Structural Instability and Fibrillar Aggregation of Non-expanded Human Ataxin-3 Revealed under High Pressure and Temperature

Abstract

Protein misfolding and formation of structured aggregates are considered to be the earliest events in the development of neurodegenerative diseases, but the mechanism of these biological phenomena remains to be elucidated. Here, we report a study of heat-and pressure-induced unfolding of human Q26 and murine Q6 ataxin-3 using spectroscopic methods. UV absorbance and fluorescence revealed that heat and pressure induced a structural transition of both proteins to a molten globule conformation. The unfolding pathway was partly irreversible and led to a protein conformation where tryptophans were more exposed to water. Furthermore, the use of fluorescent probes (8-anilino-1-naphthalenesulfonate and thioflavin T) allowed the identification of different intermediates during the process of pressure-induced unfolding. At high temperature and pressure, human Q26, but not murine Q6, underwent concentration-dependent aggregation. Fourier transform infrared and circular dichroism spectroscopy revealed that these aggregates are characterized by an increased ␤-sheet content. As revealed by electron microscopy, heat-and pressure-induced aggregates were different; high temperature treatment led to fibrillar microaggregates (8 -10-nm length), whereas high pressure induced oligomeric structures of globular shape (100 nm in diameter), which sometimes aligned to higher order suprastructures. Several intermediate structures were detected in this process. Two factors appear to govern ataxin unfolding and aggregation, the length of the polyglutamine tract and its protein context.

Polyglutamine Expansion in Ataxin-3 Does Not Affect Protein Stability

“…We found that upon incubation at pH 1.9, the acid-denatured state of Atax3(Q28), or what may be termed the "A state," displayed spectral properties similar to partially folded conformational species induced by pressure, temperature, and denaturant (24,27). Specifically, the A state showed an increased fluorescence signal, increased bis-ANS binding, and a red shift in fluorescence emission maximum ( max ) from 337 nm in the native state to 343 nm in the A state (Figs.…”

“…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).…”

Time-Course and Regional Analyses of the Physiopathological Changes Induced after Cerebral Injection of an Amyloid β Fragment in Rats

“…For comparison of ␤-amyloid aggregated peptides (A␤ and A␤ [25][26][27][28][29][30][31][32][33][34][35] ), samples that had been incubated for 4 days at 37°C were deposited onto a CaF 2 plate and the solvent was allowed to evaporate overnight at room temperature. 20 …”

The powerful high pressure tool for protein conformational studies

“…Using complementary biophysical techniques including spectroscopic methods and electron microscopy, we have shown that the pressure-induced unfolding of human ataxin-3 (Q26) was characterized by a complex mechanism, implying several steps and unfolding intermediates within the experimental pressure range (23). First, the protein started to unfold in a relatively low-pressure range (P 1/2 = 145 MPa).…”