Cloning,over expression and functional attributes of serine proteases from Oceanobacillus iheyensis O.M.A18 and Haloalkaliphilic bacterium O.M.E12

Cloning, over-expression, characterization and structural and functional analysis of two alkaline proteases from the newly isolated haloalkaliphilic bacteria: Oceanobacillus iheyensis O.M.A₁8 and Haloalkaliphilic bacterium O.M.E₁2 were carried out. The cloned protease genes were over-expressed in Escherichia coli within 6 h of the IPTG induction. The protease genes were sequenced and the sequence submitted to the GenBank with the accession numbers, HM219179 and HM219182. The recombinant proteases were active in the range of pH 8–11 and temperature 30–50 °C. The amino acid sequences of the alkaline proteases displayed hydrophobic character and stable configurations. The amino acids Asp 141, His 171 and Ser 324 formed the catalytic triad, while Ile, Leu and Ser were other amino acid moieties present in the active site. The characteristics of the recombinant proteases were compared and found to be similar to their native counterparts. On the basis of the in-silico analysis and inhibitor studies, the enzymes were confirmed as serine proteases. The study hold significance as only limited enzymes from the haloalkaliphilic bacteria have been cloned, sequenced and analyzed for the structure and function analysis.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

Inhibition of the lymphoid tyrosine phosphatase: The effect of zinc(II) ions and chelating ligand fragments on enzymatic activity

A 96-member chelator fragment library (CFL-1.1) was screened to identify inhibitors of the lymphoid tyrosine phosphatase in the absence and presence of zinc acetate. Fragments that inhibit LYP activity more potently in the presence of zinc, fragments that rescue LYP activity in the presence of inhibitory concentrations of zinc, and fragments that inhibit LYP activity independent of zinc concentration were identified. Of these, 1,2-dihydroxynaphthalene was the most potent inhibitor with an IC₅₀ value of 2.52 ± 0.06 μM after 2 h of incubation. LYP inhibition by 1,2-dihydroxynaphthalene was very similar to inhibition by 1,2-naphthoquinone (IC₅₀ = 1.10 ± 0.03 µM), indicating that the oxidized quinone species is likely the active inhibitor. The inhibition was time-dependent, consistent with covalent modification of the enzyme.     

Structure of the Human TELO2-TTI1-TTI2 Complex

Phosphatidylinositol 3-kinase-related protein kinases (PIKKs) play critical roles in various metabolic pathways related to cell proliferation and survival. The TELO2-TTI1-TTI2 (TTT) complex has been proposed to recognize newly synthesized PIKKs and to deliver them to the R2TP complex (RUVBL1-RUVBL2-RPAP3-PIH1D1) and the heat shock protein 90 chaperone, thereby supporting their folding and assembly. Here, we determined the cryo-EM structure of the TTT complex at an average resolution of 4.2 Å. We describe the full-length structures of TTI1 and TELO2, and a partial structure of TTI2. All three proteins form elongated helical repeat structures. TTI1 provides a platform on which TELO2 and TTI2 bind to its central region and C-terminal end, respectively. The TELO2 C-terminal domain (CTD) is required for the interaction with TTI1 and recruitment of Ataxia-telangiectasia mutated (ATM). The N- and C-terminal segments of TTI1 recognize the FRAP-ATM-TRRAP (FAT) domain and the N-terminal HEAT repeats of ATM, respectively. The TELO2 CTD and TTI1 N- and C-terminal segments are required for cell survival in response to ionizing radiation.     

Pseudomonas aeruginosa cholinesterase and phosphorylcholine phosphatase: two enzymes contributing to corneal infection

Choline, acetylcholine and betaine used as the sole carbon, nitrogen or carbon and nitrogen source increase cholinesterase activity in addition to phosphorylcholine phosphatase and phospholipase C activities in Pseudomonas aeruginosa. The cholinesterase activity catalyses the hydrolysis of acetylthiocholine (Km approx. 0.13 mM) and propionylthiocholine (Km approx. 0.26 mM), but not butyrylthiocholine, which is a pure competitive inhibitor (Ki 0.05 mM). Increasing choline concentrations in the assay mixture decreased the affinity of cholinesterase for acetylthiocholine, but in all cases prevented inhibition raised by high substrate concentrations. Considering the properties of these enzymes, and the fact that in the corneal epithelium there exists a high acetylcholine concentration and that Pseudomonas aeruginosa produces corneal infection, it is proposed that these enzymes acting coordinately might contribute to the breakdown of the corneal epithelial membrane.     

Optimization of integrated alkaline–extrusion pretreatment of barley straw for sugar production by enzymatic hydrolysis

In this work, an integrated one-step alkaline–extrusion process was tested as pretreatment for sugar production from barley straw (BS) biomass. The influence of extrusion temperature (T) and the ratio NaOH/BS dry matter (w/w) (R) into the extruder on pretreatment effectiveness was investigated in a twin-screw extruder at bench scale. A 2³ factorial design of experiments was used to analyze the effect of process conditions [T: 50–100 °C; R: 2.5–7.5% (w/w)] on composition and enzymatic digestibility of pretreated substrate (extrudate). The optimum conditions for a maximum glucan to glucose conversion were determined to be R = 6% and T = 68 °C. At these conditions, glucan yield reached close to 90% of theoretical, while xylan conversion was 71% of theoretical. These values are 5 and 9 times higher than that of the untreated material, which supports the great potential of this one-step combined pre-treatment technology for sugar production from lignocellulosic substrates. The absence of sugar degradation products is a relevant advantage over other traditional methods for a biomass to ethanol production process since inhibitory effect of such product on sugar fermentation would be prevented.     

PPM1D regulates p21 expression via dephoshporylation at serine 123

The cyclin-dependent kinase inhibitor p21 plays a critical role in regulating cell cycle and cell proliferation. We previously cloned the dog p21 gene and found that unlike human p21, dog p21 is expressed as 2 isoforms due to the proline-directed phosphorylation at serine 123 (S123). Here, we identified that PPM1D, also called Wip1 and a Mg²⁺-dependent phosphatase, dephosphorylates dog p21 protein at serine 123. Specifically, we showed that the level of S123-phosphorylated dog p21 is increased by a PPM1D inhibitor in a dose-dependent manner. We also showed that over-expression of PPM1D decreases, whereas knockdown of PPM1D increases, the level of S123-phosphorylated dog p21 regardless of p53. Additionally, in vitro phosphatase assay was performed and showed that phosphorylated S123 in dog p21 is dephosphorylated by recombinant rPPM1D, which contains the catalytic domain of human PPM1D (residue 1–420), but not by the phosphatase dead rPPM1D (D314A). Furthermore, dephosphorylation of S123 by rPPM1D can be abrogated by PPM1D inhibitor or by withdrawal of Mg²⁺. Finally, we showed that upon PPM1D inhibition, the level of S123-phosphorylated dog p21 was increased, concomitantly with decreased expression of cyclin A, cyclin B, Rb, and PCNA. Together, our results indicate that PPM1D functions as a phosphatase of dog p21 at serine 123 and plays a role in cell cycle control via p21.     

The HIF-2alpha dependent induction of PAP and adenosine synthesis regulates glioblastoma stem cell function through the A2B adenosine receptor

Glioblastomas are lethal tumors characterized by malignant proliferation and recurrence promoted partly by glioblastoma stem cells (GSCs). GSCs are known to be regulated by hypoxia, but the mechanisms involved in this regulation are not fully understood. We now demonstrate that hypoxia-inducible factor HIF2α and prostatic acid phosphatase (PAP) are preferentially expressed in hypoxic GSCs in comparison with non-stem tumor cells and normal neural stem cells and that PAP is regulated by HIF2α. Targeting PAP in hypoxic GSCs inhibits self-renewal and proliferation in vitro and attenuates tumor initiation potential of GSCs in vivo. Using specific adenosine receptor antagonists, we further find that the pro-proliferative role of PAP is stemmed from stimulated A2B adenosine receptors. Moreover, selective blockage of A2B receptor or knockdown of PAP or A2B on hypoxic GSCs results in significant reduction of phosphorylation of Akt and Erk-1/2. Our results demonstrate that PAP may play a pro-proliferative role in hypoxic GSCs with a HIF2α-induction pattern, which may be ascribed to stimulated A2B receptors and activated Akt and Erk-1/2 pathways. Therefore, we propose that these identified molecular regulators of GSCs in the hypoxic niche might represent promising targets for antiglioblastoma therapies.     

Substrate Selectivity in Aspergillus niger KU-8 Acid Phosphatase II Using Phosphoryl Oligosaccharides

The intracellular acid phosphatase II (ACPase II) produced by Aspergillus niger KU-8 preferentially dephosphorylates C-6 phosphate groups rather than C-3 phosphate groups of phosphoryl oligosaccharides. In this study, the kinetic parameters of ACPase II were measured. 3²-phosphoryl maltotriose and 6²-phosphoryl maltotriose, which differ only in the binding position of the phosphate group, were prepared and used as the substrates. The K _m for both substrates were similar. However, the k _cat value for the 6²-phosphoryl maltotriose was about three-fold of that for the 3²-phosphoryl maltotriose.