Polyamines as biomodulators - regulation of their contents and their physiological functions

多胺作为生物调节剂 - 其含量及其生理功能的调节

• 批准号: 11470482
• 负责人: IGARASHI Kazuei
• 金额: $ 9.47万
• 依托单位: Chiba University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B).
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470482/
• 关键词: Polyamine; Spermidine; Spermine; σ^<28>; Adenylate cyclase; Polyamine transport protein; Vacuolar membrane; OppA; NMDA受容体

1. The effects of polyamines on the synthesis of various σ subunits of RNA polymerase were studied using Western blot analysis. Synthesis of σ^<28> was stimulated 4.0-fold and that of σ^<38> was stimulated 2.3-fold by polyamines, whereas synthesis of other σ subunits was not influenced by polyamines. Stimulation of σ^<28> synthesis was due to an increase in the level of cAMP, which occurred through polyamine stimulation of the synthesis of adenylate cyclase at the level of translation. Polyamines were found to increase the translation of adenylate cyclase mRNA by facilitating the UUG codon-dependent initiation. Analysis of RNA secondary structure suggests that exposure of the Shine-Dalgamo (SD) sequence of mRNA is a prerequisite for polyamine stimulation of the UUG codon-dependent initiation.2. We identified a gene (TPO1, YLLO28w) that encodes a polyamine transport protein on the vacuolar membrane in yeast. Because the existence of other genes for a polyamine transport protein on the v … More acuolar membrane was expected, we searched sequence databases for homologues of the protein encoded by TPO1. Membrane proteins encoded by the open reading frames YGR138c (TPO2), YPR156c (TPO3) and YOR273c (TPO4) were shown to be polyamine transport proteins on the vacuolar membrane. Cells overexpressing these genes were resistant to polyamine toxicity and showed an increase in polyamine uptake activity and polyamine content in vacuoles. Furthermore, cells in which these genes were disrupted showed an increased sensitivity to polyamine toxicity and a decrease in polyamine uptake activity and polyamine content in vacuoles. Resistance to polyamine toxicity in cells overexpressing the genes was overcome by bafilomycin A_1, an inhibitor of the vacuolar H^+- ATPase. Among the four polyamine transporters, those encocded by TPO2 and TPO3 were specific for spermine, whereas those encoded by TPO1 and TPO4 recognized spermidine and spermine. These results suggest that polyamine content in the cytoplasm of yeast is elaborately regulated by several polyamine transport systems in vacuoles.3. The PotE protein can catalyze both uptake and excetion of putrescine. The Km values of putrescine for uptake and excretion are 1.8 and 73 μM, respectively. Uptake of putrescine is dependent on the membrane potential, whereas excretion involves putrescine-ornithine antiporter activity. Amino acids involved in both activities were identified using mutated PotE proteins. It was found that Cys^<62>, Trp^<201>, Trp^<292>, and Tyr^<425> were strongly involved in both activities, and that Tyr^<92>, Cys^<210>, Cys^<285>, and Cys^<286> were moderately involved in the activities. Mutations of Tyr^<78>, Trp^<90>, and Trp^<422> mainly affected uptake activity, and the Km values for putrescine uptake by these PotE mutants increased greatly, indicating that these amino acids are involved in the high affinity uptake of putrescine by PotE.Mutations of Lys^<301> and Tyr^<308> mainly affected excretion activity (putrescine-ornithine antiporter activitiy), and excretion by these mutants was not stimulated by ornithine, indicating that these amino acids are involved in the recognition of ornithine. Based on the results of competition experimentes with various putrescine analogues and the disulfide cross-linking of PotE between cytoplasmic loops and the COOH terminus, a model of the putrescine recognition site on PotE consisting of the identified amino acids is presented. Less

1。使用蛋白质印迹分析研究了多胺对RNA聚合酶各种σ亚基的合成的影响。刺激了σ^<28>的合成4.0倍，而多胺刺激了σ^<38>的合成，而其他σ亚基的合成不受多胺的影响。 σ^<28>合成的刺激是由于cAMP水平的增加，这是由于多胺刺激在翻译水平上刺激腺苷酸环化酶的合成而发生的。发现多胺通过支持UUG密码子依赖性倡议来增加腺苷酸环化酶mRNA的翻译。 RNA二级结构的分析表明，mRNA的光泽 - 达尔加莫（SD）序列暴露是对UUG密码子依赖性倡议的多胺刺激的先决条件。2。我们确定了一个基因（TPO1，YLLO28W），该基因在酵母中编码多胺转运蛋白。由于预计多胺转运蛋白的其他基因的存在预计会更多，因此我们搜索了TPO1编码的蛋白质的序列数据库。由开放式阅读框YGR138C（TPO2），YPR156C（TPO3）和YOR273C（TPO4）编码的膜蛋白显示为真空膜上的多胺转运蛋白。过表达这些基因的细胞对多胺毒性具有抗性，并且在液泡中显示出多胺摄取活性和多胺含量的增加。此外，这些基因被破坏的细胞显示出对多胺毒性的敏感性增加，多胺摄取活性和真空中多胺含量的降低。巴菲霉素A_1（一种真空h^+ATPase的抑制剂Bafilycin A_1）克服了对过表达基因的细胞中多胺毒性的抗性。在四个多胺转运蛋白中，由TPO2和TPO3所包围的那些转运蛋白特异于精子，而由TPO1和TPO4识别的精子和精子编码的那些。这些结果表明，酵母菌细胞质中的多胺含量受真空中的几种多胺传输系统的精心调节。3。 pote蛋白可以催化摄取和超过腐烂的摄取。腐烂的摄取和排泄的Km值分别为1.8和73μm。腐烂的摄取取决于膜电位，而排泄涉及腐霉氨酸氨基氨酸的抗胞药活性。使用突变的pote蛋白鉴定出参与两种活性的氨基酸。 It was found that Cys^<62>, Trp^<201>, Trp^<292>, and Tyr^<425> were strongly involved in both activities, and that Tyr^<92>, Cys^<210>, Cys^<285>, and Cys^<286> were Mutations of Tyr^<78>, Trp^<90>, and Trp^<422> mainly affected uptake activity, and the Km这些pote突变体的腐烂摄取值大大增加，表明这些氨基酸与pote的高亲和力相关。酸参与鸟氨酸的识别。基于具有各种perrescine类比的竞争实验的结果以及细胞质环与COOH末端之间的二硫键交联，提出了由识别氨基酸组成的pote上的pote蛋白识别位点的模型。较少的

项目成果

K.Igarashi and K.Kashiwagi: "Polyamines : Mysterious modulators of cellular functions. (Review)"Biochem.Biophys.Res.Commun.. 271. 559-564 (2000)

K.Igarashi 和 K.Kashiwagi：“多胺：细胞功能的神秘调节剂。（评论）”Biochem.Biophys.Res.Commun.. 271. 559-564 (2000)

K.Kashiwagi et al.: "Identification of the putrescine recognition site on polyamine transport protein PotE."J.Biol.Chem.. 275. 36007-36012 (2000)

K.Kashiwagi 等：“多胺转运蛋白 PotE 上腐胺识别位点的鉴定。”J.Biol.Chem.. 275. 36007-36012 (2000)

K. Nishimura et al.: "Gene structure and chromosomal localization of mouse S-adenosylmethionine decarboxylase"Gene. 238. 343-350 (1999)

K. Nishimura等：“小鼠S-腺苷甲硫氨酸脱羧酶的基因结构和染色体定位”基因。

H.Tomitori et al.: "Identification of a gene for a polyamine transport protein in yeast."J.Biol.Chem.. 274. 3265-3267 (1999)

H.Tomitori 等人：“酵母中多胺转运蛋白基因的鉴定。”J.Biol.Chem.. 274. 3265-3267 (1999)

H.Tomitori et al.: "Multiple polyamine transport systems on the vacuolar membrane in yeast."Biochem.J.. 353. 681-688 (2001)

H.Tomitori 等人：“酵母液泡膜上的多重多胺转运系统。”Biochem.J. 353. 681-688 (2001)