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.

Real-time signal generation methods for detection and characterization of low-abundance mutations in genomic DNA are powerful tools for cancer diagnosis and prognosis. Mutations in codon 12 of the oncogene KRAS, for example, are frequently found in several types of human cancers. We have developed a novel real-time PCR technology, FLAG (FLuorescent Amplicon Generation) and adapted it for simultaneously (i) amplifying mutated codon 12 KRAS sequences, (ii) monitoring in real-time the amplification and (iii) genotyping the exact nucleotide alteration. FLAG utilizes the exceptionally thermostable endonuclease PspGI for real-time signal generation by cleavage of quenched fluorophores from the 5′-end of the PCR products and, concurrently, for selecting KRAS mutations over wild type. By including peptide-nucleic-acid probes in the reaction, simultaneous genotyping is achieved that circumvents the requirement for sequencing. FLAG enables high-throughput, closed-tube KRAS mutation detection down to ∼0.1% mutant-to-wild type. The assay was validated on model systems and compared with allele-specific PCR sequencing for screening 27 cancer specimens. Diverse applications of FLAG for real-time PCR or genotyping applications in cancer, virology or infectious diseases are envisioned.

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.

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