The apparent deglycase activity of DJ-1 results from the conversion of free methylglyoxal present in fast equilibrium with hemithioacetals and hemiaminals
The apparent deglycase activity of DJ-1 results from the conversion of free methylglyoxal present in fast equilibrium with hemithioacetals and hemiaminals
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DOI：
10.1074/jbc.ra119.011237
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发表时间：
2019-12-06
影响因子：
4.8
通讯作者：
Utepbergenov, Darkhan
中科院分区：
文献类型：
--
作者：
Andreeva, Anna;Bekkhozhin, Zhanibek;Utepbergenov, Darkhan
编码人类蛋白DJ-1的基因的功能缺失突变导致帕金森病的早期发病，这表明DJ-1保护多巴胺能神经元。这种神经保护的分子机制尚不清楚；然而，DJ-1被认为是一种不依赖于谷胱甘肽的乙二醛酶，通过将甲基乙二醛（MGO）转化为乳酸来解毒。也有人认为，DJ-1作为脱糖酶，催化MGO与蛋白质的巯基和氨基反应形成的半硫缩醛和半氨基的水解。在本报告中，我们证明了MGO与硫醇反应的平衡常数为？500米(?1) 37岁？C，得到的半缩醛的半衰期只有12秒。这些热力学参数将决定一个显著部分的游离MGO将与溶液中的半缩醛快速平衡。我们发现，由于平衡位置的改变，DJ-1的乙草醛酶活性对游离MGO的去除迫使半缩醛立即自发分解。这种半缩醛的自发分解可能被误认为是DJ-1的脱糖苷酶活性。此外，我们证明了较高的初始半缩醛浓度与较低的DJ-1？介导的MGO转化，排除了半缩醛是DJ-1底物的可能性。CRISPR/ cas生成的DJ-1？敲除HEK293细胞表明，DJ-1不能防止细胞中急性MGO毒性或赖氨酸残基的羧甲基化。综上所述，我们的结果表明DJ-1不具有蛋白脱糖苷酶活性。
Loss-of-function mutations in the gene encoding human protein DJ-1 cause early onset of Parkinson's disease, suggesting that DJ-1 protects dopaminergic neurons. The molecular mechanisms underlying this neuroprotection are unclear; however, DJ-1 has been suggested to be a GSH-independent glyoxalase that detoxifies methylglyoxal (MGO) by converting it into lactate. It has also been suggested that DJ-1 serves as a deglycase that catalyzes hydrolysis of hemithioacetals and hemiaminals formed by reactions of MGO with the thiol and amino groups of proteins. In this report, we demonstrate that the equilibrium constant of reaction of MGO with thiols is ?500 m(?1) at 37 ?C and that the half-life of the resulting hemithioacetal is only 12 s. These thermodynamic parameters would dictate that a significant fraction of free MGO will be present in a fast equilibrium with hemithioacetals in solution. We found that removal of free MGO by DJ-1's glyoxalase activity forces immediate spontaneous decomposition of hemithioacetals due to the shift in equilibrium position. This spontaneous decomposition of hemithioacetals could be mistaken for deglycase activity of DJ-1. Furthermore, we demonstrate that higher initial concentrations of hemithioacetals are associated with lower rates of DJ-1?mediated conversion of MGO, ruling out the possibility that hemithioacetals are DJ-1 substrates. Experiments with CRISPR/Cas-generated DJ-1?knockout HEK293 cells revealed that DJ-1 does not protect against acute MGO toxicity or carboxymethylation of lysine residues in cells. Combined, our results suggest that DJ-1 does not possess protein deglycase activity.