Huntington's disease age-of-onset linked to polyglutamine aggregation nucleation

Abstract

In Huntington's Disease and related expanded CAG repeat diseases, a polyglutamine [poly(Gln)] sequence containing 36 repeats in the corresponding disease protein is benign, whereas a sequence with only 2-3 additional glutamines is associated with disease risk. Above this threshold range, longer repeat lengths are associated with earlier ages-of-onset. To investigate the biophysical basis of these effects, we studied the in vitro aggregation kinetics of a series of poly(Gln) peptides. We find that poly(Gln) peptides in solution at 37°C undergo a random coil to ␤-sheet transition with kinetics superimposable on their aggregation kinetics, suggesting the absence of soluble, ␤-sheet-rich intermediates in the aggregation process. Details of the time course of aggregate growth confirm that poly(Gln) aggregation occurs by nucleated growth polymerization. Surprisingly, however, and in contrast to conventional models of nucleated growth polymerization of proteins, we find that the aggregation nucleus is a monomer. That is, nucleation of poly(Gln) aggregation corresponds to an unfavorable protein folding reaction. Using parameters derived from the kinetic analysis, we estimate the difference in the free energy of nucleus formation between benign and pathological length poly(Gln)s to be less than 1 kcal͞mol. We also use the kinetic parameters to calculate predicted aggregation curves for very low concentrations of poly(Gln) that might obtain in the cell. The repeat-length-dependent differences in predicted aggregation lag times are in the same range as the length-dependent age-of-onset differences in Huntington's disease, suggesting that the biophysics of poly(Gln) aggregation nucleation may play a major role in determining disease onset.

A polymer physics perspective on driving forces and mechanisms for protein aggregation

“…Experiments have also shown that the tendency to form intermolecular aggregates increases with polyglutamine length. Wetzel and coworkers have carried out a series of experiments based on a variety of spectroscopic and other probes to quantify the overall rate of aggregation as a function of chain length and concentration [45,54,[65][66][67]. Their conclusions are consistent, at least qualitatively, with the expectations from polymer theory as discussed above and demonstrated in Figure 3.…”

“…The second prediction of Raos and Allegra appears to be at odds with findings in the amyloid literature, which suggest that the mechanism of aggregation proceeds through the formation of a structured nucleus, which presents a single, well-defined barrier [45]. It is noteworthy that Krishnan and Lindquist [93] have challenged this view in their recent work on the aggregation of a yeast prion.…”

“…Despite the impressive progress made by Wetzel [45,54,[65][66][67] and others [68][69][70][71][72] in the area of polyglutamine aggregation, there are significant gaps in our knowledge because not much is known about intermolecular associations and intramolecular conformational fluctuations that occur at very low protein concentrations (nM -μM). This knowledge is of utmost importance to understand the phenomenon of polyglutamine aggregation in vivo.…”

“…Points 3 and 4 emphasize that the magnitudes of spontaneous fluctuations (in conformation and concentration) and nucleus size will vary with quench depth. This is important because standard analyses of kinetic data for protein aggregation [44][45][46][47] do not account for quench-depth dependent rates of conformational fluctuations and nucleus size. These effects are lumped into pre-equilibrium constants, and if these constants are set to be small, the implicit assumption is that rates of conformational fluctuations are considerably larger that bimolecular rate constants.…”

From the test tube to the cell: Exploring the folding and aggregation of a β‐clam protein

“…The aggregation of both proteins is consistent with a nucleated-polymerization model, 26 and kinetic analysis revealed a monomeric nucleus in both cases, suggesting that a folding or unfolding reaction within a monomer was the thermodynamically unfavorable event prerequisite for aggregation. A monomeric nucleus was previously implicated in in-vitro aggregation of polyglutamine peptides, 27 making it difficult to distinguish which part of the tetra-Cys CRABP Htt53 chimera (either tetra-Cys CRABP or Htt domain) is mechanistically dominant in aggregation. Intriguingly, the diffraction pattern of isolated P39A tetra-Cys CRABP aggregates formed in vivo resembles the characteristic fingerprint pattern of ordered amyloid structures, suggesting a common microstructure for all types of aggregates.…”