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.

Expanded CAG repeats lead to debilitating neurodegenerative disorders characterized by aggregation of proteins with expanded polyglutamine (polyQ) tracts. The mechanism of aggregation involves primary and secondary nucleation steps. We show how a noncanonical member of the DNAJ-chaperone family, DNAJB6, inhibits the conversion of soluble polyQ peptides into amyloid fibrils, in particular by suppressing primary nucleation. This inhibition is mediated by a serine/threonine-rich region that provides an array of surface-exposed hydroxyl groups that bind to polyQ peptides and may disrupt the formation of the H bonds essential for the stability of amyloid fibrils. Early prevention of polyQ aggregation by DNAJB6 occurs also in cells and leads to delayed neurite retraction even before aggregates are visible. In a mouse model, brain-specific coexpression of DNAJB6 delays polyQ aggregation, relieves symptoms, and prolongs lifespan, pointing to DNAJB6 as a potential target for disease therapy and tool for unraveling early events in the onset of polyQ diseases.

Background: The origins of the inhibition of DNAJB6 against amyloid formation are unknown.Results: DNAJB6 inhibits fibril formation of the Aβ42 peptide from Alzheimer disease at low sub-stoichiometric molar ratios through strong binding to aggregated species.Conclusion: Such sequestration prevents the growth and the proliferation of the aggregates.Significance: The efficacious action of the chaperone against amyloid formation involves interactions with multiple growing aggregates.

Expanded polyglutamine (polyQ) stretches lead to protein aggregation and severe neurodegenerative diseases. A highly efficient suppressor of polyQ aggregation was identified, the DNAJB6, when molecular chaperones from the HSPH, HSPA, and DNAJ families were screened for huntingtin exon 1 aggregation in cells (Hageman et al. in Mol Cell 37(3):355–369, 2010). Furthermore, also aggregation of polyQ peptides expressed in cells was recently found to be efficiently suppressed by co-expression of DNAJB6 (Gillis et al. in J Biol Chem 288:17225–17237, 2013). These suppression effects can be due to an indirect effect of DNAJB6 on other cellular components or to a direct interaction between DNAJB6 and polyQ peptides that may depend on other cellular components. Here, we have purified the DNAJB6 protein to investigate the suppression mechanism. The purified DNAJB6 protein formed large heterogeneous oligomers, in contrast to the more canonical family member DNAJB1 which is dimeric. Purified DNAJB6 protein, at substoichiometric molar ratios, efficiently suppressed fibrillation of polyQ peptides with 45°Q in a thioflavin T fibrillation. No suppression was obtained with DNAJB1, but with the closest homologue to DNAJB6, DNAJB8. The suppression effect was independent of HSPA1 and ATP. These data, based on purified proteins and controlled fibrillation in vitro, strongly suggest that the fibrillation suppression is due to a direct protein–protein interaction between the polyQ peptides and DNAJB6 and that the DNAJB6 has unique fibrillation suppression properties lacking in DNAJB1. Together, the data obtained in cells and in vitro support the view that DNAJB6 is a peptide-binding chaperone that can interact with polyQ peptides that are incompletely degraded by and released from the proteasome.Electronic supplementary materialThe online version of this article (doi:10.1007/s12192-013-0448-5) contains supplementary material, which is available to authorized users.

The human molecular chaperone DNAJB6, an oligomeric protein with a conserved S/T-rich region, is an efficient suppressor of amyloid fibril formation by highly aggregation-prone peptides such as the Aβ and polyQ peptides associated with Alzheimer's and Huntington's disease, respectively. We previously showed that DNAJB6 can inhibit the processes through which amyloid fibrils are formed via strong interactions with aggregated forms of Aβ42 that become sequestered. Here we report that the concentration-dependent capability of DNAJB6 to suppress fibril formation in thioflavin T fluorescence assays decreases progressively with an increasing number of S/T substitutions, with an almost complete loss of suppression when 18 S/T residues are substituted. The kinetics of primary nucleation in particular are affected. No detectable changes in the structure are caused by the substitutions. Also, the level of binding of DNAJB6 to Aβ42 decreases with the S/T substitutions, as determined by surface plasmon resonance and microscale thermophoresis. The aggregation process monitored using nuclear magnetic resonance spectroscopy showed that DNAJB6, in contrast to a mutational variant with 18 S/T residues substituted, can keep monomeric Aβ42 soluble for an extended time. The inhibition of the primary nucleation is likely to depend on hydroxyl groups in side chains of the S/T residues, and hydrogen bonding with Aβ42 is one plausible molecular mechanism, although other possibilities cannot be excluded. The loss of the ability to suppress fibril formation upon S/T to A substitution was previously observed also for polyQ peptides, suggesting that the S/T residues in the DNAJB6-like chaperones have a general ability to inhibit amyloid fibril formation by different aggregation-prone peptides.