Biochemical characterization of membrane-associated septin from rat brain

Within the cell membrane there exist various microdomains (lipid rafts) in which specific lipids and proteins are assembled and these microdomains are recovered in the detergent-resistant low-density membrane fraction (DRM). Septin is a novel GTP-binding, cytoskeletal protein having various isoforms that assemble into homo- and heterooligomers and filaments. As the localization of septin 3 in DRM was found through a proteomics analysis of brain-derived DRM, the presence of other septin isoforms in DRM was studied. Western blotting analysis showed maturation-dependent enrichment of several septin isoforms in DRM prepared from synaptic plasma membrane (SPM). These isoforms were solubilized with high MgCl₂ solution and recovered as the precipitate after dialysis to low ionic solution. Three times cycling of the extraction-dialysis process resulted in the partial purification of septin complex and electron microscopic observation of this fraction revealed rod-like structures in which building units were observed. The presence of heterooligomers was shown with western blotting after the separation of the MgCl₂ extract with blue-native polyacrylamide gel electrophoresis. Immunoprecipitation assay using monoclonal anti-septin11 antibody also showed the presence of heterooligomers. These results show that septin localizes in the membrane microdomains of the SPM in adult brain and may have important roles in the membrane dynamics of neurons.     

Thrombospondin1 Deficiency Attenuates Obesity-Associated Microvascular Complications in ApoE-/- Mice

Obesity is associated with insulin resistance and the increased development of vascular complications. Previously, we have demonstrated that thrombospondin1 (TSP1) regulates macrophage function and contributes to obesity associated inflammation and insulin resistance. However, the role of TSP1 in the development of obesity associated vascular complications is not clear. Therefore, in the current study, we investigated whether TSP1 deficiency protects mice from obesity associated micro as well as macro-vascular complications in ApoE-/- mice. In this study, male ApoE-/- mice and ApoE-/-TSP1-/- mice were fed with a low-fat (LF) or a high-fat (HF) diet for 16 weeks. We found that body weight and fat mass increased similarly between the ApoE-/-TSP1-/- mice and ApoE-/- mice under HF feeding conditions. However, as compared to obese ApoE-/- mice, obese ApoE-/-TSP1-/- mice had improved glucose tolerance, increased insulin sensitivity, and reduced systemic inflammation. Aortic atherosclerotic lesion formation was similar in these two groups of mice. In contrast, albuminuria was attenuated and kidney fibrosis was reduced in obese ApoE-/-TSP1-/- mice compared to obese ApoE-/- mice. The improved kidney function in obese ApoE-/-TSP1-/- mice was associated with decreased renal lipid accumulation. Together, these data suggest that TSP1 deficiency did not affect the development of obesity associated macro-vascular complication, but attenuated obesity associated micro-vascular complications.     

Structures of septin filaments prepared from rat brain and expressed in bacteria

Septin forms a conserved family of cytoskeletal GTP-binding proteins that have diverse roles in protein scaffolding, vesicle trafficking and cytokinesis. There are 14 mammalian septin isoforms and these isoforms assemble into hetero-oligomeric rod-shaped complexes and these short filaments are the basal units to construct higher-order structures such as longer filaments, rings, gauzes or hourglasses. Septin expressed in a eukaryotic expression system forms various structures such as bundles, sheets, helixes, and rings. Septin expressed in bacteria formed hexameric short filaments and single or parallel long filaments, but no such higher order structures were observed so far. In a previous study, we showed maturation-dependent localization of septin isoforms to the lipid raft fraction of rat brain. In this study, we attempted further purification of raft-localized septin isoforms. Repeated cycles of extraction with high MgCl₂ solution and precipitation under low ionic solution were combined with several column procedures. The obtained fraction contained several septin isoforms and showed rings of bundled filaments with a diameter of ～0.4 μm. Several non-septin proteins were also detected in the fraction. We also attempted expression of septin isoforms in bacteria and found that the expressed septin complexes formed bundles of filaments. In addition to linear and curled filaments, circular bundles of thin filaments with a diameter of ～0.6 μm were also observed. These results suggest that the curvature of the bundles of septin filaments may be regulated by the regulatory factor(s) in the lipid raft.     

Overexpression of cGMP-dependent protein kinase I (PKG-I) attenuates ischemia-reperfusion-induced kidney injury

cGMP-dependent protein kinase (PKG) is a multifunctional protein. Whether PKG plays a role in ischemia-reperfusion-induced kidney injury (IRI) is unknown. In this study, using an in vivo mouse model of renal IRI, we determined the effect of renal IRI on kidney PKG-I levels and also evaluated whether overexpression of PKG-I attenuates renal IRI. Our studies demonstrated that PKG-I levels (mRNA and protein) were significantly decreased in the kidney from mice undergoing renal IRI. Moreover, PKG-I transgenic mice had less renal IRI, showing improved renal function and less tubular damage compared with their wild-type littermates. Transgenic mice in the renal IRI group had decreased tubular cell apoptosis accompanied by decreased caspase 3 levels/activity and increased Bcl-2 and Bag-1 levels. In addition, transgenic mice undergoing renal IRI demonstrated reduced macrophage infiltration into the kidney and reduced production of inflammatory cytokines. In vitro studies showed that peritoneal macrophages isolated from transgenic mice had decreased migration compared with control macrophages. Taken together, these results suggest that PKG-I protects against renal IRI, at least in part through inhibiting inflammatory cell infiltration into the kidney, reducing kidney inflammation, and inhibiting tubular cell apoptosis.