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New function of disulfide bonds in cytosolic proteins

胞质蛋白中二硫键的新功能

基本信息

批准号：
16570092
负责人：
ODANI Shoji
金额：
$ 2.05万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

A number of cytosolic proteins contain cysteine residues of unknown function. To understand the biological function of these cysteine residues and their disulfides, fatty acid-binding proteins (FABP) were chosen as model proteins. Liyer fatty acid-binding proteins (L-FABP) of rat, chick, lizard, frog, and fish possess cysteine residues at different positions in the amino acid sequence, and 3D modeling indicated that in fish L-FABP a pair of cysteine residues are located within the distance of sulfur atoms of a disulfide bond. Rat, chick and frog (Rana catesbeiana) liver FABPs were purified form the respective tissues by gel-filtration on Sephacryl columns, ion-exchange chromatography on DEAE-Sephacell, and HPLC on reversed-phase columns (octylsilane). Recombinant proteins were prepared for lizard (Anolis pukhelhis) and zebra fish (Brachydanio rerio). Lizard FABP cDNA was a gift of Prof. Morales, and zebrafish first strand cDNA library was prepared form the liver total RNA. They were li … More gated into expression vectors and transfected to E. coli BL21(DE3). Expressed proteins were purified by gel-filtration and HPLC. Mixed disulfides with glutathione and these proteins were prepared by incubation with diamide. They are defatted and lipid-binding activity was compared with untreated proteins. Lipid binding activity was measured by displacement of fluorescent probes, dansylaminoundecanoic acid and anilinonaphthalene sulfonate. Direct binding of a natural fluorescent fatty acid, cis-parinaric acid, was also measured. All these FABPs strongly bound various lipids regardless of bound glutathione. These results indicate that the purified and expressed FABPs are native and modification with glutathione does not affect biological function of the proteins, i.e., mixed disulfide formation did not alter the structure of the protein. However, it was found that mixed disulfide forms were rapidly digested with various proteinases, including intracellular proteases, regardless of the position of cysteine residues throughout the linear amino acid sequence. This strongly suggests that these mixed disulfide formation with glutathione is a process of tagging leading to proteolytic degradation of oxidatively damaged proteins. Since redox potential of a cysteine residue is under the influence of its location in the protein environment, above-mentioned mixed disulfide formation might be a determinant of life span of a protein.The nematode, Caenorhabditis elegans also has nine genes coding FABPs with or without cysteine residues. This animal is useful for examine the effects of oxidative stress as culturing the worms in medium containing hydrogen peroxide readily simulates oxidative stresses. We first prepared 9 recombinant proteins of nematode FABP 1-9, and experimental conditions for in vitro glutathionylation was established. A similar glutathionylation experiment as described above was attempted first on FABP-5, and an dramatic increase of proteolytic susceptibility was again observed. We believe cytosolic disulfide formation is a hither-to-unknown marking event for protein degradation. Less

许多胞质蛋白含有功能未知的半胱氨酸残基。为了了解这些半胱氨酸残基及其二硫键的生物学功能，选择脂肪酸结合蛋白(FABP)作为模型蛋白。大鼠、鸡、蜥蜴、蛙和鱼的Liyer脂肪酸结合蛋白(L-FABP)在氨基酸序列中具有不同位置的半胱氨酸残基，三维模拟表明，在鱼类L-FABP中，一对半胱氨酸残基位于二硫键的硫原子距离内。通过Sephacryl凝胶过滤、DEAE-Sephacell离子交换层析和反相柱(辛基硅烷)高效液相色谱分离纯化大鼠、鸡和蛙(Rana Catesbeiana)肝脏FABP。制备了蜥蜴(Anolis Pukhelis)和斑马鱼(Brachydanio Rerio)的重组蛋白。蜥蜴FABP基因是Morales教授赠送的礼物，从斑马鱼肝脏总RNA中获得了斑马鱼第一链cDNA文库。他们是li…更多克隆入表达载体，转入大肠杆菌BL21(DE3)。表达产物经凝胶过滤和高效液相色谱分离纯化。将二硫化物与谷胱甘肽混合，再与联胺孵育，制备出二硫杂蛋白。它们是脱脂的，并且与未经处理的蛋白质的脂结合活性进行了比较。通过置换荧光探针、丹磺酰亚胺十一酸和苯基苯磺酸盐来测定脂质结合活性。还测定了天然荧光脂肪酸顺式对羟基苯甲酸的直接结合力。所有这些FABP都与各种脂类强结合，而不考虑结合的谷胱甘肽。这些结果表明，纯化和表达的FABP是天然的，谷胱甘肽的修饰不影响蛋白质的生物学功能，即混合二硫键的形成没有改变蛋白质的结构。然而，研究发现，无论半胱氨酸残基在整个线性氨基酸序列中的位置如何，混合的二硫键形式都能被包括细胞内酶在内的各种蛋白酶迅速消化。这有力地表明，这些与谷胱甘肽形成的混合二硫键是一个标记过程，导致氧化损伤蛋白质的蛋白降解。由于半胱氨酸残基的氧化还原电位受其在蛋白质环境中所处位置的影响，上述混合二硫键的形成可能是蛋白质寿命的决定因素。线虫线虫也有9个基因编码含有或不含有半胱氨酸残基的FABP。这种动物对检测氧化应激的影响很有用，因为在含有过氧化氢的介质中培养蠕虫很容易模拟氧化应激。我们首先制备了9种线虫FABP 1-9重组蛋白，并建立了体外谷胱甘肽基化的实验条件。首先在FABP-5上尝试了类似于上述的谷胱甘肽基化实验，并再次观察到蛋白降解敏感性的显著增加。我们认为胞质二硫化物的形成是蛋白质降解的一种未知的标记事件。较少