Chhunchha Bhavana, Kubo Eri, Fatma Nigar, Singh Dhirendra P
Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE, USA.
Department of Ophthalmology, Kanazawa Medical University, Kanazawa, Ishikawa, Japan.
Cell Death Dis. 2017 Jan 5;8(1):e2525. doi: 10.1038/cddis.2016.424.
Aberrant Sumoylation of protein(s) in response to oxidative stress or during aging is known to be involved in etiopathogenesis of many diseases. Upon oxidative stress, Peroxiredoxin (Prdx) 6 is aberrantly Sumoylated by Sumo1, resulting in loss of functions and cell death. We identified lysines (K) 122 and 142 as the major Sumo1 conjugation sites in Prdx6. Intriguingly, the mutant Prdx6 K122/142 R (arginine) gained protective efficacy, increasing in abundance and promoting glutathione (GSH) peroxidase and acidic calcium-independent phospholipase A (aiPLA) activities. Using lens epithelial cells derived from targeted inactivation of Prdx6 gene and relative enzymatic and stability assays, we discovered dramatic increases in GSH-peroxidase (30%) and aiPLA (37%) activities and stability in the K122/142 R mutant, suggesting Sumo1 destabilized Prdx6 integrity. Prdx6LECs with EGFP-Sumo1 transduced or co-expressed with mutant TAT-HA-Prdx6K122/142 R or pGFP-Prdx6K122/142 R were highly resistant to oxidative stress, demonstrating mutant protein escaped and interrupted the Prdx6 aberrant Sumoylation-mediated cell death pathway. Mutational analysis of functional sites showed that both peroxidase and PLA active sites were necessary for mutant Prdx6 function, and that Prdx6 phosphorylation (at T177 residue) was essential for optimum PLA activity. Our work reveals the involvement of oxidative stress-induced aberrant Sumoylation in dysregulation of Prdx6 function. Mutant Prdx6 at its Sumo1 sites escapes and abates this adverse process by maintaining its integrity and gaining function. We propose that the K122/142R mutant of Prdx6 in the form of a TAT-fusion protein may be an easily applicable intervention for pathobiology of cells related to aberrant Sumoylation signaling in aging or oxidative stress.
已知蛋白质在氧化应激反应或衰老过程中发生异常的类泛素化修饰（Sumoylation）与多种疾病的发病机制有关。在氧化应激状态下，过氧化物酶体增殖物激活受体γ辅激活因子6（Peroxiredoxin，Prdx6）会被类泛素1（Sumo1）异常修饰，导致其功能丧失和细胞死亡。我们确定赖氨酸（K）122和142是Prdx6中主要的Sumo1结合位点。有趣的是，突变型Prdx6 K122/142R（精氨酸）具有保护作用，其丰度增加，并促进谷胱甘肽（GSH）过氧化物酶和酸性非钙依赖性磷脂酶A（aiPLA）的活性。利用源自Prdx6基因靶向失活的晶状体上皮细胞以及相关的酶活性和稳定性测定，我们发现K122/142R突变体中GSH过氧化物酶活性（提高30%）和aiPLA活性（提高37%）以及稳定性都有显著增加，这表明Sumo1破坏了Prdx6的完整性。转导了EGFP-Sumo1或与突变型TAT-HA-Prdx6K122/142R或pGFP-Prdx6K122/142R共表达的Prdx6晶状体上皮细胞对氧化应激具有高度抗性，这表明突变蛋白逃脱并中断了Prdx6异常类泛素化修饰介导的细胞死亡途径。功能位点的突变分析表明，过氧化物酶和PLA活性位点对于突变型Prdx6的功能都是必需的，并且Prdx6磷酸化（在T177残基处）对于最佳PLA活性至关重要。我们的研究揭示了氧化应激诱导的异常类泛素化修饰参与了Prdx6功能的失调。位于Sumo1位点的突变型Prdx6通过维持其完整性并获得功能，逃脱并减轻了这一不利过程。我们提出，以TAT融合蛋白形式存在的Prdx6 K122/142R突变体可能是一种易于应用的干预手段，用于治疗与衰老或氧化应激中异常类泛素化信号相关的细胞病理生物学。
