Decreased serum dependence in the growth of NIH3T3 cells from the overexpression of human nuclear receptor-binding SET-domain-containing protein 1 (NSD1) or fission yeast su(var)3-9, enhancer-of-zeste, trithorax 2 (SET2)

Nuclear receptor-binding SET-domain-containing protein 1 (NSD1), a culprit gene for Sotos syndrome, contains a su(var)3-9, enhancer-of-zeste, trithorax (SET) domain that is responsible for histone methyltransferase activity and other domains such as plant homeodomain (PHD) and proline-tryptophan-tryptophan-proline (PWWP) involved in protein-protein interactions in the C-terminal half of NSD1. To elucidate the function of NSD1 on cell growth, we overexpressed NSD1 in NIH3T3 cells. Cells overexpressing NSD1 grew in the presence of 2% serum, whereas vector transfected cells did not. Overexpression of the C-terminal half of NSD1 but not the N-terminal half of NSD1 also produced cell growth under low serum concentration. Furthermore, overexpression in NIH3T3 of Schizosaccharomyces pombe SET2 which has a SET domain but not PHD or PWWP domains conferred the reduced serum dependence. Thus, the SET domain of NSD1 is involved in cell growth by modulating serum dependence.     

Solution structure of the Pdp1 PWWP domain reveals its unique binding sites for methylated H4K20 and DNA

Methylation of H4K20 (Lys(20) of histone H4) plays an important role in the regulation of diverse cellular processes. In fission yeast, all three states of H4K20 methylation are catalysed by Set9. Pdp1 is a PWWP (proline-tryptophan-tryptophan-proline) domain-containing protein, which associates with Set9 to regulate its chromatin localization and methyltransferase activity towards H4K20. The structure of the Pdp1 PWWP domain, which is the first PWWP domain identified which binds to methyl-lysine at the H4K20 site, was determined in the present study by solution NMR. The Pdp1 PWWP domain adopts a classical PWWP fold, with a five-strand antiparallel β-barrel followed by three α-helices. However, it differs significantly from other PWWP domains in some structural aspects that account, in part, for its molecular recognition. Moreover, we revealed a unique binding pattern of the PWWP domain, in that the PWWP domain of Pdp1 bound not only to H4K20me3 (trimethylated Lys(20) of histone H4), but also to dsDNA (double-stranded DNA) via an aromatic cage and a positively charged area respectively. EMSAs (electrophoretic mobility-shift assays) illustrated the ability of the Pdp1 PWWP domain to bind to the nucleosome core particle, and further mutagenesis experiments indicated the crucial role of this binding activity in histone H4K20 di- and tri-methylation in yeast cells. The present study may shed light on a novel mechanism of histone methylation regulation by the PWWP domain.     

Role of the cystathionine γ lyase/hydrogen sulfide pathway in human melanoma progression

In humans, two main metabolic enzymes synthesize hydrogen sulfide (H₂S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3‐mercaptopyruvate sulfurtransferase (3‐MST), synthesizes H₂S in the presence of the substrate 3‐mercaptopyruvate (3‐MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non‐lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H₂S donors, the most active of which was diallyl trisulfide (DATS). The main pro‐apoptotic mechanisms involved were suppression of nuclear factor‐κB activity and inhibition of AKT and extracellular signal‐regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l ‐cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l ‐cysteine/CSE/H₂S pathway is involved in melanoma progression.     

Sirtuin 3-induced macrophage autophagy in regulating NLRP3 inflammasome activation

Defective autophagy of monocytes or macrophages might result in NLRP3 inflammasome activation and cause vascular metabolic inflammation. However, the mechanism underlying the initiation of the autophagy response to hyperlipidaemia remains unclear. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is sensitive to the metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated autophagy in regulating NLRP3 inflammasome activation. We determined that the inhibition of autophagy and the activation of the NLRP3 inflammasome were concomitant with reduced SIRT3 levels both in peripheral blood monocytes from obese humans and in palmitate-treated THP-1 cells. Furthermore, we demonstrated that SIRT3 could form a molecular complex with ATG5, while SIRT3 overexpression altered the acetylation of endogenous ATG5. ATG5 acetylation inhibited autophagosome maturation and induced NLRP3 inflammasome activation. In parallel, SIRT3 overexpression in THP-1 cells decreased the palmitate-induced generation of mitochondrial reactive oxygen species, restored autophagy, and attenuated NLRP3 inflammasome activation. The incubation of human aortic endothelial cells (HAECs) with macrophage-conditioned medium (MCM) induced HAEC expression of vascular cell adhesion molecule-1, intercellular adhesion molecule 1, α-smooth muscle actin, and collagen-1. The effect of MCM could be reversed by the addition of neutralizing anti-IL-1β antibody or the overexpression of SIRT3. Consistent with this, en face analyses displayed a marked increase in α-SMC-positive endothelial cells in SIRT3^?/? mice with acute hyperlipidaemia. Taken together, these findings revealed that SIRT3-deficient macrophages displayed impaired autophagy and accelerated NLRP3 inflammasome activation and endothelial dysfunction.     

Comparative assessment of the Euglena gracilis var. saccharophila variant strain as a producer of the β‐1,3‐glucan paramylon under varying light conditions

Euglena gracilis Z and a “sugar loving” variant strain E. gracilis var. saccharophila were investigated as producers of paramylon, a β‐1,3‐glucan polysaccharide with potential medicinal and industrial applications. The strains were grown under diurnal or dark growth conditions on a glucose–yeast extract medium supporting high‐level paramylon production. Both strains produced the highest paramylon yields (7.4–8 g · L^?1, respectively) while grown in the dark, but the maximum yield was achieved faster by E. gracilis var. saccharophila (48 h vs. 72 h). The glucose‐to‐paramylon yield coefficient Y_par/glu = 0.46 ± 0.03 in the E. gracilis var. saccharophila cultivation, obtained in this study, is the highest reported to date. Proteomic analysis of the metabolic pathways provided molecular clues for the strain behavior observed during cultivation. For example, overexpression of enzymes in the gluconeogenesis/glycolysis pathways including fructokinase‐1 and chloroplastic fructose‐1,6‐bisphosphatase (FBP ) may have contributed to the faster rate of paramylon accumulation in E. gracilis var. saccharophila . Differentially expressed proteins in the early steps of chloroplastogenesis pathway including plastid uroporphyrinogen decarboxylases, photoreceptors, and a highly abundant (68‐fold increase) plastid transketolase may have provided the E. gracilis var. saccharophila strain an advantage in paramylon production during diurnal cultivations. In conclusion, the variant strain E. gracilis var. saccharophila seems to be well suited for producing large amounts of paramylon. This work has also resulted in the identification of molecular targets for future improvement of paramylon production in E. gracilis , including the FBP and phosophofructokinase 1, the latter being a key regulator of glycolysis.     

NSD2 promotes ventricular remodelling mediated by the regulation of H3K36me2

Histone lysine methylation plays an important role in the regulation of ventricular remodelling. NSD2 is involved in many types of tumours through enhancing H3K36me2 expression. However, the role of NSD2 in the regulation of histone lysine methylation during ventricular remodelling remains unclear. In this study, we established cardiac hypertrophy model in C57BL/6 mice by transverse aortic constriction and found that histone lysine methylation participated in ventricular remodelling regulation via the up‐regulation of H3K27me2 and H3K36me2 expression. In addition, we constructed transgenic C57BL/6 mice with conditional knockout of NSD2 (NSD2^?/?) in the myocardium. NSD2^?/? C57BL/6 mice had milder ventricular remodelling and significantly improved cardiac function compared with wild‐type mice, and the expression of H3K36me2 but not H3K27me2 was down‐regulated. In conclusion, NSD2 promotes ventricular remodelling mediated by the regulation of H3K36me2.     

Enhanced Asymmetric Reduction of Ethyl 3‐Oxobutyrate by Baker's Yeast via Substrate Feeding and Enzyme Inhibition

The moderate enantioselectivity of wild form baker's yeast can be considerably increased either by using continuous feeding to maintain a low substrate concentration throughout the reaction, or by the selective inhibition of competing enzymatic pathways. The reduction of ethyl 3‐oxobutyrate to ethyl (S)‐3‐hydroxybutyrate was used as a model reaction. With the substrate feeding method, the enantioselectivity could be increased from 75 % to as high as 98 %. The increased selectivity originates from the much higher substrate binding constant of the (R)‐specific enzymes, so that these enzymes remain essentially inactive if a low concentration of ethyl 3‐oxobutyrate is maintained in the bioreactor. Alternatively, the enantioselectivity of baker's yeast can be improved by selectively blocking competing enzymatic pathways. It was found that vinyl acetate is a selective inhibitor for the (R)‐specific enzymes. Ethyl (S)‐3‐hydroxybutyrate with an enantiomeric excess of 98 % was obtained by pre‐incubation of baker's yeast in 100 mM of vinyl acetate solution for 1 h. These results suggest that by selecting appropriate process conditions, natural baker's yeast can be a competitive biocatalyst for the large‐scale production of chiral secondary alcohols.     

解淀粉芽胞杆菌HZ-12中ysnE参与吲哚-3-乙酸合成的代谢途径研究

【目的】吲哚-3-乙酸是调控植物生长发育和生理活动的重要激素,吲哚-3-乙酸N-乙酰转移酶YsnE在吲哚-3-乙酸合成中发挥重要作用,本研究拟解析解淀粉芽胞杆菌中YsnE参与吲哚-3-乙酸合成的代谢途径。【方法】通过基因ysnE缺失和强化表达,分析ysnE对吲哚-3-乙酸合成影响,结合吲哚-3-乙酸合成中间物(吲哚丙酮酸、吲哚乙酰胺、色胺和吲哚乙腈)添加和体外酶转化实验,解析ysnE参与吲哚-3-乙酸合成的代谢途径。【结果】明确了YsnE在解淀粉芽胞杆菌HZ-12吲哚-3-乙酸合成中发挥重要作用。发现ysnE缺失菌株中的吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈利用显著降低,揭示了YsnE主要发挥吲哚丙酮酸脱羧酶YclB和吲哚乙酰胺水解酶/腈水解酶/腈水合酶YhcX的功能,并通过参与吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径来影响吲哚-3-乙酸合成。【结论】初步揭示了YsnE通过影响吲哚丙酮酸、吲哚乙酰胺和吲哚乙腈途径参与吲哚-3-乙酸合成的代谢机理,为吲哚-3-乙酸合成途径解析和代谢工程育种构建吲哚-3-乙酸高产菌株奠定了基础。     

Activation of multiple cancer pathways and tumor maintenance function of the 3q amplified oncogene FNDC3B

FNDC3B was recently identified in an oncogenomic screen for amplified oncogenes in hepatocellular carcinoma. It is located at 3q26 and is amplified in over 20% of cancers, usually as part of a broad amplified region encompassing the entire 3q arm. Consistent with an oncogenic role in multiple cancer types, we show here that overexpression of FNDC3B is capable of malignantly transforming mammary and kidney epithelial cells in addition to hepatocytes. To explore how FNDC3B transforms cells, we determined the cellular localization of its gene product and the cancer pathways that it activates. We found that the FNDC3B oncoprotein localizes to the Golgi network, and that its correct localization is essential for its transforming function. We found that overexpression of FNDC3B induces the epithelial-to-mesenchymal transition (EMT) and activates several cancer pathways, including PI3-kinase/Akt, Rb1 and TGFβ signaling. For TGFβ signaling, we analyzed the point in the pathway at which FNDC3B operates and obtained evidence that it induces expression of all three TGFβ ligands and also promotes TGFBR1 cell-surface localization. We found that RNAi-mediated knockdown of FNDC3B in cancer cells with 3q amplification suppressed their clonogenicity and tumorigenicity, but that the same RNAi knockdown had no effect on single-copy 3q cancer cells. These results indicate that FNDC3B is an important oncogenic driver gene of the 3q amplicon, adding to the growing list of oncogenic drivers within this commonly amplified region.     

Screening of Drugs That Suppress Ste11 MAPKKK Activation in Yeast Identified a c-Abl Tyrosine Kinase Inhibitor

The yeast MAPKKK Ste11 activates three MAP kinase pathways, including pheromone signaling, osmosensing, and pseudohyphal/invasive growth pathways. We identified two chemical compounds, BTB03006 and GK03225, that suppress growth defects induced by Ste11 activation in diploid yeast cells. BTB03006, but not GK03225, was found to suppress growth defects induced by both α-factor and Ste4 G_β overexpression in the pheromone signaling pathway, suggesting that GK03225 is an osmosensing pathway-specific inhibitor. We also performed genome-wide suppressor analysis for Ste11 activation, using a yeast deletion strains collection, and identified PBS2 and HOG1, and several genes associated with chaperone functions, which represent potential target proteins of the drugs screened from Ste11 activation. GK03225 possesses an Iressa-like quinazoline ring structure, and its chemical analog, 11N-078, suppresses c-Abl human tyrosine kinase activity. These results suggest that drug screening in yeast can identify human tyrosine kinase inhibitors and other drugs for human diseases.     

13C Metabolic Flux Analysis of Escherichia coli Engineered for Gamma‐Aminobutyrate Production

Escherichia coli is engineered for γ‐aminobutyrate (GABA) production in glucose minimal medium. For this, overexpression of mutant glutamate decarboxylase (GadB) and mutant glutamate/GABA antiporter (GadC), as well as deletion of GABA transaminase (GabT), are accomplished. In addition, the carbon flux to the tricarboxylic acid cycle is engineered by the overexpression of gltA, ppc, or both. The overexpression of citrate synthase (CS), encoded by gltA, increases GABA productivity, as expected. Meanwhile, the overexpression of phosphoenolpyruvate carboxylase (PPC) causes a decrease in the rate of glucose uptake, resulting in a decrease in GABA production. The phenotypes of the strains are characterized by ¹³C metabolic flux analysis (¹³C MFA). The results reveal that CS overexpression increases glycolysis and anaplerotic reaction rates, as well as the citrate synthesis rate, while PPC overexpression causes little changes in metabolic fluxes, but reduces glucose uptake rate. The engineered strain produces 1.2 g L^?1 of GABA from glucose. Thus, by using ¹³C MFA, important information is obtained for designing metabolically engineered strains for efficient GABA production.     

The impact of acetate metabolism on yeast fermentative performance and wine quality: reduction of volatile acidity of grape musts and wines

Acetic acid is the main component of the volatile acidity of grape musts and wines. It can be formed as a by-product of alcoholic fermentation or as a product of the metabolism of acetic and lactic acid bacteria, which can metabolize residual sugars to increase volatile acidity. Acetic acid has a negative impact on yeast fermentative performance and affects the quality of certain types of wine when present above a given concentration. In this mini-review, we present an overview of fermentation conditions and grape-must composition favoring acetic acid formation, as well the metabolic pathways leading to its formation and degradation by yeast. The negative effect of acetic acid on the fermentative performance of Saccharomyces cerevisiae will also be covered, including its role as a physiological inducer of apoptosis. Finally, currently available wine deacidification processes and new proposed solutions based on zymological deacidification by select S. cerevisiae strains will be discussed.     

Elevated expression of nuclear receptor-binding SET domain 3 promotes pancreatic cancer cell growth

The nuclear receptor-binding SET domain 3 (NSD3) catalyzes methylation of histone H3 at lysine 36 (H3K36), and promotes malignant transformation and progression of human cancer. Its expression, potential functions and underlying mechanisms in pancreatic cancer are studied. Bioinformatics studies and results from local human tissues show that NSD3 is upregulated in human pancreatic cancer tissues, which is correlated with poor overall survival. In primary and established pancreatic cancer cells, NSD3 silencing (by shRNAs) or CRISPR/Cas9-induced NSD3 knockout potently inhibited cell proliferation, migration and invasion, while provoking cell cycle arrest and apoptosis. Conversely, ectopic expression of NSD3-T1232A mutation significantly accelerated proliferation, migration, and invasion of pancreatic cancer cells. H3K36 dimethylation, expression of NSD3-dependent genes (Prkaa2, Myc, Irgm1, Adam12, and Notch3), and mTOR activation (S6K1 phosphorylation) were largely inhibited by NSD3 silencing or knockout. In vivo, intratumoral injection of adeno-associated virus (AAV)-packed NSD3 shRNA potently inhibited pancreatic cancer xenograft growth in nude mice. These results suggest that elevated NSD3 could be an important driver for the malignant progression of pancreatic cancer.Subject terms: Pancreatic cancer, Oncogenes     

Lic4, a nuclear phosphoprotein that cooperates with calcineurin to regulate cation homeostasis in Saccharomyces cerevisiae

The target of the immunosuppressants cyclosporin A(CsA) and FK506 is calcineurin, a highly conserved protein phosphatase that is required for T-cell activation and the regulation of ion homeostasis in yeast. Here we identify two genes, PMR2B and LIC4 which, when overexpressed, suppress the cation-sensitive phenotype of yeast cells lacking calcineurin. PMR2B encodes a Na⁺/Li⁺-specific plasma membrane pump and is similar to PMR2A, whose expression is known to be regulated by calcineurin. LIC4 (lithium comvertas) encodes a novel 33-kDa protein with no identity to known proteins. LIC4 overexpression suppresses the Li⁺-sensitive phenotype of calcineurin mutants but not the defect in recovery from pheromone arrest or viability of calcineurin dependent mutants, indicating a specific role in cation homeostasis. Similarly, lic4 mutations increase the Li⁺ sensitivity of both wild-type and calcineurin mutant strains, and reduce expression of pmr2A in calcineurin mutant strains, indicating that calcineurin and Lic4 may regulate parallel cation homeostatic pathways. lic4 mutations also exacerbate the Li⁺-sensitive phenotype of hal3 mutant strains, and overexpression of either Lic4 or Hal3 suppresses the salt sensitivity of mutant strains lacking calcineurin, Hal3, or Lic4, either singly or in combination. Taken together, these observations suggest that calcineurin, Hal3, and Lic4 cooperatively regulate the response of yeast cells to␣cation stress. Lic4 is phosphoprotein in vivo and a calcineurin substrate in vitro. By indirect and direct immunofluorescence detection of HA- and GFP-tagged proteins, Lic4 is localized in the nucleus in wild-type cells but predominantly cytoplasmic in cells lacking calcineurin. Taken together, our findings support a model in which calcineurin and Lic4 are components of signalling cascades that regulate cation stress responses in yeast. Received: 17 August 1998 / Accepted: 7 December 1998     

G2 / M checkpoint genes of Saccharomyces cerevisiae : further evidence for roles in DNA replication and / or repair

We have cloned, sequenced and disrupted the checkpoint genes RAD17, RAD24 and MEC3 of Saccharomyces cerevisiae. Mec3p shows no strong similarity to other proteins currently in the database. Rad17p is similar to Rec1 from Ustilago maydis, a 3′ to 5′ DNA exonuclease/checkpoint protein, and the checkpoint protein Rad1p from Schizosaccharomyces pombe (as we previously reported). Rad24p shows sequence similarity to replication factor C (RFC) subunits, and the S. pombe Rad17p checkpoint protein, suggesting it has a role in DNA replication and/or repair. This hypothesis is supported by our genetic experiments which show that overexpression of RAD24 strongly reduces the growth rate of yeast strains that are defective in the DNA replication/repair proteins Rfc1p (cdc44), DNA polα (cdc17) and DNA polδ (cdc2) but has much weaker effects on cdc6, cdc9, cdc15 and CDC ⁺ strains. The idea that RAD24 overexpression induces DNA damage, perhaps by interfering with replication/repair complexes, is further supported by our observation that RAD24 overexpression increases mitotic chromosome recombination in CDC ⁺ strains. Although RAD17, RAD24 and MEC3 are not required for cell cycle arrest when S phase is inhibited by hydroxyurea (HU), they do contribute to the viability of yeast cells grown in the presence of HU, possibly because they are required for the repair of HU-induced DNA damage. In addition, all three are required for the rapid death of cdc13 rad9 mutants. All our data are consistent with models in which RAD17, RAD24 and MEC3 are coordinately required for the activity of one or more DNA repair pathways that link DNA damage to cell cycle arrest. Received: 8 April 1997 / Accepted: 10 May 1997