New function of disulfide bonds in cytosolic proteins

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化物的生物学功能，选择脂肪酸结合蛋白（FABP）作为模型蛋白。大鼠、鸡、蜥蜴、青蛙和鱼类的L-FABP在氨基酸序列的不同位置上具有半胱氨酸残基，三维建模表明鱼类L-FABP中的一对半胱氨酸残基位于二硫键的硫原子之间。采用Sephacryl凝胶过滤柱、DEAE-Sephacell离子交换柱和反相柱（辛基硅烷）高效液相色谱法从大鼠、鸡和蛙（Rana catesbeiana）肝脏中分离纯化FABP。制备蜥蜴（Anolis pukhelhis）和斑马鱼（Brachydanio rerio）的重组蛋白。斑马鱼FABP cDNA是Morales教授赠送的，从斑马鱼肝脏总RNA中制备了斑马鱼第一链cDNA文库。他们是李 ...更多信息 门控到表达载体中并转染E. coli BL21（DE3）。通过凝胶过滤和HPLC纯化表达的蛋白。混合二硫化物与谷胱甘肽和这些蛋白质的制备与二酰胺温育。将它们脱脂，并将脂质结合活性与未处理的蛋白质进行比较。脂质结合活性测定的荧光探针，丹磺酰氨基十一烷酸和苯胺萘磺酸的位移。天然荧光脂肪酸，顺式parinaric酸的直接结合，也进行了测量。所有这些FABPs强烈结合各种脂质，而不管结合的谷胱甘肽。这些结果表明，纯化和表达的FABP是天然的，用谷胱甘肽修饰不影响蛋白质的生物学功能，即，混合二硫化物的形成没有改变蛋白质的结构。然而，发现混合的二硫化物形式被各种蛋白酶（包括细胞内蛋白酶）快速消化，而不管半胱氨酸残基在整个线性氨基酸序列中的位置如何。这强烈表明，这些与谷胱甘肽的混合二硫化物形成是一个标记过程，导致氧化损伤蛋白质的蛋白水解降解。由于半胱氨酸残基的氧化还原电位受其在蛋白质环境中的位置的影响，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，秀丽隐杆线虫也有9个基因编码的FABPs有或没有半胱氨酸残基。这种动物可用于检查氧化应激的影响，因为在含有过氧化氢的培养基中培养蠕虫容易模拟氧化应激。我们首先制备了线虫FABP 1-9的9个重组蛋白，并建立了体外谷胱甘肽化的实验条件。首先在FABP-5上尝试与上述类似的谷胱甘肽化实验，再次观察到蛋白水解敏感性的显著增加。我们相信胞质二硫键的形成是一个迄今未知的蛋白质降解的标记事件。少

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