Search

Some acute exercise effects are influenced by postexercise (PEX) diet, and these diet-effects are attributed to differential glycogen resynthesis. However, this idea is challenging to test rigorously. Therefore, we devised a novel genetic model to modify muscle glycogen synthase 1 (GS1) expression in rat skeletal muscle with an adeno-associated virus (AAV) short hairpin RNA knockdown vector targeting GS1 (shRNA-GS1). Contralateral muscles were injected with scrambled shRNA (shRNA-Scr). Muscles from exercised (2-hour-swim) and time-matched sedentary (Sed) rats were collected immediately postexercise (IPEX), 5-hours-PEX (5hPEX), or 9-hours-PEX (9hPEX). Rats in 5hPEX and 9hPEX experiments were refed (RF) or not-refed (NRF) chow. Muscles were analyzed for glycogen, abundance of metabolic proteins (pyruvate dehydrogenase kinase 4, PDK4; peroxisome proliferator-activated receptor γ coactivator-1α, PGC1α; hexokinase II, HKII; glucose transporter 4, GLUT4), AMP-activated protein kinase phosphorylation (pAMPK), and glycogen metabolism-related enzymes (glycogen phosphorylase, PYGM; glycogen debranching enzyme, AGL; glycogen branching enzyme, GBE1). shRNA-GS1 versus paired shRNA-Scr muscles had markedly lower GS1 abundance. IPEX versus Sed rats had lower glycogen and greater pAMPK, and neither of these IPEX-values differed for shRNA-GS1 versus paired shRNA-Scr muscles. IPEX versus Sed groups did not differ for abundance of metabolic proteins, regardless of GS1 knockdown. Glycogen in RF-rats was lower for shRNA-GS1 versus paired shRNA-Scr muscles at both 5hPEX and 9hPEX. HKII protein abundance was greater for 5hPEX versus Sed groups, regardless of GS1 knockdown or diet, and despite differing glycogen levels. At 9hPEX, shRNA-GS1 versus paired shRNA-Scr muscles had greater PDK4 and PGC1α abundance within each diet group. However, the magnitude of PDK4 or PGC1α changes was similar in each diet group regardless of GS1 knockdown although glycogen differed between paired muscles only in RF-rats. In summary, we established a novel genetic approach to investigate the relationship between muscle glycogen and other exercise effects. Our results suggest that exercise-effects on abundance of several metabolic proteins did not uniformly correspond to differences in postexercise glycogen. [ABSTRACT FROM AUTHOR]

Introduction: McArdle disease presents clinical and genetic heterogeneity. There is no obvious association between genotype and phenotype. PYGM (muscle glycogen phosphorylase gene) mRNA expression and its association with clinical, morphological, and genetic aspects of the disease as a set have not been studied previously. Methods: We investigated genetic variation in PYGM considering the number of PTCs (premature termination codon) per sample and compared mRNA expression in skeletal muscle samples from 15 patients with McArdle disease and 16 controls to PTCs number and different aspects of the disease. Results: The main variant found was c.148C>T (PTC—premature termination codon). Patients with two PTCs showed 42% mRNA expression compared to the control group. Most cases showed an inversely proportional relation among PTCs and mRNA expression. Association between mRNA expression and other aspects of the disease showed no statistically significant difference (p> 0.05). Discussion: mRNA expression is not useful as a predictor factor for the prognosis and severity of the disease. Different mechanisms as post-transcriptional events, epigenetics factors or protein function may be involved. [ABSTRACT FROM AUTHOR]

Enzymes acting on α-L-arabinofuranosides have been extensively studied; however, the structures and functions of β-L-arabinofuranosidases are not fully understood. Three enzymes and an ABC transporter in a gene cluster of Bifidobacterium longum JCM 1217 constitute a degradation and import system of β-L-arabinooligosaccharides on plant hydroxyproline-rich glycoproteins. An extracellular β-L-arabinobiosidase (HypBA2) belonging to the glycoside hydrolase (GH) family 121 plays a key role in the degradation pathway by releasing β-1,2-linked arabinofuranose disaccharide (β-Ara2) for the specific sugar importer. Here, we present the crystal structure of the catalytic region of HypBA2 as the first three-dimensional structure of GH121 at 1.85 Å resolution. The HypBA2 structure consists of a central catalytic (α/α)6 barrel domain and two flanking (N- and C-terminal) β-sandwich domains. A pocket in the catalytic domain appears to be suitable for accommodating the β-Ara2 disaccharide. Three acidic residues Glu383, Asp515, and Glu713, located in this pocket, are completely conserved among all members of GH121; site-directed mutagenesis analysis showed that they are essential for catalytic activity. The active site of HypBA2 was compared with those of structural homologs in other GH families: GH63 α-glycosidase, GH94 chitobiose phosphorylase, GH142 β-L-arabinofuranosidase, GH78 α-L-rhamnosidase, and GH37 α,α-trehalase. Based on these analyses, we concluded that the three conserved residues are essential for catalysis and substrate binding. β-L-Arabinobiosidase genes in GH121 are mainly found in the genomes of bifidobacteria and Xanthomonas species, suggesting that the cleavage and specific import system for the β-Ara2 disaccharide on plant hydroxyproline-rich glycoproteins are shared in animal gut symbionts and plant pathogens. [ABSTRACT FROM AUTHOR]

Low temperature induces changes in plants at physiological and molecular levels, thus affecting growth and development. The Lanzhou lily (Lilium davidii, var. unicolor) is an important medicinal plant with high economic value. However, the molecular mechanisms underlying its photosynthetic and antioxidation responses to low temperature still remain poorly understood. This study subjected the Lanzhou lily to the two temperatures of 20°C (control) and 4°C (low temperature) for 24 h. Physiological parameters related to membrane integrity, photosynthesis, antioxidant system, and differentially expressed genes were investigated. Compared with control, low temperature increased the relative electrical conductivity by 43.2%, while it decreased net photosynthesis rate, ratio of variable to maximal fluorescence, and catalase activity by 47.3%, 10.1%, and 11.1%, respectively. In addition, low temperature significantly increased the content of soluble protein, soluble sugar, and proline, as well as the activity of superoxide dismutase and peroxidase. Comparative transcriptome profiling showed that a total of 238,109 differentially expressed genes were detected. Among these, 3,566 were significantly upregulated while 2,982 were significantly downregulated in response to low temperature. Gene Ontology enrichment analysis indicated that in response to low temperature, the mostly significantly enriched differentially expressed genes were mainly involved in phosphorylation, membrane and protein kinase activity, as well as photosynthesis, light harvesting, light reaction, and alpha,alpha-trehalose-phosphate synthase activity. Kyoto Encyclopedia of Genes and Genomes enrichment analysis also indicated that the most significantly enriched pathways involved ribosome biogenesis in eukaryotes, phenylalanine metabolism, circadian rhythm, porphyrin and chlorophyll metabolism, photosynthesis of antenna proteins, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, the expression patterns of 10 randomly selected differentially expressed genes confirmed the RNA-Seq results. These results expand the understanding of the physiological and molecular mechanisms underlying the response of the Lanzhou lily to low temperature stress. [ABSTRACT FROM AUTHOR]

In breast cancer, tumor hypoxia has been linked to poor prognosis and increased metastasis. Hypoxia activates transcriptional programs in cancer cells that lead to increased motility and invasion, as well as various metabolic changes. One of these metabolic changes, an increase in glycogen metabolism, has been further associated with protection from reactive oxygen species damage that may lead to premature senescence. Here we report that breast cancer cells significantly increase glycogen stores in response to hypoxia. We found that knockdown of the brain isoform of an enzyme that catalyzes glycogen breakdown, glycogen phosphorylase B (PYGB), but not the liver isoform, PYGL, inhibited glycogen utilization in estrogen receptor negative and positive breast cancer cells; whereas both independently inhibited glycogen utilization in the normal-like breast epithelial cell line MCF-10A. Functionally, PYGB knockdown and the resulting inhibition of glycogen utilization resulted in significantly decreased wound-healing capability in MCF-7 cells and a decrease in invasive potential of MDA-MB-231 cells. Thus, we identify PYGB as a novel metabolic target with potential applications in the management and/or prevention of metastasis in breast cancer. [ABSTRACT FROM AUTHOR]

The family of ribosomal proteins S1 contains about 20% of all bacterial proteins including the S1 domain. An important feature of this family is multiple copies of structural domains in bacteria, the number of which changes in a strictly limited range from one to six. In this study, the automated exhaustive analysis of 1453 sequences of S1 allowed us to demonstrate that the number of domains in S1 is a distinctive characteristic for phylogenetic bacterial grouping in main phyla. 1453 sequences of S1 were identified in 25 out of 30 different phyla according to the List of Prokaryotic Names with Standing in Nomenclature. About 62% of all records are identified as six-domain S1 proteins, which belong to phylum Proteobacteria. Four-domain S1 are identified mainly in proteins from phylum Firmicutes and Actinobacteria. Records belonging to these phyla are 33% of all records. The least represented two-domain S1 are about 0.6% of all records. The third and fourth domains for the most representative four- and six-domain S1 have the highest percentage of identity with the S1 domain from polynucleotide phosphorylase and S1 domains from one-domain S1. In addition, for these groups, the central part of S1 (the third domain) is more conserved than the terminal domains. [ABSTRACT FROM AUTHOR]

Much is known about the regulatory elements controlling the cell cycle in fission yeast (Schizosaccharomyces pombe). This regulation is mainly done by the (cyclin-dependent kinase/cyclin) complex (Cdc2/Cdc13) that activates specific target genes and proteins via phosphorylation events during the cell cycle in a time-dependent manner. However, more work is still needed to complement the existing gaps in the current fission yeast gene regulatory network to be able to overcome abnormalities in its growth, repair and development, i.e. explain many phenomena including mitotic catastrophe. In this work we complement the previously presented core oscillator of the cell cycle of fission yeast by selected phosphorylation events and study their effects on the temporal evolution of the core oscillator based Boolean network. Thereby, we attempt to establish a regulatory link between the autonomous cell cycle oscillator and the remainder of the cell. We suggest the unclear yet regulatory effect of phosphorylation on the added components, and discuss many unreported points regarding the temporal evolution of the cell cycle and its components. To better visualize the results regardless of the programming background we developed an Android application that can be used to run the core and extended model of the fission yeast cell cycle step by step. [ABSTRACT FROM AUTHOR]

Thymidine phosphorylase (TP; EC 2.4.2.4) is involved regulation of intra- or extracellular thymidine concentration, angiogenesis, cancer chemotherapy, radiotherapy, as well as tumor imaging. Although the liver is main site of pyrimidine metabolism and contains high levels of TP, nonetheless, purification and characterization of human hepatic TP has not been accomplished. We here report the purification and characterization of native human hepatic TP. The enzyme was purified to apparent homogeneity by a procedure shorter and more efficient than previously reported methods. Human hepatic TP has an apparent Kthymidine of 285 ± 55 μM. Like the enzyme from other tissues, it is highly specific to 2'-deoxyribosides. However, in contrast to TP from other normal tissues, the hepatic enzyme is active in the phosphorolysis of 5'-deoxy-5-fluorouridine, and the riboside 5-fluorouridine. Furthermore, native hepatic TP exists in different aggregates of 50 kDa subunits, with unknown aggregation factor(s) while TP from extra tissues exists as a homodimer. Isoelectric point was determined as 4.3. A total of 65 residues in the N-terminal were sequenced. The sequence of these 65 amino acids in hepatic TP has 100% sequence and location homology to the deduced amino acid sequence of the platelet derived-endothelial cell growth factor (PD-ECGF) cDNA. However, and contrary to PD-ECGF, the N-terminal of hepatic TP is blocked. The block was neither N-formyl nor pyrrolidone carboxylic acid moieties. The differences in substrate specificities, existence in multimers, and weak interaction with hydroxyapatite resin strongly suggest that hepatic TP is distinct from the enzyme in normal extrahepatic tissues. These results may have important clinical implications when TP is involved in activation or deactivation of chemotherapeutic agents in different tissues. [ABSTRACT FROM AUTHOR]

Different test systems are used to characterize the anti-aggregation efficiency of molecular chaperone proteins and of low-molecular-weight chemical chaperones. Test systems based on aggregation of UV-irradiated protein are of special interest because they allow studying the protective action of different agents at physiological temperatures. The kinetics of UV-irradiated glycogen phosphorylase b (UV-Phb) from rabbit skeletal muscle was studied at 37°C using dynamic light scattering in a wide range of protein concentrations. It has been shown that the order of aggregation with respect to the protein is equal to unity. A conclusion has been made that the rate-limiting stage of the overall process of aggregation is heat-induced structural reorganization of a UV-Phb molecule, which contains concealed damage. [ABSTRACT FROM AUTHOR]

The production of starch is essential for human nutrition and represents a major metabolic flux in the biosphere. The biosynthesis of starch in storage organs like barley endosperm operates via two main pathways using different substrates: starch synthases use ADP-glucose to produce amylose and amylopectin, the two major components of starch, whereas starch phosphorylase (Pho1) uses glucose-1-phosphate (G1P), a precursor for ADP-glucose production, to produce α-1,4 glucans. The significance of the Pho1 pathway in starch biosynthesis has remained unclear. To elucidate the importance of barley Pho1 (HvPho1) for starch biosynthesis in barley endosperm, we analyzed HvPho1 protein production and enzyme activity levels throughout barley endosperm development and characterized structure-function relationships of HvPho1. The molecular mechanisms underlying the initiation of starch granule biosynthesis, that is, the enzymes and substrates involved in the initial transition from simple sugars to polysaccharides, remain unclear. We found that HvPho1 is present as an active protein at the onset of barley endosperm development. Notably, purified recombinant protein can catalyze the de novo production of α-1,4-glucans using HvPho1 from G1P as the sole substrate. The structural properties of HvPho1 provide insights into the low affinity of HvPho1 for large polysaccharides like starch or amylopectin. Our results suggest that HvPho1 may play a role during the initiation of starch biosynthesis in barley. [ABSTRACT FROM AUTHOR]

Deficiency of methylthioadenosine phosphorylase (MTAP) supports melanoma development and progression through accumulation of its substrate 5’-methylthioadenosine (MTA), which leads amongst others to a constitutive inhibition of protein arginine methyltransferases (PRMTs) and activation of the transcription factor AP-1 via the receptor ADORA2B. Genetic association studies have also suggested that genetic polymorphism in MTAP may modulate the risk of melanoma. Here, we investigated the only globally common non-synonymous single nucleotide polymorphism (SNP) reported to date for MTAP. The SNP rs7023954 is located in exon 3 (c.166G>A), and leads to the conservative substitution of one branched-chain amino acid residue (valine) for another (isoleucine) at position 56 (p.Val56Ile). Whereas genotype frequencies in normal and primary melanoma tissues or cell lines were in Hardy-Weinberg equilibrium based on cDNA amplicon sequencing, a marked (P = 0.00019) deviation was observed in metastatic melanoma tissues and cell lines due to a deficit of heterozygotes. Enzyme assays conducted on the co-dominantly expressed alleles revealed no difference in the conversion rate of MTA to adenine and 5-methylthioribose-1-phosphate, indicating that this known enzymatic activity does not modulate the tumor suppressive function of MTAP. [ABSTRACT FROM AUTHOR]

The white-backed planthopper (WBPH), Sogatella furcifera (Horváth), is one of the serious rice pests because of its destructive feeding. The salivary glands of the WBPH play an important role in the feeding behaviour. Currently, however, very little is known about the salivary glands at the molecular level. We sequenced the salivary gland transcriptome (sialotranscripome) of adult WBPHs using the Illumina sequencing. A total of 65,595 transcripts and 51,842 unigenes were obtained from salivary glands. According to annotations against the Nr database, many of the unigenes identified were associated with the most studied enzymes in hemipteran saliva. In the present study, we identified 32 salivary protein genes from the WBPH sialotranscripome, which were categorized as those involved in sugar metabolism, detoxification, suppression of plant defense responses, immunity-related responses, general digestion, and other phytophagy processes. Tissue expression profiles analysis revealed that four of 32 salivary protein genes (multicopper oxidase 4, multicopper oxidase 6, carboxylesterase and uridine phosphorylase 1 isform X2) were primarily expressed in the salivary gland, suggesting that they played putative role in insect-rice interactions. 13 of 32 salivary protein genes were primarily expressed in gut, which might play putative role in digestive and detoxify mechanism. Development expression profiles analysis revealed that the expression level of 26 of 32 salivary protein genes had no significant difference, suggesting that they may play roles in every developmental stages of salivary gland of WBPH. The other six genes have a high expression level in the salivary gland of adult. 31 of 32 genes (except putative acetylcholinesterase 1) have no significant difference in male and female adult, suggesting that their expression level have no difference between sexes. This report analysis of the sialotranscripome for the WBPH, and the transcriptome provides a foundational list of the genes involved in feeding. Our data will be useful to investigate the mechanisms of interaction between the WBPH and the host plant. [ABSTRACT FROM AUTHOR]

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher Km value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase. [ABSTRACT FROM AUTHOR]

In a previous study we demonstrated up-regulation of the yeast GPH1 gene under conditions of phosphatidylethanolamine (PE) depletion caused by deletion of the mitochondrial (M) phosphatidylserine decarboxylase 1 (PSD1) (Gsell et al., 2013, PLoS One. 8(10):e77380. doi: ). Gph1p has originally been identified as a glycogen phosphorylase catalyzing degradation of glycogen to glucose in the stationary growth phase of the yeast. Here we show that deletion of this gene also causes decreased levels of phosphatidylcholine (PC), triacylglycerols and steryl esters. Depletion of the two non-polar lipids in a Δgph1 strain leads to lack of lipid droplets, and decrease of the PC level results in instability of the plasma membrane. In vivo labeling experiments revealed that formation of PC via both pathways of biosynthesis, the cytidine diphosphate (CDP)-choline and the methylation route, is negatively affected by a Δgph1 mutation, although expression of genes involved is not down regulated. Altogether, Gph1p besides its function as a glycogen mobilizing enzyme appears to play a regulatory role in yeast lipid metabolism. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine whether the improvement of cerebrospinal fluid (CSF) flow dynamics by CSF shunting, can suppress the oligomerization of amyloid β-peptide (Aβ), by measuring the levels of Alzheimer’s disease (AD)-related proteins in the CSF before and after lumboperitoneal shunting. Lumbar CSF from 32 patients with idiopathic normal pressure hydrocephalus (iNPH) (samples were obtained before and 1 year after shunting), 15 patients with AD, and 12 normal controls was analyzed for AD-related proteins and APLP1-derived Aβ-like peptides (APL1β) (a surrogate marker for Aβ). We found that before shunting, individuals with iNPH had significantly lower levels of soluble amyloid precursor proteins (sAPP) and Aβ38 compared to patients with AD and normal controls. We divided the patients with iNPH into patients with favorable (improvement ≥ 1 on the modified Rankin Scale) and unfavorable (no improvement on the modified Rankin Scale) outcomes. Compared to the unfavorable outcome group, the favorable outcome group showed significant increases in Aβ38, 40, 42, and phosphorylated-tau levels after shunting. In contrast, there were no significant changes in the levels of APL1β25, 27, and 28 after shunting. After shunting, we observed positive correlations between sAPPα and sAPPβ, Aβ38 and 42, and APL1β25 and 28, with shifts from sAPPβ to sAPPα, from APL1β28 to 25, and from Aβ42 to 38 in all patients with iNPH. Our results suggest that Aβ production remained unchanged by the shunt procedure because the levels of sAPP and APL1β were unchanged. Moreover, the shift of Aβ from oligomer to monomer due to the shift of Aβ42 (easy to aggregate) to Aβ38 (difficult to aggregate), and the improvement of interstitial-fluid flow, could lead to increased Aβ levels in the CSF. Our findings suggest that the shunting procedure can delay intracerebral deposition of Aβ in patients with iNPH. [ABSTRACT FROM AUTHOR]

We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and -mannose as the optimal acceptors, respectively, in the presence of the donor α--mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α--mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α--mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789. [ABSTRACT FROM AUTHOR]

Background: FAM20C is a kinase that phosphorylates secretory proteins. Previous studies have shown that FAM20C plays an essential role in the formation and mineralization of bone, dentin and enamel. The present study analyzed the loss-of-function effects of FAM20C on the health of mouse periodontal tissues. Methods: By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20Cfl/fl mice, we created 2.3 kb Col 1a1-Cre;Fam20Cfl/fl (cKO) mice, in which Fam20C was inactivated in the cells that express Type I collagen. We analyzed the periodontal tissues in the cKO mice using X-ray radiography, histology, scanning electron microscopy and immunohistochemistry approaches. Results: The cKO mice underwent a remarkable loss of alveolar bone and cementum, along with inflammation of the periodontal ligament and formation of periodontal pockets. The osteocytes and lacuno-canalicular networks in the alveolar bone of the cKO mice showed dramatic abnormalities. The levels of bone sialoprotein, osteopontin, dentin matrix protein 1 and dentin sialoprotein were reduced in the Fam20C-deficient alveolar bone and/or cementum, while periostin and fibrillin-1 were decreased in the periodontal ligament of the cKO mice. Conclusion: Loss of Fam20C function leads to periodontal disease in mice. The reduced levels of bone sialoprotein, osteopontin, dentin matrix protein 1, dentin sialoprotein, periostin and fibrillin-1 may contribute to the periodontal defects in the Fam20C-deficient mice. [ABSTRACT FROM AUTHOR]

Background: GABAA receptor (GABAAR) function is maintained by an endogenous phosphorylation mechanism for which the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase. This phosphorylation is specific to the long intracellular loop I2 of the α1 subunit at two identified serine and threonine residues. The phosphorylation state is opposed by an unknown membrane-bound phosphatase, which inhibition favors the phosphorylated state of the receptor and contributes to the maintenance of its function. In cortical nervous tissue from epileptogenic areas in patients with drug-resistant epilepsies, both the endogenous phosphorylation and the functional state of the GABAAR are deficient. Methodology/Principal Findings: The aim of this study is to characterize the membrane-bound phosphatases counteracting the endogenous phosphorylation of GABAAR. We have developed a new analytical tool for in vitro detection of the phosphatase activities in cortical washed membranes by liquid chromatography coupled to mass spectrometry. The substrates are two synthetic phosphopeptides, each including one of the identified endogenous phosphorylation sites of the I2 loop of GABAAR α1 subunit. We have shown the presence of multiple and atypical phosphatases sensitive to zinc ions. Patch-clamp studies of the rundown of the GABAAR currents on acutely isolated rat pyramidal cells using the phosphatase inhibitor okadaic acid revealed a clear heterogeneity of the phosphatases counteracting the function of the GABAAR. Conclusion/Significance: Our results provide new insights on the regulation of GABAAR endogenous phosphorylation and function by several and atypical membrane-bound phosphatases specific to the α1 subunit of the receptor. By identifying specific inhibitors of these enzymes, novel development of antiepileptic drugs in patients with drug-resistant epilepsies may be proposed. [ABSTRACT FROM AUTHOR]

Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell proliferation and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1β and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1β and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC. [ABSTRACT FROM AUTHOR]

Background: Abnormal release of neurotransmitters after microwave exposure can cause learning and memory deficits. This study investigated the mechanism of this effect by exploring the potential role of phosphorylated synapsin I (p-Syn I). Methods: Wistar rats, rat hippocampal synaptosomes, and differentiated (neuronal) PC12 cells were exposed to microwave radiation for 5 min at a mean power density of 30 mW/cm2. Sham group rats, synaptosomes, and cells were otherwise identically treated and acted as controls for all of the following post-exposure analyses. Spatial learning and memory in rats was assessed using the Morris Water Maze (MWM) navigation task. The protein expression and presynaptic distribution of p-Syn I and neurotransmitter transporters were examined via western blotting and immunoelectron microscopy, respectively. Levels amino acid neurotransmitter release from rat hippocampal synaptosomes and PC12 cells were measured using high performance liquid chromatograph (HPLC) at 6 hours after exposure, with or without synapsin I silencing via shRNA transfection. Results: In the rat experiments, there was a decrease in spatial memory performance after microwave exposure. The expression of p-Syn I (ser-553) was decreased at 3 days post-exposure and elevated at later time points. Vesicular GABA transporter (VGAT) was significantly elevated after exposure. The GABA release from synaptosomes was attenuated and p-Syn I (ser-553) and VGAT were both enriched in small clear synaptic vesicles, which abnormally assembled in the presynaptic terminal after exposure. In the PC12 cell experiments, the expression of p-Syn I (ser-553) and GABA release were both attenuated at 6 hours after exposure. Both microwave exposure and p-Syn I silencing reduced GABA release and maximal reduction was found for the combination of the two, indicating a synergetic effect. Conclusion: p-Syn I (ser-553) was found to play a key role in the impaired GABA release and cognitive dysfunction that was induced by microwave exposure. [ABSTRACT FROM AUTHOR]

We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-d-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein. [ABSTRACT FROM AUTHOR]

Boar-sperm cryopreservation is not usually performed immediately after semen collection, but rather a holding time (HT) of 4 h–30 h at 17°C is spent before starting this procedure. Taking this into account, the aim of this study was to go further in-depth into the mechanisms underlying the improving effects of HT at 17°C on boar-sperm cryotolerance by evaluating the effects of two different HTs (3 h and 24 h) on overall boar-sperm function and survival before and after cryopreservation. Given that phospho/dephosphorylation mechanisms are of utmost importance in the overall regulation of sperm function, the phosphorylation levels of serine residues (pSer) in 30 different sperm proteins after a 3 h- or 24 h-HT period were also assessed. We found that a HT of 24 h contributed to a higher sperm resistance to freeze-thawing procedures, whereas mini-array protein analyses showed that a HT of 24 h induced a significant (P<0.05) increase in pSer (from 100.0±1.8 arbitrary units in HT 3 h to 150.2±5.1 arbitrary units in HT 24 h) of HSP70 and, to a lesser extent, in protein kinases GSK3 and total TRK and in the cell-cycle regulatory protein CDC2/CDK1. In the case of HSP70, this increase was confirmed through immunoprecipation analyses. Principal component and multiple regression analyses indicated that a component explaining a percentage of variance higher than 50% in sperm cryotolerance was significantly correlated with pSer levels in HSP70. In addition, from all the parameters evaluated before freeze-thawing, only pSer levels in HSP70 resulted to be able to predict sperm cryotolerance. In conclusion, our results suggest that boar spermatozoa modulate its function during HT, at least partially, by changes in pSer levels of proteins like HSP70, and this is related to a higher cryotolerance. [ABSTRACT FROM AUTHOR]

We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-d-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein. [ABSTRACT FROM AUTHOR]

In animals, the main precursor for glycosaminoglycan and furthermore proteoglycan biosynthesis, like hyaluronic acid, is UDP-glucuronic acid, which is synthesized via the nucleotide sugar oxidation pathway. Mutations in this pathway cause severe developmental defects (deficiency in the initiation of heart valve formation). In plants, UDP-glucuronic acid is synthesized via two independent pathways. Beside the nucleotide sugar oxidation pathway, a second minor route to UDP-glucuronic acid exist termed the myo-inositol oxygenation pathway. Within this myo-inositol is ring cleaved into glucuronic acid, which is subsequently converted to UDP-glucuronic acid by glucuronokinase and UDP-sugar pyrophosphorylase. Here we report on a similar, but bifunctional enzyme from zebrafish (Danio rerio) which has glucuronokinase/putative pyrophosphorylase activity. The enzyme can convert glucuronic acid into UDP-glucuronic acid, required for completion of the alternative pathway to UDP-glucuronic acid via myo-inositol and thus establishes a so far unknown second route to UDP-glucuronic acid in animals. Glucuronokinase from zebrafish is a member of the GHMP-kinase superfamily having unique substrate specificity for glucuronic acid with a Km of 31±8 µM and accepting ATP as the only phosphate donor (Km: 59±9 µM). UDP-glucuronic acid pyrophosphorylase from zebrafish has homology to bacterial nucleotidyltransferases and requires UTP as nucleosid diphosphate donor. Genes for bifunctional glucuronokinase and putative UDP-glucuronic acid pyrophosphorylase are conserved among some groups of lower animals, including fishes, frogs, tunicates, and polychaeta, but are absent from mammals. The existence of a second pathway for UDP-glucuronic acid biosynthesis in zebrafish likely explains some previous contradictory finding in jekyll/ugdh zebrafish developmental mutants, which showed residual glycosaminoglycans and proteoglycans in knockout mutants of UDP-glucose dehydrogenase. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s−1. No carbohydrate acted as acceptor for the reverse phosphorolysis using β-d-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a kcat of 2.8 s−1; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H218O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-d-glucosylglycerol (GG) at the rate of 180 s−1. Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a kcat of 95 s−1. We propose 2-O-α-d-glucopyranosylglycerol: phosphate β-d-glucosyltransferase as the systematic name and 2-O-α-d-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates. [ABSTRACT FROM AUTHOR]

To understand the role of ubiquitous phototrophic periphyton in aquatic ecosystem on the biogeochemical cycling of organic phosphorus, the conversion and removal kinetic characteristics of organic phosphorus (Porg) such as adenosine triphosphate (ATP) were investigated in the presence of the periphyton cultured in artificial non-point source wastewater. The preliminary results showed that the periphyton was very powerful in converting Porg evidenced by the fact that inorganic phosphorus (Pinorg) content in solution increased from about 0.7 to 14.3 mg P L−1 in 48 hours in the presence of 0.6 g L−1 periphyton. This was because the periphyton could produce abundant phosphatases that benefited the conversion of Porg to Pinrog. Moreover, this conversion process was described more suitable by the pseudo-first-order kinetic model. The periphyton was also effective in removing Porg, which showed that the Porg can be completely removed even when the initial Porg concentration was as high as 13 mg P L−1 in 48 hours in the presence of 1.6 g L−1 periphyton. Furthermore, it was found that biosorption dominated the Porg removal process and exhibited the characteristics of physical adsorption. However, this biosorption process by the periphyton was significantly influenced by biomass (absorbent dosage) and temperature. This work provides insights into Porg biogeochemical circulation of aquatic ecosystem that contained the periphyton or similar microbial aggregates. [ABSTRACT FROM AUTHOR]

Background and Purpose: The targeting of therapeutics is a promising approach for the development of new cancer treatments that seek to reduce the devastating side effects caused by the systemic administration of current drugs. This study evaluates a fusion protein developed as an enzyme prodrug therapy targeted to the tumor vasculature. Cytotoxicity would be localized to the site of the tumor using a protein fusion of purine nucleoside phosphorylase (PNP) and annexin V. Annexin V acts as the tumor-targeting component of the fusion protein as it has been shown to bind to phosphatidylserine expressed externally on cancer cells and the endothelial cells of the tumor vasculature, but not normal vascular endothelial cells. The enzymatic component of the fusion, PNP, converts the FDA-approved cancer therapeutic, fludarabine, into a more cytotoxic form. The purpose of this study is to determine if this system has a good potential as a targeted therapy for breast cancer. Methods: A fusion of E. coli purine nucleoside phosphorylase and human annexin V was produced in E. coli and purified. Using human breast cancer cell lines MCF-7 and MDA-MB-231 and non-confluent human endothelial cells grown in vitro, the binding strength of the fusion protein and the cytotoxicity of the enzyme prodrug system were determined. Endothelial cells that are not confluent expose phosphatidylserine and therefore mimic the tumor vasculature. Results: The purified recombinant fusion protein had good enzymatic activity and strong binding to the three cell lines. There was significant cell killing (p<0.001) by the enzyme prodrug treatment for all three cell lines, with greater than 80% cytotoxicity obtained after 6 days of treatment. Conclusion: These results suggest that this treatment could be useful as a targeted therapy for breast cancer. [ABSTRACT FROM AUTHOR]

Matrix metalloproteinase-2 (MMP-2) is a key intra- and extra-cellular protease which contributes to several oxidative stress related pathologies. A molecular understanding of 72 kDa MMP-2 activity, directly mediated by S-glutathiolation of its cysteine residues in the presence of peroxynitrite (ONOO−) and by phosphorylation of its serine and threonine residues, is essential to develop new generation inhibitors of intracellular MMP-2. Within its propeptide and collagen binding domains there is an interesting juxtaposition of predicted phosphorylation sites with nearby cysteine residues which form disulfide bonds. However, the combined effect of these two post-translational modifications on MMP-2 activity has not been studied. The activity of human recombinant 72 kDa MMP-2 (hrMMP-2) following in vitro treatments was measured by troponin I proteolysis assay and a kinetic activity assay using a fluorogenic peptide substrate. ONOO− treatment in the presence of 30 µM glutathione resulted in concentration-dependent changes in MMP-2 activity, with 0.1–1 µM increasing up to twofold and 100 µM attenuating its activity. Dephosphorylation of MMP-2 with alkaline phosphatase markedly increased its activity by sevenfold, either with or without ONOO−. Dephosphorylation of MMP-2 also affected the conformational structure of the enzyme as revealed by circular dichroism studies, suggesting an increase in the proportion of α-helices and a decrease in β-strands compared to the phosphorylated form of MMP-2. These results suggest that ONOO− activation (at low µM) and inactivation (at high µM) of 72 kDa MMP-2, in the presence or absence of glutathione, is also influenced by its phosphorylation status. These insights into the role of post-translational modifications in the structure and activity of 72 kDa MMP-2 will aid in the development of inhibitors specifically targeting intracellular MMP-2. [ABSTRACT FROM AUTHOR]

Background: Directly comparing gene expression profiles of estrogen receptor-positive (ER+) and estrogen receptor-negative (ER−) breast cancers cannot determine whether differentially expressed genes between these two subtypes result from dysregulated expression in ER+ cancer or ER− cancer versus normal controls, and thus would miss critical information for elucidating the transcriptomic difference between the two subtypes. Principal Findings: Using microarray datasets from TCGA, we classified the genes dysregulated in both ER+ and ER− cancers versus normal controls into two classes: (i) genes dysregulated in the same direction but to a different extent, and (ii) genes dysregulated to opposite directions, and then validated the two classes in RNA-sequencing datasets of independent cohorts. We showed that the genes dysregulated to a larger extent in ER+ cancers than in ER− cancers enriched in glycerophospholipid and polysaccharide metabolic processes, while the genes dysregulated to a larger extent in ER− cancers than in ER+ cancers enriched in cell proliferation. Phosphorylase kinase and enzymes of glycosylphosphatidylinositol (GPI) anchor biosynthesis were upregulated to a larger extent in ER+ cancers than in ER− cancers, whereas glycogen synthase and phospholipase A2 were downregulated to a larger extent in ER+ cancers than in ER− cancers. We also found that the genes oppositely dysregulated in the two subtypes significantly enriched with known cancer genes and tended to closely collaborate with the cancer genes. Furthermore, we showed the possibility that these oppositely dysregulated genes could contribute to carcinogenesis of ER+ and ER− cancers through rewiring different subpathways. Conclusions: GPI-anchor biosynthesis and glycogenolysis were elevated and hydrolysis of phospholipids was depleted to a larger extent in ER+ cancers than in ER− cancers. Our findings indicate that the genes oppositely dysregulated in the two subtypes are potential cancer genes which could contribute to carcinogenesis of both ER+ and ER− cancers through rewiring different subpathways. [ABSTRACT FROM AUTHOR]

CK1δ, a member of the casein kinase 1 family, is involved in the regulation of various cellular processes and has been associated with the pathophysiology of neurodegenerative diseases and cancer. Therefore recently, interest in generating highly specific inhibitors for personalized therapy has increased enormously. However, the efficacy of newly developed inhibitors is affected by the phosphorylation state of CK1δ. Cellular kinases phosphorylating CK1δ within its C-terminal domain have been identified but still more information regarding the role of site-specific phosphorylation in modulating the activity of CK1δ is required. Here we show that Chk1 phosphorylates rat CK1δ at serine residues 328, 331, 370, and threonine residue 397 as well as the human CK1δ transcription variants 1 and 2. CK1δ mutant proteins bearing one, two or three mutations at these identified phosphorylation sites exhibited significant differences in their kinetic properties compared to wild-type CK1δ. Additionally, CK1δ co-precipitates with Chk1 from HT1080 cell extracts and activation of cellular Chk1 resulted in a significant decrease in cellular CK1δ kinase activity. Taken together, these data point towards a possible regulatory relationship between Chk1 and CK1δ. [ABSTRACT FROM AUTHOR]

Objective: The gene encoding the methionine salvage pathway methylthioadenosine phosphorylase (MTAP) is a tumor suppressor gene that is frequently inactivated in a wide variety of human cancers. In this study, we have examined if heterozygosity for a null mutation in Mtap (MtaplacZ) could accelerate tumorigenesis development in two different mouse cancer models, Eμ-myc transgenic and Pten+/−. Methods: Mtap Eμ-myc and Mtap Pten mice were generated and tumor-free survival was monitored over time. Tumors were also examined for a variety of histological and protein markers. In addition, microarray analysis was performed on the livers of MtaplacZ/+ and Mtap+/+ mice. Results: Survival in both models was significantly decreased in MtaplacZ/+ compared to Mtap+/+ mice. In Eµ-myc mice, Mtap mutations accelerated the formation of lymphomas from cells in the early pre-B stage, and these tumors tended to be of higher grade and have higher expression levels of ornithine decarboxylase compared to those observed in control Eµ-myc Mtap+/+ mice. Surprisingly, examination of Mtap status in lymphomas in Eµ-myc MtaplacZ/+ and Eµ-myc Mtap+/+ animals did not reveal significant differences in the frequency of loss of Mtap protein expression, despite having shorter latency times, suggesting that haploinsufficiency of Mtap may be playing a direct role in accelerating tumorigenesis. Consistent with this idea, microarray analysis on liver tissue from age and sex matched Mtap+/+ and MtaplacZ/+ animals found 363 transcripts whose expression changed at least 1.5-fold (P<0.01). Functional categorization of these genes reveals enrichments in several pathways involved in growth control and cancer. Conclusion: Our findings show that germline inactivation of a single Mtap allele alters gene expression and enhances lymphomagenesis in Eµ-myc mice. [ABSTRACT FROM AUTHOR]

Curcumin, a selective phosphorylase kinase inhibitor, is a naturally occurring phytochemical present in turmeric. Curcumin has been confirmed to have anti-inflammatory properties in addition to the ability to decrease the expression of pro-inflammatory cytokines in keratinocytes. The interleukin-23 (IL-23)/IL-17A cytokine axis plays a critical role in the pathogenesis of psoriasis. Here, we report that topical use of a curcumin gel formulation strongly inhibited imiquimod (IMQ)-induced psoriasis-like inflammation, the development of which was based on the IL-23/IL-17A axis. IMQ-induced epidermal hyperplasia and inflammation in BALB/c mouse ear was significantly inhibited following curcumin treatment. Real-time PCR showed that mRNA levels of IL-17A, IL-17F, IL-22, IL-1β, IL-6 and TNF-α cytokines were decreased significantly by curcumin in ear skin, an effect similar to that of clobetasol. In addition, we found that curcumin may enhance the proliferation of epidermis γδ T cells but inhibit dermal γδ T cell proliferation. We inferred that curcumin was capable of impacting the IL-23/IL-17A axis by inhibiting IL-1β/IL-6 and then indirectly down-regulating IL-17A/IL-22 production. In conclusion, curcumin can relieve the IMQ-induced psoriasis-like inflammation in a mouse model, similar to the effects of clobetasol. Therefore, we have every reason to expect that curcumin will be used in the treatment of psoriasis in the future. [ABSTRACT FROM AUTHOR]

ADP-glucose pyrophosphorylase regulates the synthesis of glycogen in bacteria and of starch in plants. The enzyme from plants is mainly activated by 3-phosphoglycerate and is a heterotetramer comprising two small and two large subunits. Here, we found that two highly conserved residues are critical for triggering the activation of the potato tuber ADP-glucose pyrophosphorylase, as shown by site-directed mutagenesis. Mutations in the small subunit, which bears the catalytic function in this potato tuber form, had a more dramatic effect on disrupting the allosteric activation than those introduced in the large subunit, which is mainly modulatory. Our results strongly agree with a model where the modified residues are located in loops responsible for triggering the allosteric activation signal for this enzyme, and the sensitivity to this activation correlates with the dynamics of these loops. In addition, previous biochemical data indicates that the triggering mechanism is widespread in the enzyme family, even though the activator and the quaternary structure are not conserved. [ABSTRACT FROM AUTHOR]

The catalogues of protein kinases, the essential effectors of cellular signaling, have been charted in Metazoan genomes for a decade now. Yet, surprisingly, using bioinformatics tools, we predicted protein kinase structure for proteins coded by five related human genes and their Metazoan homologues, the FAM69 family. Analysis of three-dimensional structure models and conservation of the classic catalytic motifs of protein kinases present in four out of five human FAM69 proteins suggests they might have retained catalytic phosphotransferase activity. An EF-hand Ca2+-binding domain in FAM69A and FAM69B proteins, inserted within the structure of the kinase domain, suggests they may function as Ca2+-dependent kinases. The FAM69 genes, FAM69A, FAM69B, FAM69C, C3ORF58 (DIA1) and CXORF36 (DIA1R), are by large uncharacterised molecularly, yet linked to several neurological disorders in genetics studies. The C3ORF58 gene is found deleted in autism, and resides in the Golgi. Unusually high cysteine content and presence of signal peptides in some of the family members suggest that FAM69 proteins may be involved in phosphorylation of proteins in the secretory pathway and/or of extracellular proteins. [ABSTRACT FROM AUTHOR]

In this study, we investigated inhibition of the phosphorylation of adenosine 5′-monophosphate-activated protein kinase (AMPK) by prothoracicotropic hormone (PTTH) in prothoracic glands of the silkworm, Bombyx mori. We found that treatment with PTTH in vitro inhibited AMPK phosphorylation in time- and dose-dependent manners, as seen on Western blots of glandular lysates probed with antibody directed against AMPKα phosphorylated at Thr172. Moreover, in vitro inhibition of AMPK phosphorylation by PTTH was also verified by in vivo experiments: injection of PTTH into day 7 last instar larvae greatly inhibited glandular AMPK phosphorylation. PTTH-inhibited AMPK phosphorylation appeared to be partially reversed by treatment with LY294002, indicating involvement of phosphatidylinositol 3-kinase (PI3K) signaling. A chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) increased both basal and PTTH-inhibited AMPK phosphorylation. Treatment with AICAR also inhibited PTTH-stimulated ecdysteroidogenesis of prothoracic glands. The mechanism underlying inhibition of PTTH-stimulated ecdysteroidogenesis by AICAR was further investigated by determining the phosphorylation of eIF4E-binding protein (4E-BP) and p70 ribosomal protein S6 kinase (S6K), two known downstream signaling targets of the target of rapamycin complex 1 (TORC1). Upon treatment with AICAR, decreases in PTTH-stimulated phosphorylation of 4E-BP and S6K were detected. In addition, treatment with AICAR did not affect PTTH-stimulated extracellular signal-regulated kinase (ERK) phosphorylation, indicating that AMPK phosphorylation is not upstream signaling for ERK phosphorylation. Examination of gene expression levels of AMPKα, β, and γ by quantitative real-time PCR (qRT-PCR) showed that PTTH did not affect AMPK transcription. From these results, it is assumed that inhibition of AMPK phosphorylation, which lies upstream of PTTH-stimulated TOR signaling, may play a role in PTTH stimulation of ecdysteroidogenesis. [ABSTRACT FROM AUTHOR]

Protein-tyrosine phosphatases (PTPs) are important regulators of signal transduction processes. Essential for the functional characterization of PTPs is the identification of their physiological substrates, and an important step towards this goal is the demonstration of a physical interaction. The association of PTPs with their cellular substrates is, however, often transient and difficult to detect with unmodified proteins at endogenous levels. Density-enhanced phosphatase-1 (DEP-1/PTPRJ) is a regulator of hematopoietic cell functions, and a candidate tumor suppressor. However, association of DEP-1 with any of its proposed substrates at endogenous levels has not yet been shown. We have previously obtained functional and biochemical evidence for a direct interaction of DEP-1 with the hematopoietic receptor-tyrosine kinase Fms-like tyrosine kinase-3 (FLT3). In the current study we have used the method of in situ proximity ligation assay (in situ PLA) to validate this interaction at endogenous levels, and to further characterize it. In situ PLA readily detected association of endogenous DEP-1 and FLT3 in the human acute monocytic leukemia cell line THP-1, which was enhanced by FLT3 ligand (FL) stimulation in a time-dependent manner. Association peaked between 10 and 20 min of stimulation and returned to basal levels at 30 min. This time course was similar to the time course of FLT3 autophosphorylation. FLT3 kinase inhibition and DEP-1 oxidation abrogated association. Consistent with a functional role of DEP-1-FLT3 interaction, stable knockdown of DEP-1 in THP-1 cells enhanced FL-induced ERK1/2 activation. These findings support that FLT3 is a bona fide substrate of DEP-1 and that interaction occurs mainly via an enzyme-substrate complex formation triggered by FLT3 ligand stimulation. [ABSTRACT FROM AUTHOR]

Phosphoinositide 3-kinase (PI3K) signaling has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand the breadth of such inhibition in odor coding, we screened a panel of odorants representing different chemical classes, as well as odorants known to occur in a natural odor object (tomato), for their ability to rapidly activate PI3K-dependent inhibitory signaling. Odorants were screened on dissociated native rat ORNs before and after pre-incubation with the PI3K-isoform specific blockers AS252424 and TGX221. Many different odorants increased their excitatory strength for particular ORNs following PI3K blockade in a manner consistent with activating PI3K-dependent inhibitory signaling in those cells. The PI3K-dependent inhibitory odorants overlapped with conventional excitatory odorants, but did not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs. [ABSTRACT FROM AUTHOR]

Changes in intracellular calcium concentration ([Ca2+]i) as well as in the phosphorylation state of proteins have been implicated in keratinocyte wound healing revealed in scratch assays. Scratching confluent HaCaT monolayers decreased the number of cells displaying repetitive Ca2+ oscillations as well as the frequency of their Ca2+-transients in cells close to the wounded area and initiated migration of the cells into the wound bed. In contrast, calyculin-A (CLA) and okadaic acid (OA), known cell permeable inhibitors of protein phosphatase-1 and 2A, increased the level of resting [Ca2+]i and suppressed cell migration and wound healing of HaCaT cells. Furthermore, neither CLA nor OA influenced how scratching affected Ca2+ oscillations. It is assumed that changes in and alterations of the phosphorylation level of Ca2+-transport and contractile proteins upon phosphatase inhibition mediates cell migration and wound healing. [ABSTRACT FROM AUTHOR]

Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation. [ABSTRACT FROM AUTHOR]

The genus Shigella infects human gut epithelial cells to cause diarrhea and gastrointestinal disorders. Like many other Gram-negative bacterial pathogens, the virulence of Shigella spp. relies on a conserved type three secretion system that delivers a handful of effector proteins into host cells to manipulate various host cell physiology. However, many of the Shigella type III effectors remain functionally uncharacterized. Here we observe that OspG, one of the Shigella effectors, interacted with ubiquitin conjugates and poly-ubiquitin chains of either K48 or K63 linkage in eukaryotic host cells. Purified OspG protein formed a stable complex with ubiquitin but showed no interactions with other ubiquitin-like proteins. OspG binding to ubiquitin required the carboxyl terminal helical region in OspG and the canonical I44-centered hydrophobic surface in ubiquitin. OspG and OspG-homologous effectors, NleH1/2 from enteropathogenic E coli (EPEC), contain sub-domains I-VII of eukaryotic serine/threonine kinase. GST-tagged OspG and NleH1/2 could undergo autophosphorylation, the former of which was significantly stimulated by ubiquitin binding. Ubiquitin binding was also required for OspG functioning in attenuating host NF-κB signaling. Our data illustrate a new mechanism that bacterial pathogen like Shigella exploits ubiquitin binding to activate its secreted virulence effector for its functioning in host eukaryotic cells. [ABSTRACT FROM AUTHOR]

p38 mitogen-activated protein kinase (MAPK) is thought to play a central role in acute and chronic inflammatory responses. Whether p38MAPK plays a pathogenic role in crescentic GN (GN) and which of its four isoforms is preferentially involved in kidney inflammation is not definitely known. We thus examined expression and activation of p38MAPK isoforms during anti-glomerular basement membrane (GBM) nephritis. Therefore, p38α conditional knockout mice (MxCre-p38αΔ/Δ) were used to examine the role of p38α in anti-GBM induced nephritis. Both wild type and MxCre-p38αΔ/Δ mice developed acute renal failure over time. Histological examinations revealed a reduced monocyte influx and less tubular damage in MxCre-p38αΔ/Δ mice, whereas glomerular crescent formation and renal fibrosis was similar. Likewise, the levels of pro- and anti-inflammatory cytokines such as TNF, IL-1 and IL-10 were similar, but IL-8 was even up-regulated in MxCre-p38αΔ/Δ mice. In contrast, we could detect strong down-regulation of chemotactic cytokines such as CCL-2, -5 and -7, in the kidneys of MxCre-p38αΔ/Δ mice. In conclusion, p38α is the primary p38MAPK isoform expressed in anti-GBM nephritis and selectively affects inflammatory cell influx and tubular damage. Full protection from nephritis is however not achieved as renal failure and structural damage still occurs. [ABSTRACT FROM AUTHOR]

Hyperthermia is widely used to treat patients with cancer, especially in combination with other treatments such as radiation therapy. Heat treatment per se activates DNA damage responses mediated by the ATR-Chk1 and ATM-Chk2 pathways but it is not fully understood how these DNA damage responses are activated and affect heat tolerance. By performing a genetic analysis of human HeLa cells and chicken B lymphoma DT40 cells, we found that heat-induced Chk1 Ser345 phosphorylation by ATR was largely dependent on Rad9, Rad17, TopBP1 and Claspin. Activation of the ATR-Chk1 pathway by heat, however, was not associated with FancD2 monoubiquitination or RPA32 phosphorylation, which are known as downstream events of ATR kinase activation when replication forks are stalled. Downregulation of ATR, Rad9, Rad17, TopBP1 or Claspin drastically reduced clonogenic cell viability upon hyperthermia, while gene knockout or inhibition of ATM kinase reduced clonogenic viability only modestly. Suppression of the ATR-Chk1 pathway activation enhanced heat-induced phosphorylation of Chk2 Thr68 and simultaneous inhibition of ATR and ATM kinases rendered severe heat cytotoxicity. These data indicate that essential factors for activation of the ATR-Chk1 pathway at stalled replication forks are also required for heat-induced activation of ATR kinase, which predominantly contributes to heat tolerance in a non-overlapping manner with ATM kinase. [ABSTRACT FROM AUTHOR]

The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 29deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (29deoxy)ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 59 hydroxyl group of adenosine and Arg43* side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl6 and Br8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser90 by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr91) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme. [ABSTRACT FROM AUTHOR]

Background: Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood. Methods: Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest. Results: In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 5'-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels. Conclusions: Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches. [ABSTRACT FROM AUTHOR]

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The twodimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions. [ABSTRACT FROM AUTHOR]