O-GlcNAcylation promotes cerebellum development and medulloblastoma oncogenesis via SHH signaling.

Sonic hedgehog (Shh) signaling plays a critical role in regulating cerebellum development by maintaining the physiological proliferation of granule neuron precursors (GNPs), and its dysregulation leads to the oncogenesis of medulloblastoma. O-GlcNAcylation (O-GlcNAc) of proteins is an emerging regulator of brain function that maintains normal development and neuronal circuitry. Here, we demonstrate that O-GlcNAc transferase (OGT) in GNPs mediate the cerebellum development, and the progression of the Shh subgroup of medulloblastoma. Specifically, OGT regulates the neurogenesis of GNPs by activating the Shh signaling pathway via O-GlcNAcylation at S355 of GLI family zinc finger 2 (Gli2), which in turn promotes its deacetylation and transcriptional activity via dissociation from p300, a histone acetyltransferases. Inhibition of OGT via genetic ablation or chemical inhibition improves survival in a medulloblastoma mouse model. These data uncover a critical role for O-GlcNAc signaling in cerebellar development, and pinpoint a potential therapeutic target for Shh-associated medulloblastoma.

O-GlcNAcylation Is Required for the Survival of Cerebellar Purkinje Cells by Inhibiting ROS Generation.

Purkinje cells (PCs), as a unique type of neurons output from the cerebellar cortex, are essential for the development and physiological function of the cerebellum. However, the intricate mechanisms underlying the maintenance of Purkinje cells are unclear. The O-GlcNAcylation (O-GlcNAc) of proteins is an emerging regulator of brain function that maintains normal development and neuronal circuity. In this study, we demonstrate that the O-GlcNAc transferase (OGT) in PCs maintains the survival of PCs. Furthermore, a loss of OGT in PCs induces severe ataxia, extensor rigidity and posture abnormalities in mice. Mechanistically, OGT regulates the survival of PCs by inhibiting the generation of intracellular reactive oxygen species (ROS). These data reveal a critical role of O-GlcNAc signaling in the survival and maintenance of cerebellar PCs.

AmyZ1: a novel α-amylase from marine bacterium Pontibacillus sp. ZY with high activity toward raw starches.

BACKGROUND: Starch is an inexpensive and renewable raw material for numerous industrial applications. However, most starch-based products are not cost-efficient due to high-energy input needed in traditional enzymatic starch conversion processes. Therefore, α-amylase with high efficiency to directly hydrolyze high concentration raw starches at a relatively lower temperature will have a profound impact on the efficient application of starch. RESULTS: A novel raw starch digesting α-amylase (named AmyZ1) was screened and cloned from a deep-sea bacterium Pontibacillus sp. ZY. Phylogenetic analysis showed that AmyZ1 was a member of subfamily 5 of glycoside hydrolase family 13. When expressed in Escherichia coli, the recombinant AmyZ1 showed high activity at pH 6.0-7.5 and 25-50 °C. Its optimal pH and temperature were 7.0 and 35 °C, respectively. Similar to most α-amylases, AmyZ1 activity was enhanced (2.4-fold) by 1.0 mM Ca2+. Its half-life time at 35 °C was also extended from about 10 min to 100 min. In comparison, AmyZ1 showed a broad substrate specificity toward raw starches, including those derived from rice, corn, and wheat. The specific activity of AmyZ1 towards raw rice starch was 12,621 ± 196 U/mg, much higher than other reported raw starch hydrolases. When used in raw starch hydrolyzing process, AmyZ1 hydrolyzed 52%, 47% and 38% of 30% (w/v) rice, corn, and wheat starch after 4 h incubation. It can also hydrolyze marine raw starch derived from Chlorella pyrenoidosa, resulting in 50.9 mg/g DW (dry weight of the biomass) of reducing sugars after 4 h incubation at 35 °C. Furthermore, when hydrolyzing raw corn starch using the combination of AmyZ1 and commercial glucoamylase, the hydrolysis rate reached 75% after 4.5 h reaction, notably higher than that obtained in existing starch-processing industries. CONCLUSIONS: As a novel raw starch-digesting α-amylase with high specific activity, AmyZ1 efficiently hydrolyzed raw starches derived from both terrestrial and marine environments at near ambient temperature, suggesting its application potential in starch-based industrial processes.

Carbohydrate-binding module assisted purification and immobilization of ß-glucosidase onto cellulose and application in hydrolysis of soybean isoflavone glycosides.

Complicated purification steps, together with the fact that ß-glucosidase has to be tolerant to ethanol restricts the application of ß-glucosidase in isoflavone aglycone hydrolyzing process. ß-Glucosidase Bgl1A(A24S/F297Y) is a promising enzyme in hydrolyzing isoflavones. In this work, six different carbohydrate-binding modules (CBMs), which were from 3 families, were fused to the C-terminal of Bgl1A(A24S/F297Y), respectively, to simplify the enzyme preparation process. The fusion proteins were expressed in Escherichia coli and adsorbed onto cellulose. The Bgl-CBM24 was found to have the highest immobilization efficiency at room temperature within 1 h adsorption. Notably, 1-g cellulose absorbs up to 254.9±5.7 U of Bgl-CBM24. Interestingly, the immobilized Bgl-CBM24 showed improved ethanol tolerance ability, with the IC50 of 35% (v/v) ethanol. Bgl-CBM24 effectively hydrolyze soybean isoflavone glycosides. The hydrolysis rate of daidzin and gemistin was 85.22±3.24% and 82.14±3.82% within 10 min, with the concentrations of daidzein and genistein increased by 6.36±0.18 mM and 3.98±0.22 mM, respectively. In the repetitive hydrolytic cycles, the concentrations of daidzein and genistein still increased by 3.07±0.24 mM and 1.94±0.34 mM in the fourth cycle with 20% (v/v) ethanol. These results suggest that the immobilized Bgl-CBM24 has excellent potential in the preparation of isoflavone aglycones.