Structural studies of perchloric acid soluble protein (PSP) from rat liver.

大鼠肝脏高氯酸可溶性蛋白（PSP）的结构研究。

基本信息

批准号：
09680594
负责人：
OKA Tatsuzo
金额：
$ 0.51万
依托单位：
Kagoshima University (1998)The University of Tokushima (1997)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680594/
关键词：
Rat Perchloric acid-soluble protein Escherichia coli Recombinant Crystallization Higher structure 蛋白質合成阻害

项目摘要

We have reported isolation and characterization of a novel perchloric acid-soluble protein (PSP) in the cytosolic fraction of rat liver. The PSP is a homodimer, each subunit consisting of 136 amino acid residues with a molecular mass of 14,149 Da. It inhibits cell-free protein synthesis in the lysate of rabbit reticulocyte in a different manner than RNase A.Recently, a 14-kDa protein which inhibit translation was characterized from human monocyte and mouse liver. The 14-kDa proteins and their mRNAs were reported to be preferentially expressed in the liver and kidney as in the case of PSP.Furthermore, the sequences of complementary DNA (cDNA) encoding the 14-kDa protein and PSP are highly similar to those of cDNA encoding a new hypothetical family (YER057c/YJGF) of small proteins with presently unknown function. Thus the sequences of PSP-like proteins are highly conserved in prokaryotic, cyanobacteria, fungi and eukaryotes. The high degree of evolutionary conservation of these proteins … More suggests that these proteins play an important role in cellular regulation.Despite the growing body of information gor PSPs, no three-dimensional structure has benn reported. In the following contribution was present the crystallization and preliminary crystallographic characterization of PSP.At first, an efficient E.coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. cDNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E.coli was expressed by IPTG, the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH2-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver.PSP from rat liver has been crystallized in a form suitable for high resolution X-ray diffraction studies. Octahedral crystals reaching 0.5 mm in size were produced by hangin drop method using polyethylene glycol (Mr=8000 Da) as precipitant. These crystals diffract to 2.44 "A" on an in-house X-ray source and to l.8"A" using a bending, agnet beamline at ESRE Grenoble. The crystals belong to the cubic space group P213 with unit cell parameters a=b=c=89.90"A" and two molecules per asymmetric unit, as indicated from VM value of 2.12"A"3/Da and self-rotation function computation. Screening for heavy-atom derivatives identified a platinum compound that binds to a single site on the protein. Furhter screening for heavy-atom derivatives indeicated a beta sheet flanled by two alpha helice. The structural similarity gave the best hit ftsz (signal recognition particle receptor), chorismate mutase and heat-shock cognate protein 70 kDa. Less

我们报道了大鼠肝脏胞质部分中一种新型高氯酸可溶性蛋白 (PSP) 的分离和表征，该 PSP 是同型二聚体，每个亚基由 136 个氨基酸残基组成，分子量为 14,149 Da，它能抑制细胞。 - 兔网织红细胞裂解液中的游离蛋白质合成方式与 RNase A 不同。最近，从人类中鉴定出一种抑制翻译的 14-kDa 蛋白质据报道，与 PSP 一样，14-kDa 蛋白及其 mRNA 在肝脏和肾脏中优先表达。此外，编码 14-kDa 蛋白和 PSP 的互补 DNA (cDNA) 序列为与编码目前未知功能的新假设小蛋白家族（YER057c/YJGF）的 cDNA 高度相似，因此 PSP 样蛋白的序列在原核生物、蓝藻细菌、真菌和真核生物中这些蛋白质的高度进化保守性表明这些蛋白质在细胞调节中发挥着重要作用。尽管PSP的信息不断增加，但尚未报道其三维结构。以下贡献是PSP的结晶和初步晶体学表征。首先，用于生产高氯酸可溶性蛋白（PSP）的高效大肠杆菌表达系统已被开发出来。将构建的编码PSP的cDNA插入诱导型细菌表达载体pGEX-4T-1中，将其导入大肠杆菌中进行IPTG表达，重组产物通过谷胱甘肽-Sepharose 4B亲和层析纯化。与预期的 NH2 末端序列相同，但本品的翻译抑制活性比从大鼠肝脏分离的正品 PSP 低 10 倍。以适合高分辨率 X 射线衍射研究的形式结晶使用聚乙二醇 (Mr=8000 Da) 作为沉淀剂，通过悬挂滴法生产尺寸达到 0.5 mm 的八面体晶体。 -house X 射线源，并使用 ESRE Grenoble 的弯曲磁力束线达到 l.8"A" 晶体属于立方空间群。 P213 具有晶胞参数 a=b=c=89.90"A"，每个不对称单元有两个分子，如 VM 值 2.12"A"3/Da 和自旋转函数计算所示，筛选出重原子衍生物。与蛋白质上的单个位点结合的铂化合物对重原子衍生物的进一步筛选表明，侧翼有两个α螺旋的β片层结构相似性最佳。 ftsz（信号识别颗粒受体）、分支酸变位酶和热休克同源蛋白 70 kDa 更少。