Structural instability and fibrillar aggregation of non-expanded human ataxin-3 revealed under high pressure and temperature.

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 beta-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

Kinetic analysis of tubulin assembly in the presence of the microtubule-associated protein TOGp.

International audienceThe microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction

Axonemal Dynein Intermediate-Chain Gene (DNAI1) Mutations Result in Situs Inversus and Primary Ciliary Dyskinesia (Kartagener Syndrome)

Kartagener syndrome (KS) is a trilogy of symptoms (nasal polyps, bronchiectasis, and situs inversus totalis) that is associated with ultrastructural anomalies of cilia of epithelial cells covering the upper and lower respiratory tracts and spermatozoa flagellae. The axonemal dynein intermediate-chain gene 1 (DNAI1), which has been demonstrated to be responsible for a case of primary ciliary dyskinesia (PCD) without situs inversus, was screened for mutation in a series of 34 patients with KS. We identified compound heterozygous DNAI1 gene defects in three independent patients and in two of their siblings who presented with PCD and situs solitus (i.e., normal position of inner organs). Strikingly, these five patients share one mutant allele (splice defect), which is identical to one of the mutant DNAI1 alleles found in the patient with PCD, reported elsewhere. Finally, this study demonstrates a link between ciliary function and situs determination, since compound mutation heterozygosity in DNAI1 results in PCD with situs solitus or situs inversus (KS)