Dissection of a Human Septin:  Definition and Characterization of Distinct Domains within Human SEPT4

Abstract

The septins are a conserved family of guanosine-5'-triphosphate (GTP)-binding proteins. In mammals they are involved in a variety of cellular processes, such as cytokinesis, exocytosis, and vesicle trafficking. Specifically, SEPT4 has also been shown to be expressed in both human colorectal cancer and malignant melanoma, as well as being involved in neurodegenerative disorders. However, many of the details of the modes of action of septins in general remain unclear, and little is known of their detailed molecular architecture. Here, we define explicitly and characterize the domains of human SEPT4. Regions corresponding to the N-terminal, GTPase, and C-terminal domains as well as the latter two together were successfully expressed in Escherichia coli in soluble form and purified by affinity and size-exclusion chromatographies. The purified domains were analyzed by circular dichroism spectroscopy, fluorescence spectroscopy, dynamic light scattering, and small-angle X-ray scattering, as well as with bioinformatics tools. Of the three major domains that comprise SEPT4, the N-terminal domain contains little regular secondary structure and may be intrinsically unstructured. The central GTPase domain is a mixed alpha/beta structure, probably based on an open beta sheet. As defined here, it is catalytically active and forms stable homodimers in vitro. The C-terminal domain also forms homodimers and can be divided into two regions, the second of which is alpha-helical and consistent with a coiled-coil structure. These studies should provide a useful basis for future biophysical studies of SEPT4, including the structural basis for their involvement in diseases such as cancer and neurodegenerative disorders.

Septin roles in tumorigenesis

“…The central GTPbinding domain is highly conserved in all human septins with 58% similarity. Between the N-terminus and GTP-binding domain is a polybasic region conserved throughout eukaryotic phylogeny that might target septins to the plasma membrane by interaction with phosphoinositides (Garcia et al, 2006). A septin unique element maps between the GTPase domain and the C-terminus.…”

Targeted Knockdown of SEPT9_v1 Inhibits Tumor Growth and Angiogenesis of Human Prostate Cancer Cells Concomitant with Disruption of Hypoxia-Inducible Factor-1 Pathway

“…Mammalian septins are evolutionary conserved proteins that form filaments associated with cytoskeleton organization, membrane dynamics, mitosis, cytokinesis, and cell cycle progression (9,10). Recently, accumulating data suggest that some septin family members also participate in the pathogenesis of neoplasia (11) and neurodegenerative diseases (12). We had shown that SEPT9_i1 interacts with HIF-1α, but not with HIF-2α, to prevent its RACK1-mediated ubiquitination and degradation (13) and that SEPT9_i1 protein activates HIF-1 transcriptional activity and increases tumor growth and angiogenesis in prostate cancer xenografts (8).…”

Simulated Annealing of Two Electron Density Solution Systems

“…This methodology was also applied to other nuclear receptors and protein systems (Fischer et al, (2003), Garcia et al (2006), Grimm et al (2006), Calgaro et al (2007), Nascimento et al (2007), Mario Oliveira Neto et al (2008), in an addition to polymers in solutions (Leite et al (2007)). For the latter, the initial part of the curve, probably due to larger particles, had to be disregarded, thus considering the polymer system as approximately monodisperse.…”