Comparative Proteomics Analysis of Engineered Saccharomyces cerevisiae with Enhanced Biofuel Precursor Production

The yeast Saccharomyces cerevisiae was metabolically modified for enhanced biofuel precursor production by knocking out genes encoding mitochondrial isocitrate dehydrogenase and over-expression of a heterologous ATP-citrate lyase. A comparative iTRAQ-coupled 2D LC-MS/MS analysis was performed to obtain a global overview of ubiquitous protein expression changes in S. cerevisiae engineered strains. More than 300 proteins were identified. Among these proteins, 37 were found differentially expressed in engineered strains and they were classified into specific categories based on their enzyme functions. Most of the proteins involved in glycolytic and pyruvate branch-point pathways were found to be up-regulated and the proteins involved in respiration and glyoxylate pathway were however found to be down-regulated in engineered strains. Moreover, the metabolic modification of S. cerevisiae cells resulted in a number of up-regulated proteins involved in stress response and differentially expressed proteins involved in amino acid metabolism and protein biosynthesis pathways. These LC-MS/MS based proteomics analysis results not only offered extensive information in identifying potential protein-protein interactions, signal pathways and ubiquitous cellular changes elicited by the engineered pathways, but also provided a meaningful biological information platform serving further modification of yeast cells for enhanced biofuel production.     

Global Gene Expression Analysis of Long-Term Stationary Phase Effects in E. coli K12 MG1655

Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect E. coli LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells.     

Functional analysis of phosphorylation on Saccharomyces cerevisiae syntaxin 1 homologues Sso1p and Sso2p

Background

The Saccharomyces cerevisiae syntaxin1 homologues Sso1p and Sso2p perform an essential function in membrane fusion in exocytosis. While deletion of either SSO1 or SSO2 causes no obvious phenotype in vegetatively grown cells, deletion of both genes is lethal. In sporulating diploid S. cerevisiae cells only Sso1p, but not Sso2p, is needed for membrane fusion during prospore membrane formation. Mass spectrometry and in vivo labeling data suggest that serines 23, 24, and 79 in Sso1p and serines 31 and 34 in Sso2p can be phosphorylated in vivo. Here we set out to assess the contribution of phosphorylation on Sso protein in vivo function.

Principal Findings

Different mutant versions of SSO1 and SSO2 were generated to target the phosphorylation sites in Sso1p and Sso2p. Basal or overexpression of phospho-mimicking or putative non-phosphorylated Sso1p or Sso2p mutants resulted in no obvious growth phenotype. However, S79A and S79E mutations caused a mild defect in the ability of Sso1p to complement the temperature-sensitive growth phenotype of sso2-1 sso1Δ cells. Combination of all mutations did not additionally compromise Sso1p in vivo function. When compared to the wild type SSO1 and SSO2, the phosphoamino acid mutants displayed similar genetic interactions with late acting sec mutants. Furthermore, diploid cells expressing only the mutant versions of Sso1p had no detectable sporulation defects. In addition to sporulation, also pseudohyphal and invasive growth modes are regulated by the availability of nutrients. In contrast to sporulating diploid cells, deletion of SSO1 or SSO2, or expression of the phospho-mutant versions of SSO1 or SSO2 as the sole copies of SSO genes caused no defects in haploid or diploid pseudohyphal and invasive growth.

Conclusions

The identified phosphorylation sites do not significantly contribute to the in vivo functionality of Sso1p and Sso2p in S. cerevisiae.     

Novel Ree1 regulates the expression of ENO1 via the Snf1 complex pathway in Saccharomyces cerevisiae

Using cDNA microarray analysis, we found that the mRNA of YJL217W and several other genes related to cell wall organization and biogenesis were up-regulated by galactose in Saccharomyces cerevisiae early during the induction process. YJL217W is also known as REE1 (Regulation of Enolase I). Both the Gal4 regulatory region and the Mac1 binding domain were found on the upstream region of REE1, and the expression of REE1 was up-regulated by galactose but not by glucose. The up-regulation of REE1 by galactose was not observed in the Δgal4 strain. From the two-hybrid analysis, we found that Ree1 physically interacted with Gal83. Furthermore, from 2-D gel electrophoresis we found that the deletion of REE1 resulted in the up-regulation of Eno1. From Western blotting, we learned that the expression of Eno1 in the Δree1 strain was different from that in wild-type strains and that Eno1 expression was not changed by glucose stimulation. Taken together, these results suggest that Ree1p functions in the galactose metabolic pathway via the Gal83 protein and that it may control the level of Eno1p, which is also affected by the Snf1 complex, in S. cerevisiae.     

Uncovering the pathogenesis and identifying novel targets of pancreatic cancer using bioinformatics approach

Pancreatic cancer is a uniformly lethal disease that can be difficult to diagnose at its early stage. Thus, our present study aimed to explore the underlying mechanism and identify new targets for this disease. The data GSE16515, including 36 tumor and 16 normal samples were available from Gene Expression Omnibus. Differentially expressed genes (DEGs) were screened out using Robust Multichip Averaging and LIMMA package. Moreover, gene ontology and pathway enrichment analyses were performed to DEGs. Followed with protein–protein interaction (PPI) network construction by STRING and Cytoscape, module analysis was conducted using ClusterONE. Finally, based on PubMed, text mining about these DEGs was carried out. Total 274 up-regulated and 93 down-regulated genes were identified as the common DEGs and these genes were discovered significantly enriched in cell adhesion and extracellular region terms, as well as ECM-receptor interaction pathway. In addition, five modules were screened out from the up-regulated PPI network with none in down-regulated network. Finally, the up-regulated genes, including MIA, MET and CEACAMS, and down-regulated genes, such as FGF, INS and LAPP, had the most references in text mining analysis. Our findings demonstrate that the up- and down-regulated genes play important roles in pancreatic cancer development and might be new targets for the therapy.     

Specific induction of indoleamine 2,3-dioxygenase by bacterial lipopolysaccharide in the mouse lung

Indoleamine 2,3-dioxygenase activity in the supernatant fractions (30,000g, 30 min) from various tissues of mice increased almost linearly after a single intraperitoneal administration of bacterial lipopolysaccharide (5 to 20 μg/mouse). The most prominent effect was observed in the lung, where both specific and total enzyme activities increased 40 to 80-fold during the first 24 h. Significant (10- to 20-fold) stimulation was also observed in the seminal vesicle, coagulating gland, colon, and caecum, and severalfold in the trachea, stomach, heart, small intestine, and spleen. Lipid A fraction, the biologically active unit in the lipopolysaccharide complex, was as active as the lipopolysaccharide preparations from either Escherichia coli or Salmonella S and R mutant strains, whereas the polysaccharide fraction was inactive under identical experimental conditions. When mice were pretreated with a series of daily injections of bacterial lipopolysaccharide, enzyme induction was no longer evident, indicating that tolerance to this agent had developed and that enzyme induction was caused by lipopolysaccharide but not by possible contaminants in the preparations. The enzyme activities from normal and lipopolysaccharide-treated mice were exclusively found in the soluble fractions of mouse lung homogenates. Other enzyme activities in the lung such as lysosomal (acid phosphatase), microsomal (prostaglandin cyclooxygenase), mitochondrial (monoamine oxidase and superoxide dismutase), and soluble enzyme activities (lipooxygenase and superoxide dismutase) were not significantly altered by this treatment. This increase in the enzyme activity with the lipopolysaccharide treatment was abolished with a simultaneous administration of cycloheximide or actinomycin D, and an immunological analysis with antibody for mouse enzyme (rabbit IgG) demonstrated that the observed increment of the enzyme activity was essentially due to an increase in the enzyme protein.     

脂多糖调控对大鼠心脏微血管内皮细胞的转录组分析

目的探索脂多糖(LPS)对大鼠心脏微血管内皮细胞(rCMECs)转录组的调节作用。方法对照组(正常培养rCMECs),LPS组(100 ng/mL LPS处理6 h的rCMECs),每组进行3个生物学重复转录组测序。得到差异基因后,使用实时定量PCR对部分差异基因mRNA的表达进行验证。分别对上调和下调基因进行GO和KEGG富集,并对差异基因进行共表达网络分析。采用独立t检验进行统计学分析。结果LPS处理后,265个基因表达上调,118个基因的表达下调。前10个最显著上调基因为:Mt2a、Cyp7b1、Sod2、Icam1、Ccl2、AC128848.1、Mt1、Cebpd、Serpinb2和Tnfrsf11b。前10个最显著下调基因为:Cavin2、Ankrd1、Edn1、Prss35、Lmod1、Dhrs3、Ttc22、Sema6a、Map2k3和Sema7a。定量PCR的结果表明Mt2a、Sod、Ccl2、Cxcl1、Icam1和Vcaml基因的表达得到了上调(P均<0.01);而Cavin2、Ankrd1、Edn1和Prss35基因表达下调(P均<0.05)。GO和KEGG富集的结果表明,上调基因与内皮细胞对炎性免疫细胞的趋化作用和黏附作用密切相关;而下调基因则是与钙离子信号和G蛋白相关通路以及内皮通透性增加有关。此外,差异基因进行共表达网络分析发现Sod2处于核心位置,提示其可能与LPS诱导的rCMECs的各种变化密切相关。结论LPS调控了rCMECs中大量与炎性免疫细胞进入心肌组织相关基因的表达。     

Regulation of Lactobacillus plantarum contamination on the carbohydrate and energy related metabolisms of Saccharomyces cerevisiae during bioethanol fermentation

During the industrial bioethanol fermentation, Saccharomyces cerevisiae cells are often stressed by bacterial contaminants, especially lactic acid bacteria. Generally, lactic acid bacteria contamination can inhibit S. cerevisiae cell growth through secreting lactic acid and competing with yeast cells for micronutrients and living space. However, whether are there still any other influences of lactic acid bacteria on yeast or not? In this study, Lactobacillus plantarum ATCC 8014 was co-cultivated with S. cerevisiae S288c to mimic the L. plantarum contamination in industrial bioethanol fermentation. The contaminative L. plantarum-associated expression changes of genes involved in carbohydrate and energy related metabolisms in S. cerevisiae cells were determined by quantitative real-time polymerase chain reaction to evaluate the influence of L. plantarum on carbon source utilization and energy related metabolism in yeast cells during bioethanol fermentation. Contaminative L. plantarum influenced the expression of most of genes which are responsible for encoding key enzymes involved in glucose related metabolisms in S. cerevisiae. Specific for, contaminated L. plantarum inhibited EMP pathway but promoted TCA cycle, glyoxylate cycle, HMP, glycerol synthesis pathway, and redox pathway in S. cerevisiae cells. In the presence of L. plantarum, the carbon flux in S. cerevisiae cells was redistributed from fermentation to respiratory and more reducing power was produced to deal with the excess NADH. Moreover, L. plantarum contamination might confer higher ethanol tolerance to yeast cells through promoting accumulation of glycerol. These results also highlighted our knowledge about relationship between contaminative lactic acid bacteria and S. cerevisiae during bioethanol fermentation.     

The Synthetic Amphipathic Peptidomimetic LTX109 Is a Potent Fungicide That Disturbs Plasma Membrane Integrity in a Sphingolipid Dependent Manner

The peptidomimetic LTX109 (arginine-tertbutyl tryptophan-arginine-phenylethan) was previously shown to have antibacterial properties. Here, we investigated the activity of this novel antimicrobial peptidomimetic on the yeast Saccharomyces cerevisiae. We found that LTX109 was an efficient fungicide that killed all viable cells in an exponentially growing population as well as a large proportion of cells in biofilm formed on an abiotic surface. LTX109 had similar killing kinetics to the membrane-permeabilizing fungicide amphotericin B, which led us to investigate the ability of LTX109 to disrupt plasma membrane integrity. S. cerevisiae cells exposed to a high concentration of LTX109 showed rapid release of potassium and amino acids, suggesting that LTX109 acted by destabilizing the plasma membrane. This was supported by the finding that cells were permeable to the fluorescent nucleic acid stain SYTOX Green after a few minutes of LTX109 treatment. We screened a haploid S. cerevisiae gene deletion library for mutants resistant to LTX109 to uncover potential molecular targets. Eight genes conferred LTX109 resistance when deleted and six were involved in the sphingolipid biosynthetic pathway (SUR1, SUR2, SKN1, IPT1, FEN1 and ORM2). The involvement of all of these genes in the biosynthetic pathway for the fungal-specific lipids mannosylinositol phosphorylceramide (MIPC) and mannosyl di-(inositol phosphoryl) ceramide (M(IP)₂C) suggested that these lipids were essential for LTX109 sensitivity. Our observations are consistent with a model in which LTX109 kills S. cerevisiae by nonspecific destabilization of the plasma membrane through direct or indirect interaction with the sphingolipids.     

Ribonuclease binase apoptotic signature in leukemic Kasumi-1 cells

Cytotoxic exogenous RNases triggering apoptotic response in malignant cells have potential as anticancer drugs; surprisingly, detailed characterization of the RNase-induced apoptosis has not been conducted so far. Here we show that a cytotoxic RNase from Bacillus intermedius (binase) induces extrinsic and intrinsic apoptotic pathways in leukemic Kasumi-1 cells. The experiments were performed using TaqMan Array Human Apoptosis 96-well Plate for gene expression analysis, and flow cytometry. Cytometric studies demonstrated dissipation of the mitochondrial membrane potential, opening of mitochondrial permeability transition pores, activation of caspases, increase of intracellular Ca²⁺ and decrease of reactive oxygen species levels. We found that expression of 62 apoptotic genes is up-regulated, including 16 genes that are highly up-regulated, and only one gene was found to be down-regulated. The highest, 16 fold increase of the expression level was observed for TNF gene. Highly up-regulated genes also include the non-canonical NF-κB signaling pathway and inflammatory caspases 1,4. The obtained results suggest that binase induces evolutionary acquired cellular response to a microbial agent and triggers unusual apoptosis pathway.