Vascular Biology Section, Boston University School of Medicine, Boston, MA, USA; Cardiology, Whitaker Cardiovascular Institute, And Boston University School of Medicine, Boston, MA, USA.
Vascular Biology Section, Boston University School of Medicine, Boston, MA, USA.
Free Radic Biol Med. 2021 Oct;174:73-83. doi: 10.1016/j.freeradbiomed.2021.07.037. Epub 2021 Jul 28.
S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway.
Exposure of HEK 293T cells to hydrogen peroxide (HO) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of HO. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism.
Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.
S-谷胱甘肽化是一种蛋白质半胱氨酸的可逆氧化修饰，在氧化还原信号中起着关键作用。谷胱甘肽还原酶 1（Glrx）是一种谷胱甘肽特异性硫转移酶，可去除蛋白质的 S-谷胱甘肽化。Glrx 虽然是一种细胞质蛋白，但可以通过去除其 S-谷胱甘肽化来激活核蛋白 Sirtuin-1（SirT1）。Glrx 缺失会导致代谢异常，并促进小鼠的受控细胞死亡和纤维化。甘油醛 3-磷酸脱氢酶（GAPDH）是糖酵解的关键酶，易受氧化修饰影响，并通过核内 SirT1/p53 途径参与细胞凋亡信号转导。我们旨在阐明 GAPDH 和谷胱甘肽还原酶 1 的 S-谷胱甘肽化在多大程度上促进 GAPDH/SirT1/p53 凋亡途径。
暴露于过氧化氢（HO）的 HEK 293T 细胞会迅速引起 GAPDH 的 S-谷胱甘肽化和核转位。核 GAPDH 在加入 HO 后 10-15 分钟达到峰值。Glrx 的过表达或氧化还原失活突变 GAPDH 抑制 S-谷胱甘肽化和核转位。核 GAPDH 与 SirT1 形成蛋白质复合物，并将 S-谷胱甘肽化交换给 SirT1 并抑制其脱乙酰酶活性。失活的 SirT1 仍与乙酰化 p53 稳定结合，并引发细胞凋亡信号转导，导致 caspase-3 的裂解。我们在人原代主动脉内皮细胞中观察到类似的效果，表明 GAPDH/SirT1/p53 途径是一种常见的凋亡机制。
富含半胱氨酸巯基的高反应性 GAPDH 可能作为一种细胞质变阻器来感知氧化应激。GAPDH 的 S-谷胱甘肽化可能将信号传递到细胞核，在细胞核中，GAPDH 将谷胱甘肽转移到核蛋白，如 SirT1，以启动细胞凋亡。Glrx 逆转 GAPDH 的 S-谷胱甘肽化并防止其核转位和细胞质-核氧化还原信号转导导致细胞凋亡。我们的数据表明，转谷胱甘肽化是细胞凋亡信号转导的关键步骤，也是细胞质 Glrx 控制核转录因子的潜在机制。
