 (dark blue, high interaction probability). The heat maps were generated for the interactions of groups of three consecutive residues in tau^4R (y‐axis) with groups of three consecutive residues in the CCT apical domains (x‐axis). Only the sequence corresponding to the R1 and R2 regions of tau^4R is shown since the interactions of the apical domains with R3 and R4 are minimal. The electrostatic potential of each apiCCT is shown on the left using a color code of red to blue for surface potential from −5 to 5 in units of kcal/mol*e⁻, respectively. The black boxes in the structures and corresponding heat maps indicate regions that differ between apiCCT3 and apiCCT7 in their interaction with tau^4R whereas the orange boxes indicate the region that interacts with tau^4R in both domains.