Self assembly of human septin 2 into amyloid filaments.

Septins are a conserved group of GTP-binding proteins that form hetero-oligomeric complexes which assemble into filaments. These are essential for septin function, including their role in cytokinesis, cell division, exocytosis and membrane trafficking. Septin 2 (SEPT2) is a member of the septin family and has been associated with neurofibrillary tangles and other pathological features of senile plaques in Alzheimer's disease. An in silico analysis of the amino acid sequence of SEPT2 identified regions with a significant tendency to aggregate and/or form amyloid. These were all observed within the GTP-binding domain. This was consistent with the experimental identification of a structure rich in ß-sheet during temperature induced unfolding transitions observed for both the full length protein and the GTP-binding domain alone. This intermediate state is characterized by irreversible aggregation and has the ability to bind Thioflavin-T, suggesting its amyloid nature. Under electron microscopy, fibers extending for several micrometers in length could be visualized. The results shown in this study support the hypothesis that single septins, when present in excess or with unbalanced stoichiometries, may be unstable and assemble into amyloid-like structures.

Urea-induced unfolding studies of free- and ligand-bound tetrameric ATP-dependent Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase. Influence of quaternary structure on protein conformational stability.

ATP-dependent phosphoenolpyruvate (PEP) carboxykinases are found in plants and microorganisms, and catalyse the reversible formation of PEP, ADP, and CO(2) from oxaloacetate plus ATP. These enzymes vary in quaternary structure although there is significant sequence identity among the proteins isolated from different sources. To help understand the influence of quaternary structure in protein stability, the urea-induced unfolding of free- and substrate-bound tetrameric Saccharomyces cerevisiae PEP carboxykinase is described and compared with the unfolding characteristics of the monomeric Escherichia coli enzyme [Eur. J. Biochem. 255 (1998) 439]. The urea-induced denaturation of S. cerevisiae PEP carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism (CD) spectra, and 1-anilino-8-naphthalenesulfonate (ANS) binding. The unfolding profiles were multi-steps, and formation of hydrophobic structures were detected. The data indicate that unfolding and dissociation of the enzyme tetramer are simultaneous events. Ligand binding, most notably PEP in the presence of MnCl(2), conferred a marked protection against urea-induced denaturation. A similar protection effect was found when N-iodoacetyl-N'-(5-sulfo-1-napthyl)ethylene diamine (1,5-I-AEDANS) was covalently bound at Cys(365), within the active site region. Refolding experiments indicated that total recovery of tertiary structure was only obtained from samples previously unfolded to less than 30%. In the presence of substrates, complete refolding was achieved from samples originally denatured up to 50%. The unfolding behaviour of S. cerevisiae PEP carboxykinase was found to be similar to that of E. coli PEP carboxykinase, however all steps take place at lower urea concentrations. These findings show that, at least for monomeric and tetrameric ATP-dependent PEP carboxykinases, quaternary structure does not contribute to protein conformational stability.