Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia.
Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Leninsky prosp. 33-2, Moscow 119071, Russia.
Biochim Biophys Acta Gen Subj. 2020 Jun;1864(6):129560. doi: 10.1016/j.bbagen.2020.129560. Epub 2020 Feb 14.
We previously showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is S-glutathionylated in the presence of HO and GSH. S-glutathionylation was shown to result in the formation of a disulfide bridge in the active site of the protein. In the present work, the possible biological significance of the disulfide bridge was investigated.
Human recombinant GAPDH with the mutation C156S (hGAPDH_C156S) was obtained to prevent the formation of the disulfide bridge. Properties of S-glutathionylated hGAPDH_C156S were studied in comparison with those of the wild-type protein hGAPDH.
S-glutathionylation of hGAPDH and hGAPDH_C156S results in the reversible inactivation of the proteins. In both cases, the modification results in corresponding mixed disulfides between the catalytic Cys152 and GSH. In the case of hGAPDH, the mixed disulfide breaks down yielding Cys152-Cys156 disulfide bridge in the active site. In hGAPDH_C156S, the mixed disulfide is stable. Differential scanning calorimetry method showed that S-glutathionylation leads to destabilization of hGAPDH molecule, but does not affect significantly hGAPDH_C156S. Reactivation of S-glutathionylated hGAPDH in the presence of GSH and glutaredoxin 1 is approximately two-fold more efficient compared to that of hGAPDH_C156S.
S-glutathionylation induces the formation of Cys152-Cys156 disulfide bond in the active site of hGAPDH, which results in structural changes of the protein molecule. Cys156 is important for reactivation of S-glutathionylated GAPDH by glutaredoxin 1.
The described mechanism may be important for interaction between GAPDH and other proteins and ligands, involved in cell signaling.
我们之前曾表明，在 HO 和 GSH 的存在下，甘油醛-3-磷酸脱氢酶 (GAPDH) 发生 S-谷胱甘肽化。S-谷胱甘肽化导致蛋白质活性位点形成二硫键。在本工作中，研究了二硫键形成的可能生物学意义。
获得了突变 C156S 的人重组 GAPDH (hGAPDH_C156S)，以防止二硫键的形成。研究了 S-谷胱甘肽化的 hGAPDH_C156S 的性质，并与野生型蛋白 hGAPDH 进行了比较。
hGAPDH 和 hGAPDH_C156S 的 S-谷胱甘肽化导致蛋白质可逆失活。在两种情况下，修饰导致催化半胱氨酸 152 和 GSH 之间形成相应的混合二硫键。对于 hGAPDH，混合二硫键分解，在活性位点生成半胱氨酸 152-半胱氨酸 156 二硫键。在 hGAPDH_C156S 中，混合二硫键稳定。差示扫描量热法表明，S-谷胱甘肽化导致 hGAPDH 分子的不稳定，但对 hGAPDH_C156S 没有显著影响。与 hGAPDH_C156S 相比，在 GSH 和谷胱甘肽还原酶 1 的存在下，S-谷胱甘肽化的 hGAPDH 的再激活效率约提高两倍。
S-谷胱甘肽化诱导 hGAPDH 活性位点中半胱氨酸 152-半胱氨酸 156 二硫键的形成，导致蛋白质分子结构发生变化。半胱氨酸 156 对于谷胱甘肽还原酶 1 对 S-谷胱甘肽化 GAPDH 的再激活很重要。
所描述的机制可能对 GAPDH 与其他参与细胞信号转导的蛋白质和配体之间的相互作用很重要。
