Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells

Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughout adipogenesis. Instead, an adipogenesis-dependent increase in O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation.     

O-glycosylation of FoxO1 increases its transcriptional activity towards the glucose 6-phosphatase gene

Mono-O-glycosylations post-translationally regulate the activity of nucleocytoplasmic proteins. We showed that glucosamine and an inhibitor of deglycosylation (PUGNAc) induced O-glycosylation of FoxO1, resulting in increased expression of a glucose-6-phosphatase reporter gene. This effect was independent of FoxO1 re-localisation, since it was also observed with constitutively nuclear FoxO1-AAA mutant. Moreover, in HepG2 cells, glucosamine and PUGNAc have a synergistic effect on the glucose-6-phosphatase reporter gene, and this effect was inhibited by FoxO1 siRNAs. Since glucose-6-phosphatase plays a key role in hepatic glucose production, our observation may be of importance with regard to glucotoxicity associated with chronic hyperglycaemia in diabetes.     

Mutation-induced reinforcement of prestin-expressing cells

The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.     

The measurement of lysosomal phospholipase A2 activity in plasma

A deficiency of lysosomal phospholipase A2 (LPLA2) causes macrophage-associated phospholipidosis, suggesting that the enzyme is important in the lipid catabolism. Because LPLA2 is secreted by macrophages, extracellular LPLA2 activity may potentially reflect a change in macrophage activation. In this report, the detection of LPLA2 activity in plasma was established by the measurement of the transacylase activity of LPLA2 under acidic conditions. No transacylase activity of LPLA2 was detected in normal human plasma when the plasma was incubated with liposomes consisting of 1,2-dioleoylphosphatidylcholine/sulfatide/N-acetylsphingosine (NAS) at pH 4.5. However, the transacylase activity in the plasma was detected when liposomes consisting of 1,2-dioleoylphosphatidylglycerol/NAS were used as a substrate. To establish the specificity of the assay, ceramide transacylase activity was detected in the plasma of wild-type mice. By contrast, the plasma obtained from LPLA2-knockout mice had no measurable transacylase activity under the same conditions. The enzymatic activity of recombinant LPLA2 was inhibited by treatment with methylarachidonylfluorophosphonate. The inhibitor also suppressed the transacylase activity observed in both normal human and wild-type mouse plasma, establishing that the transacylase activity observed in plasma is due to LPLA2. Plasma LPLA2 activity may be a useful bioassay marker for the identification of LPLA2-related disorders.     

Increased folding stability of TEM-1 beta-lactamase by in vitro selection

In vitro selections of stabilized proteins lead to more robust enzymes and, at the same time, yield novel insights into the principles of protein stability. We employed Proside, a method of in vitro selection, to find stabilized variants of TEM-1 β-lactamase from Escherichia coli. Proside links the increased protease resistance of stabilized proteins to the infectivity of a filamentous phage. Several libraries of TEM-1 β-lactamase variants were generated by error-prone PCR, and variants with increased protease resistance were obtained by raising temperature or guanidinium chloride concentration during proteolytic selections. Despite the small size of phage libraries, several strongly stabilizing mutations could be obtained, and a manual combination of the best shifted the profiles for thermal unfolding and temperature-dependent inactivation of β-lactamase by almost 20 °C to a higher temperature. The wild-type protein unfolds in two stages: from the native state via an intermediate of the molten-globule type to the unfolded form. In the course of the selections, the native protein was stabilized by 27 kJ mol^− 1 relative to the intermediate and the cooperativity of unfolding was strongly increased. Three of our stabilizing replacements (M182T, A224V, and R275L) had been identified independently in naturally occurring β-lactamase variants with extended substrate spectrum. In these variants, they acted as global suppressors of destabilizations caused by the mutations in the active site. The comparison between the crystal structure of our best variant and the crystal structure of the wild-type protein indicates that most of the selected mutations optimize helices and their packing. The stabilization by the E147G substitution is remarkable. It removes steric strain that originates from an overly tight packing of two helices in the wild-type protein. Such unfavorable van der Waals repulsions are not easily identified in crystal structures or by computational approaches, but they strongly reduce the conformational stability of a protein.     

Sumoylation of specificity protein 1 augments its degradation by changing the localization and increasing the specificity protein 1 proteolytic process

Although specificity protein 1 (Sp1) accumulation has been found in various tumor strains, its mechanism is still not very clear. Herein, we found that modification of Sp1 by SUMO-1 facilitates Sp1 degradation. Our findings revealed that, although the amounts of Sp1 and Sp1 mutant (K16R) [Sp1(K16R)] mRNA in cells were equal, the protein level of Sp1(K16R) was higher than that of wild-type Sp1. We also proved that this sumoylation site was not the residue at which ubiquitination occurred. Invitro and in vivo pull-down assays revealed that more sumoylated Sp1 was localized in the cytoplasm, and the interaction between SUMO-1-Sp1 and the proteasome subunit rpt6 in HeLa cells was enhanced. In addition, although Sp1 accumulated in the tumorous cervical tissue, it was not prone to sumoylation. Finally, by overexpression of HA (hemagglutinin)-SUMO-1-Sp1-myc, HA-Sp1-myc, and HA-Sp1(K16R), we found that modification of Sp1 by SUMO-1 was important for Sp1 proteolysis. In conclusion, modification of Sp1 by SUMO-1 altered its localization and then increased its interaction with rpt6. This interaction increased the efficiency of Sp1 proteolytic processing and ubiquitination and then resulted in Sp1 degradation. Therefore, sumoylation of Sp1 is attenuated during tumorigenesis in order to increase Sp1 stability.     

Expression patterns of peroxisome proliferator-activated receptor gamma 1 versus gamma 2, and their association with intramuscular fat in goat tissues

Intramuscular fat (IMF) shortage causes the lack of juiciness and tenderness of goat meat, while peroxisome proliferator-activated receptor gamma 1 (PPARγ1) and gamma 2 (PPARγ2) play key roles in lipid metabolism. Nevertheless, their expression patterns and the relationship with IMF have been poorly exposed. Using quantitative polymerase chain reaction (qPCR), classical Soxhlet extraction, and in situ hybridization, we demonstrated that among 13 goat tissues, expression of PPARγ1 was dramatically higher than that of PPARγ2 except for lung. We further demonstrated the expression patterns of PPARγ1 and PPARγ2 and their negative association with intramuscular fat content in three goat muscles with kids growing. Meanwhile, PPARγ expression was located in the connective tissues. These results suggest that PPARγ1 is rather active for most tissues of goat, and closely related with the muscular fat metabolism during early postnatal life, but a more direct proof remains to be provided.     

Monitoring of Fasciola Species Contamination in Water Dropwort by cox1 Mitochondrial and ITS-2 rDNA Sequencing Analysis

Fascioliasis, a food-borne trematode zoonosis, is a disease primarily in cattle and sheep and occasionally in humans. Water dropwort (Oenanthe javanica), an aquatic perennial herb, is a common second intermediate host of Fasciola, and the fresh stems and leaves are widely used as a seasoning in the Korean diet. However, no information regarding Fasciola species contamination in water dropwort is available. Here, we collected 500 samples of water dropwort in 3 areas in Korea during February and March 2015, and the water dropwort contamination of Fasciola species was monitored by DNA sequencing analysis of the Fasciola hepatica and Fasciola gigantica specific mitochondrial cytochrome c oxidase subunit 1 (cox1) and nuclear ribosomal internal transcribed spacer 2 (ITS-2). Among the 500 samples assessed, the presence of F. hepatica cox1 and 1TS-2 markers were detected in 2 samples, and F. hepatica contamination was confirmed by sequencing analysis. The nucleotide sequences of cox1 PCR products from the 2 F. hepatica-contaminated samples were 96.5% identical to the F. hepatica cox1 sequences in GenBank, whereas F. gigantica cox1 sequences were 46.8% similar with the sequence detected from the cox1 positive samples. However, F. gigantica cox1 and ITS-2 markers were not detected by PCR in the 500 samples of water dropwort. Collectively, in this survey of the water dropwort contamination with Fasciola species, very low prevalence of F. hepatica contamination was detected in the samples.     

Impairment of the activity of the xenobiotic-metabolizing enzymes arylamine N-acetyltransferases 1 and 2 (NAT1/NAT2) by peroxynitrite in mouse skeletal muscle cells

Reactive nitrogen species and their by-products, such as peroxynitrite, modulate many physiological functions of skeletal muscle. Peroxynitrite generation occuring under specific conditions, such as inflammation, may also lead to skeletal muscle dysfunction and pathologies. Arylamine N-acetyltransferases (NATs) are xenobiotic-metabolizing enzymes (XMEs) involved in the detoxification and/or metabolic activation of several drugs and chemicals. In addition to other XMEs, such as gluthatione S-transferases or cytochromes P450, NAT enzymes are expressed in skeletal muscle. We show here that functional NAT1 and NAT2 isoforms are expressed in mouse myotubes and that peroxynitrite may impair their activity in these cells. We show that this inactivation is likely due to the irreversible modification of NATs catalytic cysteine residue in vivo. Our results suggest that peroxynitrite-dependent inactivation of muscle XMEs such as NATs may contribute to muscle dysfunction by impairing the biotransformation activity of this key cellular defense enzyme system.