Temperature-Dependent, Irreversible Formation of Amyloid Fibrils by a Soluble Human Ataxin-3 Carrying a Moderately Expanded Polyglutamine Stretch (Q36)

Abstract

The protein ataxin-3 is responsible for Machado-Joseph disease/spinocerebellar ataxia type 3, a neurodegenerative disorder caused by the presence of an expanded polyglutamine tract. A previous investigation [Bevivino, A. E., and Loll, P. J. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 11955-11960] showed that a nonexpanded ataxin-3 (Q27) was fully soluble, whereas an expanded form (Q78) gave rise to amyloid fibrils. Here, we report investigations on three forms of ataxin-3 (i.e., human nonexpanded (Q26), moderately expanded (Q36) ataxins-3, and the murine protein (Q6)). Far-UV circular dichroism spectra at room temperature were substantially similar, with a relatively high helical content. On heating to 96 degrees C, human Q26 and murine proteins did not display large structural changes, nor did they undergo any precipitation, which highlights their amazing heat-resistance. In contrast, human Q36 ataxin-3 underwent a progressive increase in the beta-sheet and a concomitant decrease in helical content when the temperature was shifted from 37 to 80 degrees C, followed by the irreversible formation of aggregates above 80 degrees C. They were shown to consist of amyloid fibrils, as supported by both electron microscopy images and the typical spectral shift displayed by Congo red when it was added to the protein at growing temperatures. We also found that protein precipitation could be prevented by mixing the dye with Q36 ataxin-3 prior to heating, which also confirms that the precipitates do represent authentic amyloid fibrils. In contrast, other compounds structurally related to Congo red did not exert significant effects. Our observations suggest that the temperature of the observed transition is inversely related to the length of the expansion. Finally, we suggest that antiamyloidogenic compounds might be selected on the basis of their ability to block or retard human Q36 ataxin-3 precipitation on heat-treatment.

Polyglutamine Expansion in Ataxin-3 Does Not Affect Protein Stability

“…1. In accordance with previous studies (22,25,35,36), we observed preferential binding of the polyglutamine-specific antibody 1C2 for the longer variant, which lies at the cusp of toxicity in Machado-Joseph disease, although nonpathological proteins could also be recognized (Fig. 1C).…”

“…In light of this, and given that ataxin-3 fibrillization does not necessarily proceed via a partially unfolded state, it is possible that the solubility of the protein may be a key element involved in the process. This is also consistent with difficulties experienced by ourselves and others in producing satisfactory quantities of expanded ataxin-3 variants in soluble form (19,25) and also the well documented insolubility of polyglutamine peptides alone and their rapid propensity to form fibrils even at nonpathological lengths (20,23).…”

“…In the case of ataxin-3, it has been shown that a variant containing 78 glutamine residues will readily fibrillize under native conditions (19), whereas shorter variants are also capable of forming fibrillar aggregates under partially denaturing conditions (24,25). In the current study, we examined the behavior and propensity of ataxin-3 of different lengths to form fibrils.…”

“…Recent work from our laboratory and others have shown that destabilization of the native state of nonpathological variants of ataxin-3 by various stresses such as chemical denaturation and heat can result in the formation of fibrillar aggregates (24,25,27). Based on various results, we and others have proposed that in the pathological state, the elongation of the polyglutamine tract disrupts the stability of the native conformation of the ataxin-3 (19,24).…”

“…In vitro, expanded forms of polyglutamine-containing proteins and peptides have been shown to form fibrils very readily (17,(19)(20)(21)(22). It has also been shown that nonpathological length proteins can also form fibrils, albeit generally at slower rates or under specific solution conditions (23)(24)(25)(26).…”