Multi-enzyme Machinery for Chitin Degradation in the Chitinolytic Bacterium Chitiniphilus shinanonensis SAY3T.

The chitinolytic bacterium, Chitiniphilus shinanonensis SAY3T was examined to characterize its chitin-degrading enzymes in view of its potential to convert biomass chitin into useful saccharides. A survey of the whole-genome sequence revealed 49 putative genes encoding polypeptides that are thought to be related to chitin degradation. Based on an analysis of the relative quantity of each transcript and an assay for chitin-degrading activity of recombinant proteins, a chitin degradation system driven by 19 chitinolytic enzymes was proposed. These include sixteen endo-type chitinases, two N-acetylglucosaminidases, and one lipopolysaccharide monooxygenase that catalyzes the oxidative cleavage of glycosidic bonds. Among the 16 chitinases, ChiL was characterized by its remarkable transglycosylation activity. Of the two N-acetylglucosaminidases (ChiI and ChiT), ChiI was the major enzyme, corresponding to > 98% of the total cellular activity. Surprisingly, a chiI-disrupted mutant was still able to grow on medium with powdered chitin or GlcNAc dimer. However, its growth rate was slightly lower compared to that of the wild-type SAY3. This multi-enzyme machinery composed of various types of chitinolytic enzymes may support SAY3 to efficiently utilize native chitin as a carbon and energy source and may play a role in developing an enzymatic process to decompose and utilize abundant chitin at the industrial scale.

Redox-dependent Regulation of Gluconeogenesis by a Novel Mechanism Mediated by a Peroxidatic Cysteine of Peroxiredoxin.

Peroxiredoxin is an abundant peroxidase, but its non-peroxidase function is also important. In this study, we discovered that Tsa1, a major peroxiredoxin of budding yeast cells, is required for the efficient flux of gluconeogenesis. We found that the suppression of pyruvate kinase (Pyk1) via the interaction with Tsa1 contributes in part to gluconeogenic enhancement. The physical interactions between Pyk1 and Tsa1 were augmented during the shift from glycolysis to gluconeogenesis. Intriguingly, a peroxidatic cysteine in the catalytic center of Tsa1 played an important role in the physical Tsa1-Pyk1 interactions. These interactions are enhanced by exogenous H2O2 and by endogenous reactive oxygen species, which is increased during gluconeogenesis. Only the peroxidatic cysteine, but no other catalytic cysteine of Tsa1, is required for efficient growth during the metabolic shift to obtain maximum yeast growth (biomass). This Tsa1 function is separable from the peroxidase function as an antioxidant. This is the first report to demonstrate that peroxiredoxin has a novel nonperoxidase function as a redox-dependent target modulator and that pyruvate kinase is modulated via an alternative mechanism.

Transcription factor Nrf1 negatively regulates the cystine/glutamate transporter and lipid-metabolizing enzymes.

Liver-specific Nrf1 (NF-E2-p45-related factor 1) knockout mice develop nonalcoholic steatohepatitis. To identify postnatal mechanisms responsible for this phenotype, we generated an inducible liver-specific Nrf1 knockout mouse line using animals harboring an Nrf1(flox) allele and a rat CYP1A1-Cre transgene (Nrf1(flox/flox)::CYP1A1-Cre mice). Administration of 3-methylcholanthrene (3-MC) to these mice (Nrf1(flox/flox)::CYP1A1-Cre+3MC mice) resulted in loss of hepatic Nrf1 expression. The livers of mice lacking Nrf1 accumulated lipid, and the hepatic fatty acid (FA) composition in such animals differed significantly from that in the Nrf1(flox/flox)::CYP1A1-Cre control. This change was provoked by upregulation of several FA metabolism genes. Unexpectedly, we also found that the level of glutathione was increased dramatically in livers of Nrf1(flox/flox)::CYP1A1-Cre+3MC mice. While expression of glutathione biosynthetic enzymes was unchanged, xCT, a component of the cystine/glutamate antiporter system x(c)(-), was significantly upregulated in livers of Nrf1(flox/flox)::CYP1A1-Cre+3MC mice, suggesting that Nrf1 normally suppresses xCT. Thus, stress-inducible expression of xCT is a two-step process: under homeostatic conditions, Nrf1 effectively suppresses nonspecific transactivation of xCT, but when cells encounter severe oxidative/electrophilic stress, Nrf1 is displaced from an antioxidant response element (ARE) in the gene promoter while Nrf2 is recruited to the ARE. Thus, Nrf1 controls both the FA and the cystine/cysteine content of hepatocytes by participating in an elaborate regulatory network.

Transcription factor NF-E2-related factor 1 impairs glucose metabolism in mice.

Nrf1 (NF-E2-related factor 1) is a basic region leucine zipper-type transcription factor belonging to the CNC (cap-'n'-collar) family. Major pathophysiological contribution of Nrf1 remains unclear. As single nucleotide polymorphism rs3764400 in 5'-flanking region of NRF1 gene appears to associate with obesity, in this study, we focused on the Nrf1 function on metabolism. We found that the risk C allele of rs3764400 increased NRF1 gene transcriptional activity compared with the T allele in hepatoma cell lines. Therefore, we newly established Nrf1 transgenic (Nrf1-Tg) mouse lines and examined roles that Nrf1 plays on the obesity and metabolism. Unexpectedly, Nrf1 over-expression repressed bodyweight gain in both lean and diet-induced obesity mice. Of note, Nrf1-Tg mice showed rise in blood glucose levels; Nrf1 strongly reduced glucose infusion rate in euglycemic-hyperinsulinemic clamp test and increased blood glucose levels in insulin tolerance test, indicating that Nrf1 induces insulin resistance in mice. Nrf1 repressed insulin-regulated glycolysis-related gene expression and gave rise to loss of glucose-6-phosphate and fructose-6-phosphate contents in liver. Consistently, Nrf1 heterozygote improved impaired glucose regulations in diet-induced obesity model. These results showed that Nrf1 contributes to metabolic regulation, which gain-of-function develops diabetes mellitus in mice.

Isocitrate dehydrogenase mutation is frequently observed in giant cell tumor of bone.

Giant cell tumors of bone (GCTB) are benign and locally destructive tumors that include osteoclast-type multinuclear giant cells. No available treatment is definitively effective in curing GCTB, especially in surgically unresectable cases. Isocitrate dehydrogenase (IDH) mutations have been reported not only in gliomas and acute myeloid leukemias, but also in cartilaginous tumors and osteosarcomas. However, IDH mutations in GCTB have not been investigated. The IDH mutations are remarkably specific to arginine 132 (R132) in IDH1 and arginine 172 (R172) or arginine 140 (R140) in IDH2; IDH1/2 mutations are known to convert α-ketoglutarate to oncometabolite R(-)-2-hydroxyglutarate. We recently reported that the most frequent IDH mutation in osteosarcomas is IDH2-R172S, which was detected by MsMab-1, a multispecific anti-IDH1/2 mAb. Herein, we newly report the IDH mutations in GCTB, which were stained by MsMab-1 in immunohistochemistry. DNA direct sequencing and subcloning identified IDH mutations of GCTB as IDH2-R172S (16 of 20; 80%). This is the first report to describe IDH mutations in GCTB, and MsMab-1 can be anticipated for use in immunohistochemical determination of IDH1/2 mutation-bearing GCTB.