Characterization of a novel substrate of mammalian thioredoxin reductase 1

我们鉴定了一种含有半胱氨酸残基的大鼠脑蛋白，该蛋白对H2O2氧化敏感。该蛋白全长14kda，由123个氨基酸组成，包含一个Cys-X-X-Cys基序（Cys-Pro-Asp-Cys），该基序在包括硫氧还蛋白（Trx）在内的硫醇二硫氧化还原酶超家族成员中是保守的。由于新发现的14-KDa蛋白在大小上与Trx相似，因此将其命名为硫氧还蛋白相关蛋白（TRP14）。BLAST同源性搜索结果显示，TRP14同源物广泛分布于细菌、酵母、线虫、昆虫、植物和哺乳动物中，其TRP14同源物的Trp-Cys-Pro-Asp-Cys基序完全相同。免疫印迹分析表明，TRP14与Trx一样是一种普遍存在的蛋白。除肾外，TRP14在各组织中的表达水平远低于Trx，其细胞分布与Trx互补。TRP14 （-0.257 V）和Trx （-0.274 V）的还原电位相似。对两个保守的半胱氨酸残基（Cys43和Cys46）突变体的研究以及对含有cys的肽的直接分析表明，与Trx的情况一样，保守的半胱氨酸残基在过氧化氢氧化后形成分子内二硫键。生成的二硫化物可以被胞质Trx还原酶（TrxR1）还原，但不能被线粒体Trx还原酶（TrxR2）还原，而Trx是TrxR1和TrxR2同样好的底物。TrxR1的还原反应中，TRP14的底物性能略好于Trx (TRP14和Trx的Kms分别为8.3 uM和10.8 uM)，两者的vmax几乎相同（TRP14和Trx的vmax分别为32.4 uM/min和30.8 uM/min）。Trx作为供氢体，对核糖核苷酸还原酶、蛋氨酸亚砜还原酶和过氧化物还毒素具有催化作用，是一种一般的蛋白质二硫还原酶，可以降低胰岛素的二硫键，促进核糖核酸酶的重折叠。用纯化的重组TRP14测试了TRP14替代Trx支持这五种反应的能力。TRP14不能支持这五种反应中的任何一种。鉴于它们相似的还原电位，这种特异性是令人惊讶的。为了寻找依赖TRP14提供还原等量物的蛋白，我们将GST-TRP14融合蛋白固定在Sepharose凝胶上。TrxR1被发现与trp14结合的Sepharose柱特异性结合。我们的研究结果表明，TRP14是一种二硫氧化还原酶，能够通过TrxR1接收NADPH的电子。TRP14在还原级联中的下游靶点仍有待确定。

We identified a rat brain protein containing cysteine residues that are sensitive to the oxidation by H2O2. This 14-kDa protein, consisted of 123 amino acids, contains a Cys-X-X-Cys motif (Cys-Pro-Asp-Cys), which is conserved among the members of thiol-disulfide oxidoreductase superfamily that include thioredoxin (Trx). Because the newly discovered 14-KDa protein is similar to Trx in size, it was named thioredoxin-related protein (TRP14). The BLAST homology search showed that TRP14 homologues are distributed in wide range of organisms including bacteria, yeast, nematode, insect, plant and mammalian, and their Trp-Cys-Pro-Asp-Cys motifs are completely identical. Immunoblot analysis indicates that TRP14 is a ubiquitous protein like Trx. TRP14 was expressed in much lower level than Trx in every tissue except in kidney and its cell distribution was complimentary to that of Trx.The reduction potentials were similar for TRP14 (-0.257 V) and Trx (-0.274 V). Studies with the mutants of the two conserved cysteine residues (Cys43 and Cys46) and direct analysis of the Cys-containing peptides revealed that as in the case of Trx, the conserved cysteine residues form an intramolecular disulfide linkage upon oxidation by hydrogen peroxide. The resulting disulfide can be reduced by the cytosolic Trx reductase (TrxR1) but not by the mitochondrial Trx reductase (TrxR2), whereas Trx is an equally good substrate for TrxR1 and TrxR2. For the reduction reaction by TrxR1, TRP14 was slightly better substrate than Trx (the Kms for TRP14 and Trx were 8.3 uM and 10.8 uM, respectively) and their Vmaxs were nearly similar (the Vmaxs for TRP14 and Trx were 32.4 uM/min and 30.8 uM/min, respectively). Trx serves as a hydrogen donor for the catalytic function of ribonucleotide reductase, methionine sulfoxide reductase and peroxiredoxins, and as a general protein-disulfide reductase that reduce the disulfide bond of insulin and facilitates refolding of ribonuclease. The ability of TRP14 to support the five reactions in replacement of Trx was tested using purified recombinant TRP14. TRP14 could not support any of the five reactions. This specificity is surprising in view of their similar reduction potentials. As an effort to search for proteins that depend on TRP14 for the supply of reducing equivalents, GST-TRP14 fusion protein was immobilized to Sepharose gels. TrxR1 was found to bind specifically to the TRP14-bound Sepharose column. Our results suggest that TRP14 is a disulfide-oxidoreductase that is capable of receiving electrons from NADPH via TrxR1. The downstream target of TRP14 in the reduction cascade remains to be identified.