顶头孢霉中己糖激酶编码基因Achka的功能研究

己糖激酶在真菌中广泛存在,参与葡萄糖磷酸化等生理过程。本文从头孢菌素产生菌顶头孢霉中克隆并鉴定了一个己糖激酶编码基因,命名为Achka。通过RT‐PCR证明Achka含有4个内含子,其推测的编码蛋白含有484个氨基酸,分子量为53.7k Da。在顶头孢霉中敲除Achka后发现,突变株在以果糖、蔗糖或甘露糖为唯一碳源的培养基上生长受到严重限制。我们在大肠杆菌中异源表达了Achka,并对表达的重组蛋白AcHKA进行了分离纯化。酶学动力学分析表明,AcHKA对果糖的最大反应速率要大于葡萄糖,但是却对葡萄糖有更高的亲和力。上述结果进一步证明AcHKA为己糖激酶,在顶头孢霉体内主要负责果糖、蔗糖和甘露糖等的代谢。     

野生型和ospk1突变体水稻幼苗根对外源糖分的吸收和响应

植物根系如何响应环境因子变化是植物发育和营养吸收研究的重要科学问题。丙酮酸激酶OsPK1在根部的表达主要在根尖成熟区和根毛区,其表达水平变化有可能影响水稻对外源糖分的吸收。采用日本晴和水稻突变体ospk1,通过改变1/2 MS培养基中蔗糖含量,探索水稻幼苗对外源糖分的吸收和响应。通过GC-MS的方法检测了水稻幼苗叶片、叶鞘和根中蔗糖、葡萄糖、果糖和半乳糖的含量。发现根与培养基中糖分接触能明显提高幼苗中的糖含量。并且这些幼苗的根系长度大于那些不加蔗糖的培养基培养的幼苗,表明外源糖分被吸收后能促进根的伸长。OsPK1表达下调影响了糖代谢和外源糖分的吸收。半定量RT-PCR结果显示,幼苗根与糖分的直接接触明显上调根中OsPIP2;4,OsPIP2;5和OsTIP2;1三个水孔蛋白基因的表达。     

休眠期间油桃花芽碳水化合物代谢及其相关基因的表达变化

以十年生大田和三年生盆栽‘曙光’油桃花芽为材料,分别测定了其休眠期碳水化合物含量、糖代谢相关基因的季节性表达及低温处理下相关基因的表达变化,旨在探讨碳水化合物及低温与休眠的关系。结果表明：休眠期间可溶性糖（主要是蔗糖）含量逐渐增加,淀粉含量则呈相反趋势。糖代谢相关基因表达明显不同：腺苷二磷酸葡萄糖焦磷酸化酶基因口GPase）无明显变化;组氨酸H3基因（HisH3）和己糖激酶I基因（胱,）在进入内休眠前有明显上升,蔗糖合酶基因（SuSy）则与之相反;尿苷二磷酸葡萄糖焦磷酸化酶（UGPase）表达总体上呈上调趋势,在进入内休眠后稍有下调。表明进入内休眠后,依赖HKl的糖信号转导途径起重要作用。在4℃处理后,与细胞分裂有关的基因HisH3含量急剧升高,而后下降,说明细胞分裂的减少并不是休眠期间抑制生长的原因;UGPase表现出与内休眠期一致的变化趋势,说明对低温有一定的适应性。     

重组荞麦胰蛋白酶抑制剂通过下调己糖激酶Ⅱ抑制HepG2细胞生长

糖酵解过度活跃是肿瘤细胞能量代谢的显著特征。抑制过度糖酵解已经成为一种新的癌症疗法。重组荞麦胰蛋白酶抑制剂 (recombinant buckwheat trypsin inhibitor, rBTI)可以通过上调磷酸酶及张力蛋白同源基因 (PTEN) 进而抑制HepG2细胞增殖。有关rBTI对肿瘤细胞能量代谢的影响仍未见报道。本研究中的MTT和ATP检测分析表明,rBTI以剂量依赖性方式抑制细胞活力及胞内ATP含量。qRT-PCR和Western印迹分析表明,rBTI处理HepG2细胞后,己糖激酶Ⅱ转录显著下调,但是糖酵解过程中的其他酶及葡萄糖转运蛋白基因在转录水平未发生显著变化,同时己糖激酶Ⅱ蛋白水平的表达也显著下调。酶活性分析也表明,rBTI能显著降低己糖激酶的活性。进一步分析表明, rBTI使细胞内PTEN转录及表达水平明显上调,己糖激酶Ⅱ转录和p-AKT,p-mTOR、己糖激酶Ⅱ的表达下调。当PTEN抑制剂phen存在时,可阻断rBTI诱导的己糖激酶 Ⅱ表达下降,表明rBTI能通过上调PTEN进而影响己糖激酶Ⅱ的表达。免疫荧光及Western印迹分析显示,rBTI作用后减弱了己糖激酶 Ⅱ在线粒体的定位,导致己糖激酶Ⅱ与线粒体电压依赖性阴离子通道蛋白 (voltage-dependent anion channel, VDAC) 分离,促使己糖激酶Ⅱ从线粒体转位到细胞质,降低糖酵解的效率。上述结果证明,rBTI对肿瘤细胞能量代谢的调控作用主要通过抑制PI3K/AKT信号通路,下调己糖激酶Ⅱ的表达并影响空间定位,进而抑制肿瘤细胞糖酵解过程,导致癌细胞生长受到抑制。     

达乌尔黄鼠育肥过程和冬眠期白色脂肪组织糖代谢相关基因的差异表达

为研究贮脂类冬眠动物育肥过程和冬眠期糖代谢的机制,使用第二代转录组测序(RNA-Seq)技术,检测了达乌尔黄鼠起始育肥期、快速育肥期、育肥完成期和冬眠期4个阶段血糖含量和白色脂肪组织中与糖代谢途径相关基因的表达情况。结果显示,起始育肥期、快速育肥期、育肥完成期的血糖浓度无组间差异,但均高于冬眠期。与起始育肥期相比,快速育肥期的果糖-1,6-二磷酸酶基因表达下调了8.3倍,己糖激酶、醛缩酶和烯醇化酶等基因表达上调;育肥完成期与快速育肥期相比,果糖-1,6-二磷酸酶基因表达上调了9.6倍,醛缩酶、磷酸烯醇式丙酮酸羧化酶和柠檬酸合酶等基因表达下调1.2-2倍;冬眠期己糖激酶、丙酮酸脱氢酶E1、醛缩酶、柠檬酸合酶和6-磷酸脱氢酶等基因的表达均明显下调。结果表明,己糖激酶等基因表达上调可能与达乌尔黄鼠快速育肥期的糖代谢增强直接相关;醛缩酶和柠檬酸合酶等基因表达下调可能是动物在育肥完成期发生糖代谢降低的原因;入眠后,己糖激酶、丙酮酸脱氢酶等基因的低表达可能是调控糖代谢降至极低的主要机制。达乌尔黄鼠在冬眠前的活跃期既已启动糖代谢通路在分子水平上的主动调控。     

蔗糖调节拟南芥花青素的生物合成

为了探讨糖在花青素合成过程中的调节作用,采用蔗糖和其代谢糖（葡萄糖 和果糖）组合处理拟南芥幼苗.实验结果表明,60 mmol/L蔗糖处理显著提高拟南芥 幼苗的花青素、还原糖含量,并上调花青素合成相关基因（CHS, FLS-1, DFR, LDOX, BANYULS）的转录,对叶绿素含量和UGT78D2基因的转录无影响;20 mmol/L 葡萄糖+20 mmol/L果糖处理,对花青素、叶绿素和还原糖的含量无影响,对花青素 合成相关基因转录影响不一;20 mmol/L蔗糖+20 mmol/L葡萄糖+20 mmol/L果糖处 理后,花青素和还原糖含量介于前两个处理之间,也上调花青素合成相关基因的转 录;但和蔗糖处理组相比,上调UGT78D2基因转录,下调FLS-1基因转录.在不同处 理组之间,花青素含量变化和还原糖含量变化趋势相同,有可能糖在调节花青素 合成的同时也调节还原糖含量.因此,蔗糖既可以通过蔗糖特异信号途径,也可以 和其代谢糖通过其他途径共同调节拟南芥花青素的生物合成.     

脱落酸和己糖激酶抑制剂对高表达C4-PEPC转基因稻苗耐旱性的影响

为了研究高表达转玉米C₄-磷酸烯醇式丙酮酸羧化酶（phosphoenolpyruvate carboxylase,PEPC）基因水稻（PC）的耐旱性机制,本研究以PC和未转基因野生型原种kitaake为材料,分别在光照和黑暗预处理24 h,其中光照处理中光强为600 μmol·m^-2·s^-1,预处理后稻苗再在15％聚乙二醇-6000模拟干旱胁迫下,同时使用药理学的方法,通过加入脱落酸和己糖激酶的专一性抑制剂100 μmol·L^-1去甲二氢愈创木酸和10 mmol·L^-1葡萄糖胺,观察两种水稻4~5叶期稻苗耐旱表现。结果发现,与WT水稻相比,在PEG-6000处理后,经过光预处理的PC水稻叶片相对含水量下降较少,始终比WT的高,而且丙二醛含量则较少,脯氨酸诱导增加,表现耐旱;而经过暗预处理后PC植株显著降低这个优势,表明光预处理有利于PC耐旱性的表现;黑暗预处理均显著下调了2供试材料植株叶片中可溶性糖的含量,而对可溶性蛋白的含量影响不显著;而光预处理后PC水稻叶片内可溶性糖含量比WT增加,而在黑暗预处理则PC的显著低于WT的,其中葡萄糖胺对光预处理下PC的可溶性糖含量的下调作用最显著;暗预处理逆转或消除了NO,H₂O₂和钙离子含量变化趋势,这些变化与暗预处理减少了两材料叶片蔗糖和葡萄糖含量变化同步;光暗预处理对两材料的PEPC酶活性的差异影响不大,表明外源玉米C₄-PEPC在水稻中是组成型表达。可见PC叶片可部分通过糖组分,参与内源糖介导ABA和HXK信号途径,缓解干旱处理对叶片的伤害,稳定光合能力。     

花色形成与花生长的调控

结合笔者的研究结果,对光、糖和GAs在花生长及花色形成中的作用和可能的调节机制进行了综述。光通过光受体介导的高辐照度反应(HIR)和光合作用调控花生长及花色素苷合成;糖作为碳源和渗透调节因子,影响花瓣细胞的生长及花色素苷积累,依赖己糖激酶的信号途径可能在糖的调控中起作用;GAs通过调节特异基因的转录间接地诱导花色素苷合成途径中结构基因的表达。     

己糖激酶-Ⅱ与肿瘤的糖代谢

己糖激酶作为糖酵解途径的第一个关键酶,在肿瘤细胞中有高度表达,并使肿瘤细胞表现出高度的糖分解代谢表型。该介绍己糖激酶一Ⅱ基因的组成特点及其在肿瘤细胞中的表达特征等。     

光和糖对水稻Rubisco活化酶基因表达的影响

水稻黄化苗在光照2h内其Rubisco。活化酶的mRNA和蛋白量明显增加,然后维持在相对稳定的水平。光对水稻Rubisco活化酶的基因表达的诱导作用主要在转录水平上。Rubisco活化酶主要在绿叶中表达,这与Rubisco基因表达的器官特异性完全一致。用等渗葡萄糖喂养成熟的水稻叶片1h,促使水稻Rubisco大、小亚基和Rubisco活化酶可翻译mRNA含量下降。同样蔗糖对Rubisco小亚基和Rubisco活化酶的表达也有抑制,其作用弱于葡萄糖。     

Structural basis for subunit assembly in UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae

UDP-glucose is the universal activated form of glucose, employed in all organisms for glucosyl transfer reactions and as precursor for various activated carbohydrates. In animal and fungal metabolism, UDP-glucose is required for utilization of galactose and for the synthesis of glycogen, the major carbohydrate storage polymer. The formation of UDP-glucose is catalyzed by UDP-glucose pyrophosphorylase (UGPase), which is highly conserved among eukaryotes. Here, we present the crystal structure of yeast UGPase, Ugp1p. Both in solution and in the crystal, Ugp1p forms homooctamers, which represent the enzymatically active form of the protein. Ugp1p subunits consist of three domains, with the active site presumably located in the central SpsA GnT I core (SGC) domain. The association in the octamer is mediated by contacts between left-handed beta-helices in the C-terminal domains, forming a toroidal solenoid structure in the core of the complex. The catalytic domains attached to this scaffold core do not directly contact each other, consistent with simple Michaelis-Menten kinetics found for Ugp1p. Conservation of hydrophobic residues at the subunit interfaces suggests that all fungal and animal homologs form this quarternary structure arrangement in contrast to monomeric plant UGPases, which have charged residues at these positions. Implications of this oligomeric arrangement for regulation of UGPase activity in fungi and animals are discussed.     

Regulation of glucose partitioning by PAS kinase and Ugp1 phosphorylation

The ability of cells to recognize and respond to specific metabolic deficiencies is required for all forms of life. We have uncovered a system in the yeast S. cerevisiae that, in response to a perceived deficiency in cell wall glucan, alters partitioning of glucose toward glucan synthesis and away from glycogen synthesis. The paralogous yeast PAS kinases Psk1 and Psk2 phosphorylate UDP-glucose pyrophosphorylase (Ugp1), the primary producer of UDP-glucose, the glucose donor for glucan biosynthesis. Unexpectedly, phosphorylation of Ugp1 does not affect its catalytic activity but instead alters the terminal destination of the UDP-glucose it generates. Phosphorylated Ugp1 is required for intensive glucan production, and inability to phosphorylate Ugp1 is associated with a weak cell wall, decreased glucan content, and increased glycogen content. We provide data indicating that phosphorylation by Psk1 or Psk2 targets Ugp1 to the cell periphery, where the UDP-glucose it produces is in proximity to the site of glucan synthesis. We propose that regulation of glucose partitioning by altered enzyme and substrate localization is a rapid and potent response to metabolic deficiency.     

Interactive effects of phosphate deficiency, sucrose and light/dark conditions on gene expression of UDP-glucose pyrophosphorylase in Arabidopsis

The effects of inorganic phosphate (Pi) status, light/dark and sucrose on expression of UDP-glucose pyrophosphorylase (UGPase) gene (Ugp), which is involved in sucrose/ polysaccharides metabolism, were investigated using Arabidopsis wild-type (wt) plants and mutants impaired in Pi and carbohydrate status. Generally, P-deficiency resulted in increased Ugp expression and enhanced UGPase activity and protein content, as found for wt plants grown on P-deficient and complete nutrient solution, as well as for pho1 (P-deficient) mutants. Ugp was highly expressed in darkened leaves of pho1, but not wt plants; daily light exposure enhanced Ugp expression both in wt and pho mutants. The pho1 and pho2 (Pi-accumulating) mutations had little or no effect on leaf contents of glucose and fructose, regardless of light/dark conditions, whereas pho1 plants had much higher levels of sucrose and starch in the dark than pho2 and wt plants. The Ugp was up-regulated when leaves were fed with sucrose in wt plants, but the expression in pho2 background was much less sensitive to sucrose supply than in wt and pho1 plants. Expression of Ugp in pgm1 and sex1 mutants (impaired in starch/sugar content) was not dependent on starch content, and not tightly correlated with soluble sugar status. Okadaic acid (OKA) effectively blocked the P-starvation and sucrose-dependent expression of Ugp in excised leaves, whereas staurosporine (STA) had only a small effect on both processes (especially in -P leaves), suggesting that P-starvation and sucrose effects on Ugp are transmitted by pathways that may share similar components with respect to their (in) sensitivity to OKA and STA. The results of this study suggest that Ugp expression is modulated by an interaction of signals derived from P-deficiency status, sucrose content and dark/light conditions, and that light/sucrose and P-deficiency may have additive effects on Ugp expression.     

<Emphasis Type="Italic">FLOURY ENDOSPERM8</Emphasis>, encoding the UDP-glucose pyrophosphorylase 1, affects the synthesis and structure of starch in rice endosperm

Cereal opaque-kernel mutants are ideal genetic materials for studying the mechanism of starch biosynthesis and amyloplast development. Here we isolated and identified two allelic floury endosperm 8 (flo8) mutants of rice, named flo8-1 and flo8-2. In the flo8 mutant, the starch content was decreased and the normal physicochemical features of starch were altered. Map-based cloning and subsequent DNA sequencing analysis revealed a single nucleotide substitution and an 8-bp insertion occurred in UDP-glucose pyrophosphorylase 1 (Ugp1) gene in flo8-1 and flo8-2, respectively. Complementation of the flo8-1 mutant restored normal seed appearance by expressing full length coding sequence of Ugp1. RT-qPCR analysis revealed that Ugp1 was ubiquitously expressed. Mutation caused the decreased UGPase activity and affected the expression of most of genes associated with starch biosynthesis. Meanwhile, western blot and enzyme activity analyses showed the comparability of protein levels and enzyme activity of most tested starch biosynthesis related genes. Our results demonstrate that Ugp1 plays an important role for starch biosynthesis in rice endosperm.     

Recombinant Arabidopsis SQD1 converts udp-glucose and sulfite to the sulfolipid head group precursor UDP-sulfoquinovose in vitro

The sulfolipid sulfoquinovosyldiacylglycerol is a component of plant photosynthetic membranes and represents one of the few naturally occurring sulfonic acids with detergent properties. Sulfolipid biosynthesis involves the transfer of sulfoquinovose, a 6-deoxy-6-sulfoglucose, from UDP-sulfoquinovose to diacylglycerol. The formation of the sulfonic acid precursor, UDP-sulfoquinovose, from UDP-glucose and a sulfur donor is proposed to be catalyzed by the bacterial SQDB proteins or the orthologous plant SQD1 proteins. To investigate the underlying enzymatic mechanism and to elucidate the de novo synthesis of sulfonic acids in biological systems, we developed an in vitro assay for the recombinant SQD1 protein from Arabidopsis thaliana. Among different possible sulfur donors tested, sulfite led to the formation of UDP-sulfoquinovose in the presence of UDP-glucose and SQD1. An SQD1 T145A mutant showed greatly reduced activity. The UDP-sulfoquinovose formed in this assay was identified by co-chromatography with standards and served as substrate for the sulfolipid synthase associated with spinach chloroplast membranes. Approximate K(m) values of 150 microm for UDP-glucose and 10 microm for sulfite were established for SQD1. Based on our results, we propose that SQD1 catalyzes the formation of UDP-sulfoquinovose from UDP-glucose and sulfite, derived from the sulfate reduction pathway in the chloroplast.     

Gender,mutant p53 and PML: A growing “affaire” in tumor suppression and oncogenesis

The inability to coordinate cellular metabolic processes with the cellular and organismal nutrient environment leads to a variety of disorders, including diabetes and obesity. Nutrient-sensing protein kinases, such as AMPK and mTOR, play a pivotal role in metabolic regulation and are promising therapeutic targets for the treatment of disease. In this Extra View, we describe another member of the nutrient-sensing protein kinase group, PAS kinase, which plays a role in the regulation of glucose utilization in both mammals and yeast. PAS kinase deficient mice are resistant to high fat diet-induced weight gain, insulin resistance, and hepatic triglyceride hyperaccumulation, suggesting a role for PAS kinase in the regulation of glucose and lipid metabolism in mammals. Likewise, PAS kinase deficient yeast display altered glucose partitioning, favoring glycogen biosynthesis at the expense of cell wall biosynthesis. As a result, PAS kinase deficient yeast are sensitive to cell wall perturbing agents. This partitioning of glucose in response to PAS kinase activation is due to phosphorylation of Ugp1, the enzyme primarily responsible for UDP-glucose production. The two yeast PAS kinase homologs, Psk1 and Psk2, are activated by two stimuli, cell integrity stress and nonfermentative carbon sources. We review what is known about yeast PAS kinase and describe a genetic screen that may help elucidate pathways involved in PAS kinase activation and function.     

Genetic transformation using Agrobacterium rhizogenes

UDP-glucose pyrophosphorylase (EC 2.7.7.9) has been highly purified from the plant fraction of soybean ( Glycine max L. Merr. cv Williams) nodules. The purified enzyme gave a single polypeptide band following sodium docecyl sulphate polyacryla-mide gel electrophoresis, but was resolved into three bands of activity in non-denaturing gels. The enzyme appeared to be a monomer of molecular weight between 30 and 40 kDa. UDP-glucose pyrophosphorylase had optimum activity at pH 8.5 and displayed typical hyperbolic kinetics. The enzyme had a requirement for divalent metal ions, and was highly specific for the substrates pyrophosphate and UDP-glucose in the pyrophosphorolysis direction, and glucose-1-phosphate and UTP in the direction of UDP-glucose synthesis. The K_m values were 0.19 m M and 0.07 m M for pyrophosphate and UDP-glucose, respectively, and 0.23 m M and 0.11 m M for glucose-1-phosphate and UTP. The maximum velocity in the pyrophosphorolysis direction was almost double that for the reverse reaction. UDP-glucose pyrophosphorylase did not appear to be subject to a high degree of fine control, and activity in vivo may be regulated mainly by the availability of the substrates.