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学技术与我们的多胺需要的酿酒酵母突变体一起，以发现哪些基因和蛋白质被亚精胺补充剂上调或下调多胺缺乏的文化。 我们发现了大量基因和蛋白质的变化。 因此，我们现在集中在亚精胺后的最早变化 初步研究表明，参与碳水化合物代谢的基因占优势。45个基因在亚精胺诱导的早期（30 min）被诱导表达，其中25%与酵母糖代谢相关（如GCY 1、GLK 1、GPH 1、GSY 2、HXK 1、PGM 2、SOL 4、TDH 1、TPS 2、TSL 1、UGP 1）。 我们也正在进行微阵列研究与我们的大肠杆菌突变体，缺乏所有参与多胺生物合成的酶。为了这个目的，我们已经开发了恒化器的使用，因为我们认为，这是非常重要的，不要有结果复杂的亚精胺添加后的生长速率的变化。 赖氨酸和精氨酸营养缺陷型也被构建，以允许使用SILAC技术进行蛋白质组学研究。 本研究对E.大肠杆菌对谷胱甘肽亚精胺的生理功能特别关注。如前所述，E.大肠杆菌细胞和细胞内的谷胱甘肽的非常大的百分比转化为谷胱甘肽亚精胺在对数生长结束。在目前的工作中，我们已经集中在形成和功能，这种衍生物在pharmically增长的文化。谷胱甘肽亚精胺在大肠杆菌中的作用尚不清楚。大肠杆菌，虽然它已被证明是至关重要的锥虫。E.可获得缺乏谷胱甘肽亚精胺合成酶/酰胺酶（GSP）基因的大肠杆菌。我们比较了gsp基因缺失在大肠杆菌中的表型效应。大肠杆菌与野生型菌株在各种生长条件下（空气，95%氧气，温度和铜毒性），并发现突变体和野生型之间没有差异。在我们目前的工作中，我们进行了详细的微阵列研究，比较野生型和gsp突变株（在NIDDK微阵列设施的帮助下）。 我们已经标准化了从E. coli总RNA，并将富集的mRNA用于该比较。 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，约160个基因在突变细胞中上调超过2倍，其中包括嘌呤和嘧啶核苷酸生物合成、精氨酸和腐胺代谢的基因。gsp突变体中约有120个基因下调2倍以上。与grxA（谷氧还蛋白1，一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一起下调的是metalochaperone、钼、铜、锌和银离子转运基因。 详细的蛋白质组学研究也已经进行（与Eric安德森博士合作 NIDDK质谱部分）使用SILAC技术比较两种菌株的蛋白质组成。 为此，我们构建了在lysA和argA基因中含有缺失的菌株。最近，我们从这种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.