Septin structure and filament assembly.

Septins are able to polymerize into long apolar filaments and have long been considered to be a component of the cytoskeleton alongside intermediate filaments (which are also apolar in nature), microtubules and actin filaments (which are not). Their central guanosine triphosphate (GTP)-binding domain, which is essential for stabilizing the filament itself, is flanked by N- and C-terminal domains for which no direct structural information is yet available. In most cases, physiological filaments are built from a number of different septin monomers, and in the case of mammalian septins this is most commonly either three or four. Comprehending the structural basis for the spontaneous assembly of such filaments requires a deeper understanding of the interfaces between individual GTP-binding domains than is currently available. Nevertheless, in this review we will summarize the considerable progress which has been made over the course of the last 10 years. We will provide a brief description of each structure determined to date and comment on how it has added to the body of knowledge which is rapidly growing. Rather than simply repeat data which have already been described in the literature, as far as is possible we will try to take advantage of the full set of information now available (mostly derived from human septins) and draw the reader's attention to some of the details of the structures themselves and the filaments they form which have not be commented on previously. An additional aim is to clarify some misconceptions.

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.

Endocytosis of pulchellin and its recombinant B-chain into K-562 cells: binding and uptake studies.

Most of the type 2 ribosome-inactivating proteins (RIPs) are toxins formed by an RNA-N-glycosidase A-chain polypeptide linked to a lectin B-chain by a single disulfide bond. Members of this protein class vary greatly in cytotoxity, correlating more with B-chain diversity rather than to A-chain differences. Pulchellin is a type 2 ribosome-inactivating protein toxin found in the seeds of Abrus pulchellus tenuiflorus. Recombinant pulchellin B-Chain (rPBC) has been previously produced as inclusion bodies in Escherichia coli and successfully refolded recovering biological activity. New approaches for using this kind of protein as a biotechnological tool require a better understanding of cell targeting, binding, uptake, intracellular routing and delivery. In this work, cell adhesion experiments were used to determine the interaction of rPBC with mammalian cells. Fluorescence and confocal microscopy revealed the intracellular localization and trafficking. Subcellular sorting of the native pulchellin could also be determined. The results support that the endosomal internalization pathway and the retrograde transport through the Golgi apparatus might be used by both native protein and rPBC.