Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation

Abstract

It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation.

Figure 1. Schematic illustration of an aggregation reaction network.

(a) This network is made up by the microscopic steps in the aggregation mechanism of an amyloidogenic protein (blue), and the several possible interactions between a molecular chaperone (green) and the different protein species present in the system; (b) Depending on the target species, different microscopic steps can be affected.

Figure 2. Molecular chaperones can affect individual microscopic steps in the aggregation process.

(a–c) Numerical integration of the master equations illustrates how perturbations of specific microscopic aggregation events modify in characteristic ways the global kinetic profiles. The analysis of the changes in the macroscopic profiles provides quantitative information on the microscopic events altered by the molecular chaperones. (d–f) To demonstrate the experimental consequences of the different mechanisms described in (a–c) we consider the aggregation kinetics of the Aβ42 peptide and the prion protein Ure2p of in the absence and presence of different molecular chaperones at increasing concentrations. (d) DNAJB6 inhibits strongly the primary nucleation rate of unseeded 3 μM Aβ42 aggregation reactions in phosphate buffer pH 8.0 at 37 °C (ref. 31); (e) a member of the Hsp70 family (Ssa1) suppresses specifically the fibril elongation rate in the aggregation process of 30 μM Ure2p in Tris buffer pH 7.5 at 30 °C (ref. 32); (f) a molecular chaperone belonging to the Brichos family (proSP-C Brichos) inhibits specifically the secondary nucleation rate in the aggregation of 3 μM Aβ42 in phosphate buffer pH 8.0 at 37 °C (ref. 33). The molecular equivalents of chaperones as well as the microscopic rate constants as a function of molecular chaperone concentration, evaluated by fitting the experimental data with Equation 1, are shown in the inserts. The continuous line in the inset in (e) corresponds to the evaluation of the binding equilibrium constant according to Equation 4.

Figure 3. Identification of the target species interacting with different molecular chaperones.

Two specific sets of data taken from the literature are re-analysed in the frame of the novel kinetic model developed here. The reaction profiles at different molecular chaperone concentrations are compatible with binding to the fibril ends in the case of the Ure2p-Saa1 system (a,b) and to the fibril surface in the case of the Aβ42-Brichos system (c,d). The global fitting was performed by applying the set of equations reported in the Supplementary Equations 5–24.

Figure 4. Molecular chaperones can affect simultaneously several microscopic steps in the aggregation process.

Reaction profiles of the aggregation of 3 μM Aβ42 in the absence and presence of (a) αB-crystallin and (b) a member of the Brichos family (Bri2-Brichos) in phosphate buffer pH 8.0 at 37 °C. The continuous lines represent the integrated rate laws. The molecular equivalents of chaperones as well as the microscopic rate constants as a function of the molecular chaperone concentration relative to Aβ42 are reported in (c) and in (d) relative to the values in the absence of the molecular chaperone.

Figure 5. Summary of the diverse modes of action.

Overview of the variety of diverse microscopic mechanisms through which molecular chaperones can suppress amyloid formation, as revealed by the kinetic analysis presented in this work. As we demonstrate, molecular chaperones have evolved to exploit in a variety of ways the different opportunities to modulate protein aggregation offered by the binding to different protein species (Fig. 1).