Physiologic Functions of Polyamines

在以前的报告中，我们已经描述了使用微阵列和蛋白质组技术与我们的需要多胺的酿酒酵母突变体一起来寻找在缺乏多胺的培养物中补充亚精胺上调或下调哪些基因和蛋白质。我们发现了大量基因和蛋白质的变化。因此，我们现在专注于补充亚精胺后最早的变化，初步研究表明，参与碳水化合物代谢的基因占优势。在早期时间点(30min)，有45个基因被亚精胺诱导，其中25%与酵母中的碳水化合物代谢有关(如GCY1、GLK1、GPH1、GSY2、HXK1、PGM2、SOL4、TDH1、TPS2、TSL1、UGP1)。 我们还在对我们的大肠杆菌突变株进行微阵列研究，这些突变株缺乏与多胺生物合成有关的所有酶。为此，我们开发了恒化器的用途，因为我们认为，不让结果因添加亚精胺后生长速度的变化而复杂化是非常重要的。赖氨酸和精氨酸营养缺陷菌也已被构建，以允许使用SILAC技术进行蛋白质组学研究。 我们目前在大肠杆菌中的研究特别关注谷胱甘肽亚精胺的生理功能。正如我们之前所展示的，在对数生长结束时，大肠杆菌细胞中的所有亚精胺和非常大比例的细胞内谷胱甘肽被转化为谷胱甘肽亚精胺。在目前的工作中，我们集中在对数生长的培养中这种导数的形成和功能。谷胱甘肽亚精胺在大肠杆菌中的功能尚不清楚，尽管已经证明它在锥虫体内具有至关重要的作用。目前已发现一种缺乏谷胱甘肽亚精胺合成酶/酰胺酶(GSP)基因的突变体。我们比较了在不同生长条件(空气、95%氧气、温度和铜毒)下GSP基因缺失在大肠杆菌和野生型菌株中的表型效应，发现突变体和野生型菌株之间没有差异。在我们目前的工作中，我们进行了详细的微阵列研究，比较了野生型和GSP突变株(在NIDDK微阵列设备的帮助下)。我们已经标准化了一种从大肠杆菌总RNA中去除rRNA的技术，并使用丰富的mRNA进行了比较。最近的研究结果表明，GSP基因的缺失有很大的影响。与野生型细胞相比，突变细胞中约有160个基因上调了2倍以上，其中包括嘌呤和嘧啶核苷酸生物合成、精氨酸和腐胺代谢基因。在GSP突变体中，大约有120个基因下调了2倍以上。金属香草酮、钼、铜、锌和银离子转运基因与GRXA(一种核糖核苷酸还原酶的氧化还原辅酶)和亚硝酸盐转运蛋白NIRC一起下调。还进行了详细的蛋白质组学研究(与NIDDK质谱科的Eric Anderson博士合作)，使用SILAC技术比较了两个菌株的蛋白质组成。为此，我们构建了含有Lysa和Arga基因缺失的菌株。最近，我们从这一质谱分析中获得了数据，并发现8-10个肽在突变体中上调或下调。

In previous reports we have described the use of microarray and proteomic techniques together with our polyamine-requiring mutants of Saccharomyces cerevisiae to find which gene and proteins are upregulated or downregulated by spermidine supplementation to polyamine -deficient cultures. We found changes in a very large number of genes and proteins. Therefore we have now focused on the earliest changes after spermidine supplementation, and preliminary studies indicate a preponderance of genes involved in carbohydrate metabolism. Forty five genes are induced by spermidine at an early time points (30 min), among those 25% are related to carbohydrate metabolism in yeast (such as GCY1, GLK1, GPH1, GSY2, HXK1, PGM2, SOL4, TDH1, TPS2, TSL1, UGP1). We are also carrying out microarray studies with our Escherichia coli mutants that lack all of the enzymes involved in polyamine-biosynthesis.. For this purpose we have developed the use of a chemostat since we feel that it is very important not to have the results complicated by changes in the growth rates after spermidine addition. Lysine and arginine auxotrophs have also being constructed to permit the use of the SILAC technique for proteomic studies. Our current studies in E. coli have been particularly concerned with the physiologic function of glutathionylspermidine. As we have previously shown, all of the spermidine of the E. coli cells and a very large percentage of the intracellular glutathione are converted to glutathionylspermidine at the end of the logarithmic growth. In the current work we have concentrated on the formation and function of this derivative in logarithmically growing cultures. Nothing is known about the function of glutathionylspermidine in E. coli, although it has been shown to be of critical importance in trypanosomes. A mutant of E. coli is available that lacks the gene for glutathionylspermidine synthetase/amidase (gsp). We have compared the phenotypic effects of the gsp gene deletion in E. coli with a wild type strain in various growth conditions (air, 95% oxygen, temperature and copper toxicity) and found no difference between the mutant and the wild type. In our current work we have carried out detailed microarray studies, comparing the wild type and gsp mutant strain (with the help of the NIDDK Microarray facility). We have standardized a technique to remove rRNA from E. coli total RNA and used the enriched mRNA for this comparison. The recent results show a large effect of deletion of gsp gene. Around 160 genes were upregulated more than 2-fold in mutant cells as compared to wild type cells, which include genes for purine and pyrimidine nucleotide biosynthesis, arginine and putrescine metabolism. Approximately 120 genes were down-regulated more than 2-fold in gsp mutant. The genes for metalochaperone, molybdenum, copper, zinc and silver ion transport were down-regulated together with grxA (glutaredoxin 1, a redox coenzyme for ribonucleotide reductase), and nitrite transporter nirC. Detailed proteomic studies have also been carried out (in collaboration with Dr. Eric Anderson of the NIDDK Mass Spectrometry section) comparing the protein composition of the two strains using the SILAC technique. For this purpose we have constructed strains containing deletions in the lysA and argA genes. Very recently we have obtained data from this mass spectroscopy analysis, and found 8-10 peptides are upregulated or downregulated in the mutant.