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: 9310260
• 负责人: DHIRENDRA P SINGH
• 金额: $ 37.63万
• 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/9310260
• 关键词: Age; Aging; Antioxidants; Applications Grants; Attenuated; Binding; Biological; Biological Assay; Cataract; Cell Aging; Cell Death; Cell Line; Cell Survival; Cells; Collaborations; Complementary DNA; Crystalline Lens; Cytoprotection; DNA-Binding Proteins; Data; Degenerative Disorder; Dimerization; Disease; Elderly; Enzyme-Linked Immunosorbent Assay; Enzymes; Epithelial Cells; Etiology; Event; Failure; Genes; Genetic Transcription; Goals; Homeostasis; In Vitro; Injury; Lead; Letters; Link; Mediating; Modeling; Molecular; Monitor; Mus; Outcome; Oxidation-Reduction; Oxidative Stress; Pathogenicity; 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; ataxia telangiectasia mutated protein; base; biodegradable polymer; cell age; cell injury; cellular targeting; cellular transduction; design; dimer; experimental study; human subject; in vivo; lens; mRNA Expression; mutant; novel therapeutics; overexpression; particle; prevent; promoter; protein expression; public health relevance; 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）：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（包括白内障）发病的主要因素。由于局部抗氧化能力决定了细胞靶点对氧化应激的易感性，因此确定过氧化氧还蛋白6 （Prdx6）等保护性分子对氧化应激的调控影响将是至关重要的。通过靶向灭活Prdx6基因，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发生，这表明Prdx6在晶状体防御中起关键作用。然而，Prdx6活性降低的机制尚不清楚。最近，在氧化应激水平和翻译后修饰之间发现了非常有趣的联系，蛋白质的Sumoylation，从而调节蛋白质的表达，活性和稳定性。晶状体持续暴露在环境压力下。ROS在细胞内不断产生，如果不去除，会导致细胞氧化还原状态的失调。初步研究发现，Prdx6的表达和活性被Sumoylation解除调控，导致Prdx6的保护活性丧失。我们最初的研究表明(a) Prdx6是Sumo (Small ubiqin -like Modifier)1的靶标，Prdx6的summoylation降低了其活性和细胞损伤，这一点可以通过Prdx6缺陷细胞面临氧化应激时过度表达Sumo1和Prdx6得到证明；(b)氧化应激和衰老的LECs中Sumo1特异性蛋白酶（Senp）1失活，Sumo1表达升高；(c) Prdx6的激活剂Sp1的sumo化可减弱其激活电位；(d)在Sumo1位点将蛋白转导结构域（TAT）连接的Prdx6K122R/K142R突变体传递到LECs/晶状体可增强细胞对氧化应激的保护作用。我们推测，在氧化应激/衰老过程中，异常的summoylation过程会使Prdx6的功能失调，而Prdx6及其反激活子Sp1的异常summo酰化会使Prdx6的功能出错，导致细胞功能失调，进而可能导致白内障的形成。本研究的总体目标是通过三个具体目标来描述sumoyl化介导的异常信号导致Prdx6的表达和活性降低，并揭示Prdx6活性丧失引发的致病过程：(1)记录和研究氧化应激和衰老对Prdx6 sumoyl化以及Prdx6蛋白完整性和保护活性的影响；(2)确定Sp1 summoylation /deSumoylation对氧化应激细胞或衰老细胞Prdx6基因转录的影响，并将Prdx6-/-缺陷的LECs作为衰老模型；(3)评估在Sumo1位点突变的TAT-linked Prdx6在体外氧化应激下保护细胞/晶状体的潜力。Shumiya白内障大鼠将用于测试tat相关的Prdx6在预防/延缓白内障形成进程中的作用。这些研究应该允许我们设计新的治疗方法，以减弱在老年人和涉及生理和环境压力的情况下导致损害的有害信号，并最终预防与氧化应激或衰老相关的疾病。