Abstract In previous time-resolved microarray-based expression profiling, we identified 32 genes encoding putative transcription factors, signaling components, and unknown proteins that are rapidly and robustly induced by phytochrome (phy)-mediated light signals. Postulating that they are the most likely to be direct targets of phy signaling and to function in the primary phy regulatory circuitry, we examined the impact of targeted mutations in these genes on the phy-induced seedling deetiolation process in Arabidopsis thaliana. Using light-imposed concomitant inhibition of hypocotyl and stimulation of cotyledon growth as diagnostic criteria for normal deetiolation, we identified three major mutant response categories. Seven (22%) lines displayed statistically significant, reciprocal, aberrant photoresponsiveness in the two organs, suggesting disruption of normal deetiolation; 13 (41%) lines displayed significant defects either unidirectionally in both organs or in hypocotyls only, suggesting global effects not directly related to photomorphogenic signaling; and 12 (37%) lines displayed no significant difference in photoresponsiveness from the wild type. Potential reasons for the high proportion of rapidly light-responsive genes apparently unnecessary for the deetiolation phenotype are discussed. One of the seven disrupted genes displaying a significant mutant phenotype, the basic helix-loop-helix factor–encoding PHYTOCHROME-INTERACTING FACTOR3-LIKE1 gene, was found to be necessary for rapid light-induced expression of the photomorphogenesis- and circadian-related PSEUDO-RESPONSE REGULATOR9 gene, indicating a regulatory function in the early phy-induced transcriptional network.
Abstract Objective: This study identified to provide a comprehensive understanding of genome-wide expression patterns of synovial tissue from rheumatoid arthritis (RA) patients to investigate the potential mechanism RA occurrence and development. Methods: The transcription profiles of 9 RA and 15 control (osteoarthritis OA) synovial tissue were generated by RNA-Seq. Gene set enrichment analysis (GSEA) was used to analyze all detected genes and differentially expressed genes (DEGs) were identified by DESeq. To further analyze the DEGs, Gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed. The protein-protein interaction (PPI) network of DEGs were constructed by STRING and the hub genes were identified by topology clustering with MCODE-Cytoscape. The most important hub genes were validated by quantitative real time polymerase chain reaction (qRT-PCR). Results: A total of 17736 genes were detected and 651 DEGs were identified. For the GSEA, significantly enriched gene sets positively correlated with the RA group were CD40 signaling over-activation, Th1 cytotoxic module in C2, over-activation of immune response, adaptive immune response in C5, In C7, the up-regulation of the gene set of effective versus memory CD8 T cell is related to RA group. The down-regulation of gene set of naïve versus effective CD8 T cell is related to RA group.in C7. Biology process enrichment analysis showed that the DEGs were significantly enriched for signal transduction (P=1.52×10 -08 ), immune response (P=1.94×10 -22 ) and inflammatory response (P=1.11×10 -11 ). Molecule function enrichment analysis revealed over-represented calcium ion binding (P=8.61×10 -03 ), receptor binding (P=7.03×10 -05 ) and chemokine activity (P=4.15×10 -15 ). The DEGs were significantly enriched for plasma membrane (P=2.26×10 -20 ), integral component of membrane (P=7.79×10 -07 ), extracellular region (P=3.43×10 -16 ) in cellular component. The KEGG pathway analysis showed that the DEGs were enriched in the cytokine-cytokine receptor interaction (P=6.86×10 -21 ), chemokine signaling pathway (P=2.03×10 -11 ), systemic lupus erythematosus(P=9.23×10 -07 ), T cell receptor signaling pathway (P=6.59×10 -06 ) and rheumatoid arthritis (P=3.24×10 -05 ). We confirmed RA over-expressed PPI network hub genes included CXCL13 , CXCL6 , CCR5 , CXCR5 , CCR2 , CXCL3 , CXCL10 and RA down-regulated hub genes included SSTR1 . Conclusions: The study identified and verified the DEGs between RA and OA synovial tissue which highlighted the activity of a subset of chemokine genes, thereby providing novel insights into the molecular mechanisms of RA pathogenesis and identified potential diagnostic and therapeutic targets for RA.
d-Xylose is the most abundant hemicellulosic monomer on earth, but wild-type Saccharomyces cerevisiae has very limited d-xylose uptake capacity. We conducted bioprospecting for new sugar transporters from the d-xylose-consuming filamentous fungus Trichoderma reesei and identified three candidates belonging to the major facilitator superfamily. When they were expressed in yeast and assayed for d-xylose uptake, one of them, Xltr1p, had d-xylose transport activity that was more efficient than that of Gal2p, an endogenous yeast transporter. Site-directed mutagenesis was used to examine the functional contributions of 13 amino acid residues for the uptake of d-xylose, and these experiments identified particular amino acids that function distinctly in d-xylose vs glucose transport (e.g., F300). Excitingly, the yeast strain expressing the N326FXltr1p variant was able to carry a "high efficiency" transport for d-xylose but was nearly unable to utilize glucose; in contrast, the strain with the F300AXltr1p variant grew on glucose but lost d-xylose transport activity.
Arabidopsis nucleoside diphosphate kinase 2 (NDPK2) is a component in the phytochrome-mediated light signaling. In the present study, its autophosphorylation was investigated. Acid-stable and alkali-stable phosphorylated residues were analyzed under two different conditions. Results revealed that NDPK2 is phosphorylated only on its active histidine residue His197 and the presence of serine/threonine phosphorylation is an experimental artifact due to the harsh condition applied in the treatment of the phosphorylated protein sample. To resolve the controversy of whether serine/threonine phosphorylation of NDPK occurs as has been suggested by other NDPK studies, NDPK2 putative phosphorylation site mutants were generated and examined. No serine/threonine phosphorylation was identified in NDPK2 or implicated in its enzymatic activity. Further studies indicated that the low enzymatic activity and autophosphorylation level of NDPK2 mutant S199A are shown to be due to a damaged H-bonding with the active histidine residue His197 in the nucleotide-binding pocket. In addition, NDPK2 Kpn loop mutant T182A was found to possess an extremely low enzymatic activity and almost no autophosphorylation, suggesting the importance of the oligomeric states of NDPK2 in NDPK2 functioning.
Abstract Saccharomyces cerevisiae is a robust host for heterologous protein expression. The efficient expression of cellulases in S. cerevisiae is important for the consolidated bioprocess that directly converts lignocellulose into valuable products. However, heterologous proteins are often N -hyperglycosylated in S. cerevisiae , which may affect protein activity. In this study, the expression of three heterologous proteins, β-glucosidase, endoglucanase and cellobiohydrolase, was found to be N -hyperglycosylated in S. cerevisiae . To block the formation of hypermannose glycan, these proteins were expressed in strains with deletions in key Golgi mannosyltransferases (Och1p, Mnn9p and Mnn1p), respectively. Their extracellular activities improved markedly in the OCH1 and MNN9 deletion strains. Interestingly, truncation of the N -hypermannose glycan did not increase the specific activity of these proteins, but improved the secretion yield. Further analysis showed OCH1 and MNN9 deletion up-regulated genes in the secretory pathway, such as protein folding and vesicular trafficking, but did not induce the unfolded protein response. The cell wall integrity was also affected by OCH1 and MNN9 deletion, which contributed to the release of secretory protein extracellularly. This study demonstrated that mannosyltransferases disruption improved protein secretion through up-regulating secretory pathway and affecting cell wall integrity and provided new insights into glycosylation engineering for protein secretion.
Secretory protein
Heterologous
Endoplasmic-reticulum-associated protein degradation
Microbial production of monoterpenes provides a promising substitute for traditional chemical-based methods, but their production is lagging compared with sesquiterpenes. Geraniol, a valuable monoterpene alcohol, is widely used in cosmetic, perfume, pharmaceutical and it is also a potential gasoline alternative. Previously, we constructed a geraniol production strain by engineering the mevalonate pathway together with the expression of a high-activity geraniol synthase. In this study, we further improved the geraniol production through reducing the endogenous metabolism of geraniol and controlling the precursor geranyl diphosphate flux distribution. The deletion of OYE2 (encoding an NADPH oxidoreductase) or ATF1 (encoding an alcohol acetyltransferase) both involving endogenous conversion of geraniol to other terpenoids, improved geraniol production by 1.7-fold or 1.6-fold in batch fermentation, respectively. In addition, we found that direct down-regulation of ERG20 expression, the branch point regulating geranyl diphosphate flux, does not improve geraniol production. Therefore, we explored dynamic control of ERG20 expression to redistribute the precursor geranyl diphosphate flux and achieved a 3.4-fold increase in geraniol production after optimizing carbon source feeding. Furthermore, the combination of dynamic control of ERG20 expression and OYE2 deletion in LEU2 prototrophic strain increased geraniol production up to 1.69 g/L with pure ethanol feeding in fed-batch fermentation, which is the highest reported production in engineered yeast. An efficient geraniol production platform was established by reducing the endogenous metabolism of geraniol and by controlling the flux distribution of the precursor geranyl diphosphate. The present work also provides a production basis to synthesis geraniol-derived chemicals, such as monoterpene indole alkaloids.
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