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.采用Western blot分析研究多胺对RNA聚合酶各σ亚基合成的影响。多胺刺激σ^<28>的合成4.0倍，并且刺激σ^<38>的合成2.3倍，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，其通过在翻译水平上多胺刺激腺苷酸环化酶的合成而发生。研究发现多胺通过促进 UUG 密码子依赖性起始来增加腺苷酸环化酶 mRNA 的翻译。 RNA二级结构分析表明，暴露mRNA的Shine-Dalgamo (SD)序列是多胺刺激UUG密码子依赖性起始的先决条件。2．我们鉴定了酵母液泡膜上编码多胺转运蛋白的基因（TPO1，YLLO28w）。由于预期无泡膜上存在多胺转运蛋白的其他基因，因此我们在序列数据库中搜索了 TPO1 编码的蛋白质的同源物。由开放阅读框 YGR138c (TPO2)、YPR156c (TPO3) 和 YOR273c (TPO4) 编码的膜蛋白被证明是液泡膜上的多胺转运蛋白。过度表达这些基因的细胞对多胺毒性具有抵抗力，并表现出多胺摄取活性和液泡中多胺含量的增加。此外，这些基因被破坏的细胞对多胺毒性的敏感性增加，多胺摄取活性和液泡中多胺含量降低。巴弗洛霉素A_1（一种液泡H^+-ATP酶抑制剂）克服了过度表达该基因的细胞对多胺毒性的抵抗力。在四种多胺转运蛋白中，TPO2和TPO3编码的转运蛋白对精胺具有特异性，而TPO1和TPO4编码的转运蛋白则识别亚精胺和精胺。这些结果表明，酵母细胞质中的多胺含量受到液泡中多个多胺转运系统的精心调控。 3． PotE 蛋白可以催化腐胺的摄取和排出。腐胺的摄取和排泄的 Km 值分别为 1.8 和 73 μM。腐胺的摄取取决于膜电位，而排泄涉及腐胺-鸟氨酸逆向转运蛋白活性。使用突变的 PotE 蛋白鉴定了参与这两种活性的氨基酸。结果发现，Cys^<62>、Trp^<201>、Trp^<292> 和 Tyr^<425> 强烈参与这两种活动，Tyr^<92>、Cys^<210>、Cys^<285> 和 Cys^<286> 中度参与这些活动。 Tyr^<78>、Trp^<90>、Trp^<422>突变主要影响摄取活性，这些PotE突变体对腐胺摄取的Km值大幅增加，表明这些氨基酸参与了PotE对腐胺的高亲和力摄取。Lys^<301>和Tyr^<308>突变主要影响排泄活性 （腐胺-鸟氨酸逆向转运蛋白活性），并且这些突变体的排泄不受鸟氨酸的刺激，表明这些氨基酸参与了鸟氨酸的识别。基于各种腐胺类似物的竞争实验结果以及PotE在细胞质环和COOH末端之间的二硫键交联，提出了由已识别氨基酸组成的PotE上腐胺识别位点的模型。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)

Yoshida, M.et al.: "Polyamine enhancement of the synthesis of adenylate cyclase at the translational level and the consequential stimulation of the synthesis of the RNA polymerase σ^<28> subunit."J.Biol.Chem.. 276(in press). (2001)

Yoshida, M.等人：“多胺在翻译水平上增强腺苷酸环化酶的合成，从而刺激 RNA 聚合酶 σ^<28> 亚基的合成。”J.Biol.Chem.. 276（in (2001)

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)

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

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

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)