Physiologic Functions of Polyamines

多胺的生理功能

• 批准号: 7967211
• 负责人: Herbert Tabor
• 金额: $ 29.52万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7967211
• 关键词: Amines; Anabolism; Biotin; Caenorhabditis elegans; Cell division; Cell physiology; Cells; Collaborations; Core Facility; DDR2 gene; Defect; Development; Differentiation and Growth; Embryonic Development; Enzymes; Escherichia coli; Gel; Gene Expression; Genes; Growth; Harvest; Laboratories; Lasers; Life; Mass Spectrum Analysis; Methionine; Molecular; National Institute of Diabetes and Digestive and Kidney Diseases; Oocytes; Peptides; Physiological; Polyamines; Proteome; Putrescine; RNA; Retrotransposon; Role; Running; Sampling; Scanning; Signal Transduction; Spermidine; Spermidine Synthase; Spermine; Staining method; Stains; Structure; Supplementation; System; Techniques; Time; Yeasts; arginyllysine; biological adaptation to stress; computerized; feeding; gag Gene Products; in vivo; interest; mutant; nucleic acid metabolism; polyamine oxidase; research study; response; time interval

The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well established in vivo. In our first studies we performed microarray experiments with a spermidine synthase/ spermine oxidase mutant (deleted spe3 deleted fms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10-5 M) or spermine (10-5 M) compared to a control culture containing 10-8 M spermidine. 247 genes were up-regulated >2-fold, and 11 genes were up-regulated more than 10-fold after spermidine addition. Functional categorization of the genes showed induction of transport related genes, and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were down-regulated more than 2-fold, and 6 genes were down-regulated more than 8-fold after spermidine addition. A majority of the down-regulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only few genes (18) were significantly responsive to spermine. Thus, these results demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. In our current experiments we studied the effect of spermidine addition in a yeast double mutant deleted in both spe1 and spe2 genes. We have compared the early (30 min and 60 min) and late (120 min and 180 min) responses to spermidine additions to avoid any complications due to changes in hypusinated eIF5A since we found that the modified eIF5A is unchanged up to 180 min after spermidine addition. This polyamine auxotroph has the advantage over our earlier studies since it does not accumulate decarboxylated S-adenosylmethionine or putrescine and thus the effect of gene expression will be mainly due to change in spermidine concentration. As spermine didnt show much of an effect in earlier studies, we focused on the response to spermidine. Yeast cultures were harvested at different time intervals intervals after addition of 10-5 M spermidine and RNA was isolated and microarray signals were compared at each time point with control cultures (total 27 arrays). 176 genes were up or down regulated within 30-60 min after spermidine (10-5 M) addition while the expressin of 313 genes changed within 120-180 min. Expression of 40 genes were changed at all time points from 30 min to 180 min. Some of these genes were ADH5, BNA1, MDH2, OPI1, RPL22B, RPS22B, TDH1 HTB2, TOF2, QCR10, 7 and 9, RIP1, ROX1, GAD1, CTR1, DDR2, GDH3, several HSPs, GSP2, MBF1, NPL3 and OPT1. The most interesting sets of up-regulated genes are genes encoding the retrotransposons TyB, TyA Gag-proteins. We have also compared the whole proteome of the yeast polyamine auxotroph (spe1 spe2) grown in two different concentrations (low 10-8 M and high 10-5 M) of spermidine by 2D peptide arrays. Samples were run on 2D gels, and were scanned with a laser densitometer and differentially expressed peptides were detected by computerized comparison of the two gels. 195 differentially expressed peptides were noted from these study. The samples were reanalyzed by staining with coomassie stain and we are currently analyzing by mass spectrometry 20 differentially expressed peptides in collaboration with the NIDDK core facility. The multi-gene E. coli polyamine mutant has been used to feed C. elegans polyamine mutant (odc-1) in a different study in collaboration with Dr. Kevin OConnell of our laboratory. Initial studies have shown developmental defect during L1 to L4 larval development and embryonic lethality in C. elegans due to polyamine deficiency. E. coli grown in 10-5 to 10-6 M putrescine can rescue the C. elegans from this larval defect. Recently, Dr. OConnell has developed a mutant in combination with odc-1 and an oocyte developmental marker to study the developmental defect in detail.

天然存在的多胺腐胺、亚精胺或精胺普遍存在于所有细胞中。虽然多胺在细胞分裂和生长中具有突出的调节作用，但精确的分子和细胞功能在体内还没有很好地建立。在我们的第一个研究中，我们进行了基因芯片实验与亚精胺合酶/精胺氧化酶突变体（删除spe 3删除fms 1）菌株，以调查酵母基因的反应，补充亚精胺或精胺。表达分析鉴定了与含有10-8 M亚精胺的对照培养物相比，响应于添加过量亚精胺（10-5 M）或精胺（10-5 M）的基因。247个基因在添加亚精胺后上调>2倍，11个基因在添加亚精胺后上调超过10倍。基因的功能分类显示，诱导运输相关基因，和基因参与蛋氨酸，精氨酸，赖氨酸，NAD和生物素的生物合成。268个基因表达下调2倍以上，6个基因表达下调8倍以上。大多数下调的基因参与核酸代谢和各种应激反应。相比之下，只有少数基因（18个）对精胺有显著反应。因此，这些结果表明，亚精胺在调节酵母中的基因表达比精胺更重要的作用。 在我们目前的实验中，我们研究了亚精胺添加在酵母双突变体中的spe 1和spe 2基因缺失的影响。 我们比较了对亚精胺添加的早期（30分钟和60分钟）和晚期（120分钟和180分钟）反应，以避免由于羟腐胺赖氨酸化eIF 5A变化引起的任何并发症，因为我们发现，亚精胺添加后修饰的eIF 5A在180分钟内没有变化。这种多胺营养缺陷型与我们早期的研究相比具有优势，因为它不积累脱羧的S-腺苷甲硫氨酸或腐胺，因此基因表达的影响将主要是由于亚精胺浓度的变化。由于精胺在早期的研究中没有显示出太大的影响，我们专注于对亚精胺的反应。在加入10-5 M亚精胺后，在不同的时间间隔收获酵母培养物，分离RNA，并在每个时间点将微阵列信号与对照培养物（总共27个阵列）进行比较。在30 ~ 180 min内有40个基因表达发生变化，其中ADH 5、BNA 1、MDH 2、OPI 1、RPL 22 B、RPS 22 B、TDH 1、HTB 2、TOF 2、QCR 10、7和9、RIP 1、ROX 1、GAD 1、CTR 1、DDR2、GDH 3、几种HSP、GSP 2、MBF 1、NPL 3和OPT 1。最有趣的上调基因组是编码逆转录转座子TyB、TyA GAG蛋白的基因。我们还比较了整个蛋白质组的酵母多胺营养缺陷型（spe 1 spe 2）生长在两个不同浓度（低10-8 M和高10-5 M）的亚精胺的二维肽阵列。将样品在2D凝胶上运行，并用激光密度计扫描，并通过两种凝胶的计算机比较检测差异表达的肽。从这些研究中观察到195个差异表达的肽。通过考马斯染色对样品进行再分析，我们目前正在与NIDDK核心设施合作，通过质谱分析20种差异表达的肽。 多基因E. coli多胺突变体已被用于喂养C. elegans多胺突变体（odc-1）在另一项研究中与我们实验室的Kevin OConnell博士合作。初步研究表明，在L1至L4幼虫发育和胚胎致死C。多胺缺乏导致的线虫病E.在10 ~（-5）~ 10 ~（-6）M腐胺中生长的大肠杆菌能拯救C. elegans从这个幼虫缺陷。最近，OConnell博士开发了一种突变体与odc-1和卵母细胞发育标记物相结合，以详细研究发育缺陷。