Regulation of polyamine contents in cells and their effects on protein synthesis

细胞内多胺含量的调节及其对蛋白质合成的影响

• 批准号: 07457534
• 负责人: IGARASHI Kazuei
• 金额: $ 4.86万
• 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: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07457534/
• 关键词: Polyamine; Spermine; Spermidine; Putrescine; Polyamine transprt system; Polyamine binding site; 蛋白質合成; 輸送; ATPase; プロテインキナーゼ

1.PotD protein is a periplasmic binding protein and the primary receptor of the polyamine transport system. The crystal structure of PotD in complex with spermidine has been solved at 2.5-* resolution. The PotD protein consists of two domains with an alternating beta-alpha-beta topology. The polyamine binding site is in a central cleft lying in the interface between the domains. Spermidine binding sites on PotD were studied by measuring polyamine transport activities of right-side-out membrane vesicles with mutated PotD proteins prepared by site-directed mutagenesis of the potD gene and by measuring polyamine binding activities of these mutated PotD proteins. It was found that Trp-34, Thr-35, Glu-36, Tyr-37, Ser-83, Tyr-85, Asp-168, Glu-171, Trp-229, Trp-255, Asp-257, Tyr-293, and Gln-327 of PotD protein were involved in the binding to spermidine, and that Glu-171, Trp-225, and Asp-257 were more strongly involved in the binding of spermidine to PotD protein than the other amino acids l … More isted above.2.Polyamine stimulation of the synthesis of oligopeptide-binding protein (OppA) was shown to occur mainly at the level of translation by measuring OppA synthesis and its mRNA level. Several artificial oppA genes were constructed by site-directed mutagenesis. These synthesize different kinds of OppA mRNAs : mRNAs differing in the size of 5'-untranslated region (5'-UTR) ; mRNAs having the Shine-Dalgarno (SD) sequence in a different position ; mRNAs having dirrerent secondary structure in the region of the SD sequence ; and fusion mRNAs consisting of the 5'-UTR of OppA mRNA and the open reading frame of beta-galactosidase. By measuring the synthesis of OppA or beta-galactosidase from these mRANs, we found that the 171-nucleotide 5'-UTR and 145 nucleotides of the ORF OppA mRNA are involved in the polyamine stimulation of OppA synthesis. When the secondary structure of the above region of OppA mRNA was analyzed by optimal computer folding, it was shown that the degree of polyamine stimulation of OppA protein synthesis was dependent on the structure of the SD sequence in addition to its position. Loose base pairing of the SD sequence with other regions of the mRNA caused strong polyamine stimulation, while intense base pairing of the SD sequence with other regions of the mRNA resulted in insignificant or weak polyamine stimulation. Less

1.PotD蛋白是一种质周结合蛋白，是多胺转运系统的主要受体。在2.5 *分辨率下对pod与亚精胺配合物的晶体结构进行了解析。PotD蛋白由两个具有交替- α - β拓扑结构域组成。多胺结合位点位于两个结构域之间界面的中心裂缝中。通过位点定向诱变制备的突变pod蛋白，并通过测量这些突变pod蛋白的多胺结合活性，研究了pod上亚精胺的结合位点。结果表明，pod蛋白的Trp-34、Thr-35、Glu-36、Tyr-37、Ser-83、Tyr-85、Asp-168、Glu-171、Trp-229、Trp-255、Asp-257、Tyr-293和Gln-327参与了亚精胺与pod蛋白的结合，其中，与其他氨基酸相比，Glu-171、Trp-225和Asp-257参与亚精胺与pod蛋白结合的作用更强。通过对寡肽结合蛋白（OppA）合成及其mRNA水平的测定，发现多胺刺激主要发生在翻译水平。利用定点诱变技术构建了多个人工oppA基因。它们合成不同种类的OppA mrna: 5'-非翻译区（5'-UTR）大小不同的mrna；Shine-Dalgarno （SD）序列位于不同位置的mrna；在SD序列区域具有不同二级结构的mrna；并融合由OppA mRNA的5'-UTR和β -半乳糖苷酶的开放阅读框组成的mRNA。通过测量这些mRANs合成OppA或β -半乳糖苷酶，我们发现171个核苷酸的5'-UTR和145个核苷酸的ORF OppA mRNA参与了多胺刺激OppA合成。通过优化计算机折叠对OppA mRNA上述区域的二级结构进行分析，发现多胺对OppA蛋白合成的刺激程度除了依赖于SD序列的位置外，还依赖于其结构。当SD序列与mRNA其他区域碱基配对较松时，多胺刺激作用较强；当SD序列与mRNA其他区域碱基配对较强时，多胺刺激作用不明显或较弱。少

项目成果

S.Sugiyama et al.: "Crystal structure of PotD,the primary receptor of the polyamine transport system in Escherichia coli." J.Biol.Chem.271. 9519-9525 (1996)

S.Sugiyama 等人：“PotD 的晶体结构，大肠杆菌中多胺转运系统的主要受体。”

Sugiyama, S., Vassylyev, D.G., Matsushima, M., Kashiwagi, K., Igarashi, K., and Morikawa, K.: "Crystal structure of PotD,the primary receptor of the polyamine transport system in Escherichia coli." J.Biol.Chem.271. 9519-9525 (1996)

Sugiyama, S.、Vassylyev, D.G.、Matsushima, M.、Kashiwagi, K.、Igarashi, K. 和 Morikawa, K.：“PotD 的晶体结构，大肠杆菌中多胺转运系统的主要受体。”

Nozaki, T., Nishimura, K., Michael, A.J., Maruyama, T., Kakinuma, Y., and Igarashi, K.: "A second gene encoding a putative serine/threonine protein kinase which enhances spermine uptake in Saccharomyces cerevisiae." Biochem.Biophys.Res.Commun.228. 452-458

Nozaki, T.、Nishimura, K.、Michael, A.J.、Maruyama, T.、Kakinuma, Y. 和 Igarashi, K.：“编码假定的丝氨酸/苏氨酸蛋白激酶的第二个基因可增强酿酒酵母中精胺的摄取。

J.Fukuchi et al.: "Decrease in cell viability due to the accumulation of spermidine in spermidine acetyltransferase-deficient mutant of Escherichia coli." J. Biol. Chem.270. 18831-18835 (1995)

J.Fukuchi 等人：“由于亚精胺乙酰转移酶缺陷型大肠杆菌突变体中亚精胺的积累，导致细胞活力下降。”

K.Igarashi et al.: "Molecular mechanism of polyamine stimulation of the synthesis of oligopeptide binding protein." J.Biol.Chem.(in press). (1997)

K.Igarashi 等人：“多胺刺激寡肽结合蛋白合成的分子机制”。