Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary.
Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, 4056, Basel, Switzerland.
BMC Biol. 2020 Feb 26;18(1):19. doi: 10.1186/s12915-020-0749-y.
The lumen of the endoplasmic reticulum (ER) acts as a cellular Ca store and a site for oxidative protein folding, which is controlled by the reduced glutathione (GSH) and glutathione-disulfide (GSSG) redox pair. Although depletion of luminal Ca from the ER provokes a rapid and reversible shift towards a more reducing poise in the ER, the underlying molecular basis remains unclear.
We found that Ca mobilization-dependent ER luminal reduction was sensitive to inhibition of GSH synthesis or dilution of cytosolic GSH by selective permeabilization of the plasma membrane. A glutathione-centered mechanism was further indicated by increased ER luminal glutathione levels in response to Ca efflux. Inducible reduction of the ER lumen by GSH flux was independent of the Ca-binding chaperone calreticulin, which has previously been implicated in this process. However, opening the translocon channel by puromycin or addition of cyclosporine A mimicked the GSH-related effect of Ca mobilization. While the action of puromycin was ascribable to Ca leakage from the ER, the mechanism of cyclosporine A-induced GSH flux was independent of calcineurin and cyclophilins A and B and remained unclear.
Our data strongly suggest that ER influx of cytosolic GSH, rather than inhibition of local oxidoreductases, is responsible for the reductive shift upon Ca mobilization. We postulate the existence of a Ca- and cyclosporine A-sensitive GSH transporter in the ER membrane. These findings have important implications for ER redox homeostasis under normal physiology and ER stress.
内质网（ER）的腔作为细胞内的钙库和氧化蛋白折叠的场所，这一过程由还原型谷胱甘肽（GSH）和谷胱甘肽二硫化物（GSSG）氧化还原对控制。尽管 ER 腔中的钙耗竭会引起 ER 向更还原的平衡快速且可逆地转变，但潜在的分子基础仍不清楚。
我们发现钙动员依赖性 ER 腔还原对 GSH 合成的抑制或通过质膜的选择性通透化对细胞质 GSH 的稀释敏感。谷胱甘肽中心机制进一步表明，钙外流会导致 ER 腔中谷胱甘肽水平升高。通过 GSH 流诱导的 ER 腔还原与以前涉及该过程的钙结合伴侣钙网蛋白无关。然而，通过嘌呤霉素打开易位通道或添加环孢菌素 A 模拟了钙动员的 GSH 相关效应。虽然嘌呤霉素的作用可归因于 ER 内钙的漏出，但环孢菌素 A 诱导的 GSH 流的机制与钙调神经磷酸酶和亲环素 A、B 无关，其机制仍不清楚。
我们的数据强烈表明，细胞质 GSH 向 ER 的内流，而不是局部氧化还原酶的抑制，是钙动员时发生还原转变的原因。我们假设 ER 膜上存在一种 Ca 和环孢菌素 A 敏感的 GSH 转运蛋白。这些发现对正常生理学和 ER 应激下 ER 氧化还原稳态具有重要意义。
