Regulation and Function of Prdx6 in eye lens during Aging & Oxidative Stress

描述(由申请人提供)：环境压力和衰老过程中细胞防御能力的下降是许多年龄相关疾病的主要病因，包括白内障。由于局部抗氧化能力决定了细胞靶标对氧化应激的敏感性，因此确定过氧化还原蛋白6(Prdx6)等保护性分子的调节对氧化应激的影响将是至关重要的，反之亦然。利用Prdx6基因的靶向失活，我们发现老化的晶状体上皮细胞(LECs)和Prdx6缺陷的LECs或眼晶状体容易发生细胞死亡或白内障形成，表明Prdx6在晶状体防御中起着关键作用。然而，Prdx6活性降低的潜在机制尚不清楚。最近，在氧化应激水平和翻译后修饰，即蛋白质的苏莫化(S)之间发现了非常有趣的联系，从而调节了蛋白质的表达、活性和稳定性。人工晶状体经常暴露在环境压力下。ROS在细胞内不断产生，如果不被清除，会导致细胞氧化还原状态的放松调控。初步研究表明，Prdx6的表达和活性受到Sumoyl的破坏，导致Prdx6‘S保护活性的丧失。我们的初步研究表明：(A)Prdx6是SUMO(小泛素样修饰物)1的靶标，Prdx6的Sumoylating降低了其活性和细胞损伤，Prdx6缺失的细胞过度表达SUMO1和面临氧化应激的Prdx6；(B)在面临氧化应激和老化的LEC中，SUMO1特异的蛋白酶(Senp)1失活，SUMO1增加；(C)Prdx6的激活剂Sp1的Sumoyl降低了其激活潜力；(D)将SUMO1位点的蛋白转导结构域(TAT)连接的Prdx6K122R/K142R突变体运送到晶状体上皮细胞/晶状体可增强细胞对氧化应激的保护作用。我们假设，在氧化应激/衰老过程中，Prdx6功能的异常相加过程放松了对Prdx6功能的调节(S)，并且随着Prdx6及其反式激活因子Sp1的异常相加过程，Prdx6的功能发生了错误，导致细胞功能障碍，进而可能导致白内障的形成。本研究的总体目标是阐明Prdx6表达和活性降低的异常信号转导途径，并通过三个具体目标揭示Prdx6活性丧失所引发的致病过程：(1)记录和研究氧化应激和衰老对Prdx6总化和Prdx6蛋白完整性和保护活性的影响；(2)确定Sp1的总和/去总化对面临氧化应激或衰老的细胞和Prdx6/缺失的LECs中Prdx6基因转录的影响，作为衰老的模型；(3)评估SUMO1位点突变的TAT连锁Prdx6在体外保护面临氧化应激的细胞/晶状体的潜力。Shumiya白内障大鼠将被用来测试与TAT相关的Prdx6在预防/延缓白内障形成方面的作用。这些研究应该让我们能够设计新的疗法，以减弱在老年人和涉及生理和环境压力的情况下导致侮辱的有害信号，并最终总体上预防与氧化应激或衰老相关的疾病。

DESCRIPTION (provided by applicant): Environmental stress and a decline in cellular defense during aging are major factors in etiopathogenesis of many age-linked disorders including cataract. Since local antioxidant capacity determines the susceptibility of a cellular target to oxidative stress, it will be crucial to determine the impact of regulation of a protectiv molecule like Peroxiredoxin6 (Prdx6) on oxidative stress and vice versa. Using targeted inactivation of Prdx6 gene, we have shown that aging lens epithelial cells (LECs) and Prdx6-deficient LECs or eye lenses are vulnerable to cell death or cataractogenesis, suggesting that Prdx6 plays a pivotal role in lens defense. However, mechanisms underlying the reduced activity of Prdx6 are not known. Recently, very intriguing links have been found among oxidative stress levels and a posttranslational modification, Sumoylation of protein(s) and thereby the modulation of protein expression, activity and stability. Ocular lens is constantly exposed to environmental stress. ROS are constantly produced intracellularly, if not removed, leads to deregulation of cellular redox status. Initial studies revealed that Prdx6 expression and activity are deregulated by Sumoylation, leading to loss of Prdx6's protective activity. The rationale for the present proposal is derived from our initial studies showing that (a) Prdx6 is a target for Sumo (Small Ubiqitin-like Modifier)1, and Sumoylation of Prdx6 reduces its activity and cell injury, as evidenced by Prdx6-deficent cells overexpressing Sumo1 and Prdx6 facing oxidative stress; (b) Sumo1-specific protease (Senp)1 is inactivated and Sumo1 is increased in LECs facing oxidative stress and aging LECs; (c) Sumoylation of Sp1, activator of Prdx6, attenuates its activation potential; (d) Delivery of protein transduction domain (TAT)-linked Prdx6K122R/K142R mutant at Sumo1 sites to LECs/lenses provides enhanced cytoprotection against oxidative stress. We hypothesize that aberrant Sumoylation process deregulates Prdx6 function(s) during oxidative stress/aging, and that with aberrant Sumoylation of Prdx6 and its transactivator Sp1, Prdx6 functions go awry, leading to cell malfunction and, in turn, may lead to cataract formation. The overall goal of this proposal is to delineate Sumoylation-mediated-aberrant signaling that causes reduced expression and activity of Prdx6, as well as to uncover the pathogenic processes initiated by loss of Prdx6 activity through three specific aims: (1) Document and examine the effect of oxidative stress and aging on Prdx6 Sumoylation and on Prdx6 protein integrity and protective activity; (2) Determine the effect of Sumoylation/deSumoylation of Sp1 on Prdx6 gene transcription in cells facing oxidative stress or aging cell and Prdx6-/--deficient LECs as a model for aging; (3) Evaluate the potential of TAT-linked Prdx6 mutated at Sumo1 sites in protecting cells/lenses facing oxidative stress in vitro. The Shumiya cataract rat will be used to test TAT-linked Prdx6 in preventing/delaying the progression of cataract formation. The studies should allow us to design new therapies to attenuate the deleterious signaling that causes insults in the elderly and in situations involving physiological and environmental stress, and ultimately to prevent diseases associated with oxidative stress or aging, in general.