THIOREDOXIN PEROXIDASE AS A MODULATOR OF TUMOR NECROSIS FACTOR-A SIGNALING PATHW

硫氧还蛋白过氧化物酶（TPx）是过氧化物酶家族中的一种新型过氧化物酶。Tpx含有两个直接参与催化循环的必需半胱氨酸残基。Tpx以头尾二聚体形式存在，其催化功能需要来自两个亚基的半胱氨酸残基。为了研究Tpx的细胞功能，我们通过将两个保守的半胱氨酸残基突变为丝氨酸残基来制备Tpx的显性负突变形式。如预期的，突变体多肽与野生型形成二聚体，使野生型的活性丧失。野生型突变型TPx在NIH 3 T3细胞中的过表达部分地减少了响应于TNF α产生的H2 O2的瞬时增加，而突变型TPx的过表达增强了H2 O2响应，表明TPx直接参与了细胞内H2 O2水平的调节。然后，我们研究了它们的表达对TNF α诱导的NF-κ B和MAP激酶的活化以及HeLa细胞凋亡的影响。野生型的过表达导致IkappaB的磷酸化和随后NF κ B的转录活性的抑制，而突变体表达具有相反的效果。在表达突变体的细胞中，由TNF α和环己酰胺处理引发的细胞凋亡显著加速，而野生型酶则抑制细胞凋亡。此外，TPx调节FLICE的激活，FLICE是TNFa诱导的细胞凋亡中最上游的半胱天冬酶。野生型Tpx的表达不影响ERK活性，而显性突变表达抑制ERK激活。这些结果表明，TPx可能作为第二信使H2 O2的生理调节剂产生的响应TNFalpah和不同的信号级联表现出不同的敏感性细胞内H2 O2。- 硫氧还蛋白过氧化物酶，过氧化物氧还蛋白，过氧化氢，TNF-α，NF-κ B，细胞凋亡

Thioredoxin peroxidase(TPx) is a new type of peroxidase that belongs to peroxiredoxin family. Tpx contains two essential cysteine residues that directly participate in catalytic cycle. Tpx exists as a head-tail dimer and its catalytic function requires cysteine residues from both subunits. To study cellular function of Tpx, we prepared a dominant negative mutant form of Tpx by mutating the two conserved cysteine residues to serine residues. As expected, the mutant polypeptide formed a dimer with wild type, incapacitating the activity of wild type. Overexpression of wild-type mutant TPx in NIH3T3 cells partially reduced the transient increase of H2O2 generated in response to TNFalpha, whereas overexpression of mutant TPx potentiated the H2O2 response, indicating that TPx is directly involved in regulation of the intracellular level of H2O2. We then investigated the effect of their expression on TNFalpha-induced activation of NF-kB and MAP kinases and apoptosis of HeLa cells. Overexpression of wild type resulted in a suppression of the phosphorylation of IkappaB and subsequently the transcriptional activity of NFkappaB, whereas mutant expression had opposite effects. The apoptosis, triggered by TNFalpha and cyclohexamide treatment, was dramatically accelerated in cells expressing the mutant, whereas it was inhibited by wild-type enzyme. Furthermore, TPx modulated the activation of FLICE, the most upstream caspase in TNFalpha-induced apoptosis. Expression of wild-type Tpx did not affect ERK activity, whereas dominant mutant expression inhibited ERK activation. These results suggest that TPx may function as a physiological modulator of second messenger H2O2 produced in response to TNFalpah and that different signaling cascades exhibit different sensitivity to intracellular H2O2. - thioredoxin peroxidase, peroxiredoxin, hydrogen peroxide, TNF-alpha, NF-kappaB, apoptosis