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

Prdx6在眼晶状体老化过程中的调控及功能

• 批准号: 8894009
• 负责人: DHIRENDRA P SINGH
• 金额: $ 36.87万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8894009
• 关键词: Age; Aging; Antioxidants; Applications Grants; Attenuated; Binding; Biological Assay; Cataract; Cell Aging; Cell Death; Cell Line; Cell Survival; Cells; Collaborations; Complementary DNA; Crystalline Lens; Cytoprotection; DNA-Binding Proteins; Data; Degenerative Disorder; Disease; Elderly; Enzyme-Linked Immunosorbent Assay; Enzymes; Epithelial Cells; Etiology; Event; Failure; Figs - dietary; Genes; Genetic Transcription; Goals; Health; Homeostasis; In Vitro; Injury; Lead; Letters; Link; Mediating; Modeling; Molecular; Monitor; Mus; Mutate; Outcome; Oxidation-Reduction; Oxidative Stress; Peptide Hydrolases; Physiological; Play; Post-Translational Protein Processing; Predisposition; Process; Proteins; Protocols documentation; Publishing; Rattus; Regulation; Repression; Resistance; Role; Signal Transduction; Site; Stress; Testing; Therapeutic; Tissues; Trans-Activators; Transactivation; Transfection; Translating; Work; age related; base; cell age; cell injury; cellular targeting; design; human subject; in vivo; lens; mRNA Expression; mutant; overexpression; particle; prevent; promoter; protein expression; research study; sumo1 gene

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.

描述（由申请人提供）：衰老过程中的环境压力和细胞防御能力下降是许多年龄相关疾病（包括白内障）发病机制的主要因素。由于局部抗氧化能力决定了细胞靶点对氧化应激的敏感性，因此确定保护性分子如过氧化物酶6（Prdx 6）对氧化应激的调节作用至关重要，反之亦然。利用Prdx 6基因的靶向失活，我们已经表明，老化的透镜上皮细胞（LEC）和Prdx 6缺陷的LEC或眼晶状体容易发生细胞死亡或白内障，这表明Prdx 6在透镜防御中起着关键作用。然而，Prdx 6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（蛋白质的SUMO化，从而调节蛋白质的表达、活性和稳定性）之间发现了非常有趣的联系。眼用透镜不断地暴露于环境应力。细胞内不断产生ROS，如果不清除，会导致细胞氧化还原状态失调。最初的研究表明，Prdx 6的表达和活性被Sumoylation去调控，导致Prdx 6的保护活性丧失。本提案的基本原理来自我们的初步研究，表明（a）Prdx 6是Sumo的目标（小泛素样修饰物）1，并且Prdx 6的Sumoylation降低其活性和细胞损伤，如通过Prdx 6缺陷细胞过度表达Sumo 1和Prdx 6面对氧化应激所证明的;（B）Sumo 1特异性蛋白酶（Senp）1在面临氧化应激的LEC和老化LEC中失活并且Sumo 1增加;（c）Prdx 6的激活剂Sp1的Sumo化减弱其激活潜力;（d）在Sumo 1位点处将蛋白转导结构域（达特）连接的Prdx 6 K122 R/K142 R突变体递送至LEC/晶状体提供增强的针对氧化应激的细胞保护。我们假设，在氧化应激/衰老过程中，异常的Sumoylation过程使Prdx 6功能失调，并且在Prdx 6及其反式激活因子Sp1的异常Sumoylation的情况下，Prdx 6功能出错，导致细胞功能障碍，进而可能导致白内障形成。本提案的总体目标是描述导致Prdx 6表达和活性降低的Sumoylation介导的异常信号传导，以及通过三个具体目标揭示由Prdx 6活性丧失引发的致病过程：（1）记录和检查氧化应激和衰老对Prdx 6 Sumoylation和对Prdx 6蛋白完整性和保护活性的影响;（2）确定Sp1的Sumoylation/deSumoylation对面临氧化应激或衰老细胞的细胞中Prdx 6基因转录的影响，以及作为衰老模型的Prdx 6-/-缺陷型晶状体;（3）评估TAT连接的Prdx 6在Sumo 1位点突变的潜力在体外保护面临氧化应激的细胞/晶状体。Shumiya白内障大鼠将用于测试TAT连锁的Prdx 6在预防/延迟白内障形成的进展中的作用。这些研究应该使我们能够设计新的治疗方法，以减弱在老年人和涉及生理和环境压力的情况下引起损伤的有害信号，并最终预防与氧化应激或衰老相关的疾病。