Moreno Mari-Luz, Escobar Javier, Izquierdo-Álvarez Alicia, Gil Anabel, Pérez Salvador, Pereda Javier, Zapico Inés, Vento Máximo, Sabater Luis, Marina Anabel, Martínez-Ruiz Antonio, Sastre Juan
Department of Physiology, School of Pharmacy, University of Valencia, 46100 Burjasot (Valencia), Spain.
Department of Physiology, School of Pharmacy, University of Valencia, 46100 Burjasot (Valencia), Spain; Division of Neonatology, University Hospital Materno-Infantil La Fe, 46026 Valencia, Spain.
Free Radic Biol Med. 2014 May;70:265-77. doi: 10.1016/j.freeradbiomed.2014.01.009. Epub 2014 Jan 20.
Glutathione oxidation and protein glutathionylation are considered hallmarks of oxidative stress in cells because they reflect thiol redox status in proteins. Our aims were to analyze the redox status of thiols and to identify mixed disulfides and targets of redox signaling in pancreas in experimental acute pancreatitis as a model of acute inflammation associated with glutathione depletion. Glutathione depletion in pancreas in acute pancreatitis is not associated with any increase in oxidized glutathione levels or protein glutathionylation. Cystine and homocystine levels as well as protein cysteinylation and γ-glutamyl cysteinylation markedly rose in pancreas after induction of pancreatitis. Protein cysteinylation was undetectable in pancreas under basal conditions. Targets of disulfide stress were identified by Western blotting, diagonal electrophoresis, and proteomic methods. Cysteinylated albumin was detected. Redox-sensitive PP2A and tyrosine protein phosphatase activities diminished in pancreatitis and this loss was abrogated by N-acetylcysteine. According to our findings, disulfide stress may be considered a specific type of oxidative stress in acute inflammation associated with protein cysteinylation and γ-glutamylcysteinylation and oxidation of the pair cysteine/cystine, but without glutathione oxidation or changes in protein glutathionylation. Two types of targets of disulfide stress were identified: redox buffers, such as ribonuclease inhibitor or albumin, and redox-signaling thiols, which include thioredoxin 1, APE1/Ref1, Keap1, tyrosine and serine/threonine phosphatases, and protein disulfide isomerase. These targets exhibit great relevance in DNA repair, cell proliferation, apoptosis, endoplasmic reticulum stress, and inflammatory response. Disulfide stress would be a specific mechanism of redox signaling independent of glutathione redox status involved in inflammation.
谷胱甘肽氧化和蛋白质谷胱甘肽化被认为是细胞氧化应激的标志，因为它们反映了蛋白质中的硫醇氧化还原状态。我们的目的是分析硫醇的氧化还原状态，并确定实验性急性胰腺炎（作为与谷胱甘肽耗竭相关的急性炎症模型）胰腺中混合二硫键和氧化还原信号的靶点。急性胰腺炎时胰腺中的谷胱甘肽耗竭与氧化型谷胱甘肽水平或蛋白质谷胱甘肽化的任何增加均无关。胰腺炎诱导后，胰腺中的胱氨酸和同型胱氨酸水平以及蛋白质半胱氨酸化和γ-谷氨酰半胱氨酸化显著升高。在基础条件下，胰腺中未检测到蛋白质半胱氨酸化。通过蛋白质印迹、对角线电泳和蛋白质组学方法确定了二硫键应激的靶点。检测到半胱氨酸化白蛋白。氧化还原敏感的PP2A和酪氨酸蛋白磷酸酶活性在胰腺炎中降低，而N-乙酰半胱氨酸可消除这种降低。根据我们的研究结果，二硫键应激可被认为是与蛋白质半胱氨酸化、γ-谷氨酰半胱氨酸化以及半胱氨酸/胱氨酸对的氧化相关的急性炎症中一种特定类型的氧化应激，但不伴有谷胱甘肽氧化或蛋白质谷胱甘肽化的变化。确定了两种类型的二硫键应激靶点：氧化还原缓冲剂，如核糖核酸酶抑制剂或白蛋白，以及氧化还原信号硫醇，其中包括硫氧还蛋白1、APE1/Ref1、Keap1、酪氨酸和丝氨酸/苏氨酸磷酸酶以及蛋白质二硫键异构酶。这些靶点在DNA修复、细胞增殖、凋亡、内质网应激和炎症反应中具有重要意义。二硫键应激将是一种独立于参与炎症的谷胱甘肽氧化还原状态的氧化还原信号传导的特定机制。
