Septin 7 forms a complex with CD2AP and nephrin and regulates glucose transporter trafficking

Podocytes are insulin-sensitive and take up glucose in response to insulin. This requires nephrin, which interacts with vesicle-associated membrane protein 2 (VAMP2) on GLUT4 storage vesicles (GSVs) and facilitates their fusion with the plasma membrane. In this paper, we show that the filament-forming GTPase septin 7 is expressed in podocytes and associates with CD2-associated protein (CD2AP) and nephrin, both essential for glomerular ultrafiltration. In addition, septin 7 coimmunoprecipitates with VAMP2. Subcellular fractionation of cultured podocytes revealed that septin 7 is found in both cytoplasmic and membrane fractions, and immunofluorescence microscopy showed that septin 7 is expressed in a filamentous pattern and is also found on vesicles and the plasma membrane. The filamentous localization of septin 7 depends on CD2AP and intact actin organization. A 2-deoxy-d-glucose uptake assay indicates that depletion of septin 7 by small interfering RNA or alteration of septin assembly by forchlorfenuron facilitates glucose uptake into cells and further, knockdown of septin 7 increased the interaction of VAMP2 with nephrin and syntaxin 4. The data indicate that septin 7 hinders GSV trafficking and further, the interaction of septin 7 with nephrin in glomeruli suggests that septin 7 may participate in the regulation of glucose transport in podocytes.     

Evolution of a family of metazoan active-site-serine enzymes from penicillin-binding proteins: a novel facet of the bacterial legacy

Background  

Bacterial penicillin-binding proteins and β-lactamases (PBP-βLs) constitute a large family of serine proteases that perform essential functions in the synthesis and maintenance of peptidoglycan. Intriguingly, genes encoding PBP-βL homologs occur in many metazoan genomes including humans. The emerging role of LACTB, a mammalian mitochondrial PBP-βL homolog, in metabolic signaling prompted us to investigate the evolutionary history of metazoan PBP-βL proteins.     

Expression and purification of the mitochondrial serine protease LACTB as an N-terminal GST fusion protein in Escherichia coli

LACTB is a mammalian mitochondrial protein sharing sequence similarity to the beta-lactamase/penicillin-binding protein family of serine proteases that are involved in bacterial cell wall metabolism. The physiological role of LACTB is unclear. In this study we have subcloned the cDNA of mouse LACTB (mLACTB) and produced recombinant mLACTB protein in Escherichia coli. When mLACTB was expressed as an N-terminal GST fusion protein (GST-mLACTB), full-length GST-mLACTB protein was recovered by glutathione-agarose affinity chromatography as determined by MALDI-TOF mass spectrometry and immunoblotting. Expression of mLACTB as a C-terminal GST fusion protein or with either an N- or C-terminal His6-tag resulted in proteolytic degradation of the protein and we were not able to detect full-length mLACTB. Analysis of GST-mLACTB by Fourier transform infrared spectrometry revealed the presence of alpha-helices, beta-sheets and turns, consistent with a well-defined secondary structure. These results show that mLACTB can be expressed as a GST fusion protein in E. coli and suggest that GST-mLACTB was properly folded.     

Ebselen enhances insulin sensitivity and decreases oxidative stress by inhibiting SHIP2 and protects from inflammation in diabetic mice

Ebselen, a multifunctional organoselenium compound, has been recognized as a potential treatment for diabetes-related disorders. However, the underlying mechanisms whereby ebselen regulates metabolic pathways remain elusive. We discovered that ebselen inhibits lipid phosphatase SHIP2 (Src homology 2 domain-containing inositol-5-phosphatase 2), an emerging drug target to ameliorate insulin resistance in diabetes. We found that ebselen directly binds to and inhibits the catalytic activity of the recombinant SHIP2 phosphatase domain and SHIP2 in cultured cells, the skeletal muscle and liver of the diabetic db/db mice, and the liver of the SHIP2 overexpressing (SHIP2-Tg) mice. Ebselen increased insulin-induced Akt phosphorylation in cultured myotubes, enhanced insulin sensitivity and protected liver tissue from lipid peroxidation and inflammation in the db/db mice, and improved glucose tolerance more efficiently than metformin in the SHIP2-Tg mice. SHIP2 overexpression abrogated the ability of ebselen to induce glucose uptake and reduce ROS production in myotubes and blunted the effect of ebselen to inhibit SHIP2 in the skeletal muscle of the SHIP2-Tg mice. Our data reveal ebselen as a potent SHIP2 inhibitor and demonstrate that the ability of ebselen to ameliorate insulin resistance and act as an antioxidant is at least in part mediated by the reduction of SHIP2 activity.