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Gene Regulation by and of LEDGF

LEDGF 的基因调控

基本信息

批准号：
8204533
负责人：
DHIRENDRA P SINGH
金额：
$ 35.28万
依托单位：
UNIVERSITY OF NEBRASKA MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-03-01 至 2013-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8204533
关键词：
Acquired Immunodeficiency Syndrome Affect Affinity Age Aging Amino Acids Antibodies Apoptosis Attenuated Autoimmune Process Binding Bioinformatics Biological Assay C-terminal Cataract Cell Death Cell Survival Cell physiology Cells Cellular Stress Chronic stress Collaborations Competence Complementary DNA Coupled Crystallins Cytoprotection DNA Binding DNA Binding Domain DNA-Protein Interaction Data Deletion Mutation Disease Drug Formulations Electrophoretic Mobility Shift Assay Elements Epithelial Cells Figs - dietary Funding Future Gene Expression Gene Expression Regulation Genes Genetic Transcription Goals Grant Growth Growth Factor Growth Factor Gene Heat Stress Disorders Heat shock proteins Heat-Shock Proteins 70 Heat-Shock Response Heat-Shock Transcription Factor 2 Heating Helix-Turn-Helix Motifs Homeostasis Impairment Investigation Letters Literature Malignant Neoplasms Maps Measures Mediating Methods Modification Molecular Molecular Structure Monitor Mutagenesis Mutate N-terminal Nature Oxidative Stress Peptide Hydrolases Phosphorylation Phosphorylation Site Physiological Physiological Processes Point Mutation Post-Translational Protein Processing Predisposition Principal Investigator Process Proteins Published Comment Publishing Qualifying RNA Reaction Reagent Reporting Research Research Personnel Resistance Resources Role Running Sampling Signal Transduction Site Site-Directed Mutagenesis Small Interfering RNA Stress Stretching Structure-Activity Relationship Suggestion System TNF gene Therapeutic Time Transactivation Transfection Ubiquitin Work acute stress age related cell type combat experience expression vector gel mobility shift assay gene interaction heat-shock factor 1 insight interest knock-down lens lens epithelium-derived growth factor mutant novel overexpression programs promoter protein function protein protein interaction research study response satisfaction stoichiometry stress protein transcription factor vector yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Environmental stress is a major cause of age related cataract (ARC). Acute or chronic stresses provoke multiple cellular reactions, including activation of the heat shock or stress protein response. Lens epithelial cell-derived growth factor (LEDGF) is a stress-inducible transcription factor having pro-survival, cytoprotective function. Overall, LEDGF achieves this function by transactivating the stress-associated genes such as heat shock protein genes, for which LEDGF binds to the heat shock element (HSE) and stress element (STRE) present in the promoter of these genes. The specific molecular mechanism(s) by which LEDGF exerts its cytoprotective action under cellular stress is poorly understood, but various possibilities may exist, such as protein-protein and protein-DNA interactions involving LEDGF, its post-translational modifications, and the level of LEDGF gene expression. Our preliminary data show that (a) LEDGF binds with trimeric HSF1 during stress that cooperatively transactivates the heat shock genes - an interaction that leads cytoprotection; (b) LEDGF and HSF1 interaction is essential for cell survival under stress, as Hsf1 depleted cells, despite displaying higher LEDGF expression, are more prone to stress-induced cell death than are wild type cells; and (c) SUMO-1 modification of LEDGF, a transcription factor, and sumoylation of transcriptional proteins leads to gene modulation; a mechanism involved in cellular signaling. These data suggest the existence of molecular interaction between LEDGF and HSF1. More specifically, how and when these two transcriptional proteins interact, how LEDGF expression is upregulated, and how its sumoylation affects transcription of heat shock genes remain to be understood. Therefore, we hypothesize that during stress and/or normal physiological conditions, LEDGF, a multidomain protein, exerts its cytoprotective role through multiple modes of action, viz. by DNA binding activity, protein modification by sumoylation and protein-protein interaction, and by its expression levels. The following specific aims will be pursued: 1) to map and determine the sites of SUMO-1 modification in LEDGF protein and to define its functional consequences for the DNA-binding and transactivation activity of LEDGF, 2) to define and characterize interacting domains of LEDGF and HSF1 and determine underlying mechanism(s) of their interaction during stress, 3) to demonstrate the effect of LEDGF and HSF1 interaction and/or their sumoylation in transactivation of heat shock genes, hsp27 or 1B-crystallin, and 4) to demonstrate that LEDGF and HSF1 interaction is necessary for stress-induced cellular cytoprotection. These studies will reveal new information concerning the molecular modes of LEDGF function under cellular stress in lens epithelial cells leading to future formulation of therapeutic strategy to combat age- related impairment of lens function, and other disorders in general such as Cancer and AIDS. Cloned LEDGF is a novel growth and survival factor, and acts as transcription factor. Despite the known regulatory function of LEDGF, only limited investigation has been made into the molecular structure-function relationship of LEDGF's domains. The research proposed in current grant will disclose the functionality of LEDGF domains; that will provide important insights into how LEDGF functions at the cellular and molecular levels, how it regulates cellular survival and other physiological processes, how these processes go awry in various disease states, and how the expression levels of LEDGF are regulated in cells facing stress. Thus accomplishing these goals in turn will provide opportunity to manipulate this molecule to cure and control various age associated diseases including cataract as well as other diseases, such as cancer, AIDS and autoimmune disoders, in general.

描述（由申请人提供）：环境压力是年龄相关性白内障（ARC）的主要原因。急性或慢性应激会引发多种细胞反应，包括热休克或应激蛋白反应的激活。透镜上皮细胞衍生生长因子（LEDGF）是一种应激诱导的转录因子，具有促生存、细胞保护等功能。总体而言，LEDGF通过反式激活应激相关基因如热休克蛋白基因来实现该功能，其中LEDGF结合存在于这些基因的启动子中的热休克元件（HSE）和应激元件（STRE）。LEDGF在细胞应激下发挥其细胞保护作用的具体分子机制知之甚少，但可能存在各种可能性，如涉及LEDGF的蛋白质-蛋白质和蛋白质-DNA相互作用、其翻译后修饰和LEDGF基因表达水平。我们的初步数据显示（a）LEDGF在应激期间与三聚体HSF 1结合，其协同反式激活热休克基因-导致细胞保护的相互作用;（B）LEDGF和HSF 1的相互作用对于应激下的细胞存活是必需的，因为Hsf 1耗尽的细胞尽管显示出更高的LEDGF表达，但比野生型细胞更倾向于应激诱导的细胞死亡;和（c）LEDGF（一种转录因子）的SUMO-1修饰和转录蛋白的SUMO化导致基因调节;一种涉及细胞信号传导的机制。这些数据表明LEDGF和HSF 1之间存在分子相互作用。更具体地说，这两种转录蛋白如何以及何时相互作用，LEDGF表达如何上调，以及其sumoylation如何影响热休克基因的转录仍有待了解。因此，我们假设在应激和/或正常生理条件下，LEDGF，一种多结构域蛋白，通过多种作用模式发挥其细胞保护作用，即通过DNA结合活性，通过类小泛素化和蛋白质-蛋白质相互作用的蛋白质修饰，以及通过其表达水平。将努力实现以下具体目标：1）定位和确定LEDGF蛋白中SUMO-1修饰的位点，并确定其对LEDGF的DNA结合和反式激活活性的功能后果，2）确定和表征LEDGF和HSF 1的相互作用结构域，并确定它们在应激期间相互作用的潜在机制，3）证明LEDGF和HSF 1相互作用和/或它们的类小泛素化在热休克基因hsp 27或1B-晶状体蛋白的反式激活中的作用，和4）证明LEDGF和HSF 1相互作用对于应激诱导的细胞保护是必需的。这些研究将揭示关于在透镜上皮细胞中在细胞应激下LEDGF功能的分子模式的新信息，从而导致未来制定治疗策略以对抗与年龄相关的透镜功能损伤以及一般的其它病症如癌症和AIDS。克隆的LEDGF是一种新的生长和存活因子，并作为转录因子。尽管已知LEDGF的调节功能，但仅对LEDGF结构域的分子结构-功能关系进行了有限的研究。目前的研究将揭示LEDGF结构域的功能;这将为LEDGF如何在细胞和分子水平上发挥作用提供重要的见解，它如何调节细胞存活和其他生理过程，这些过程如何在各种疾病状态下出错，以及如何在面临压力的细胞中调节LEDGF的表达水平。因此，实现这些目标反过来将提供机会来操纵这种分子以治疗和控制各种年龄相关疾病，包括白内障以及其他疾病，例如癌症、AIDS和自身免疫性疾病。

项目成果

期刊论文数量（22）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Peroxiredoxin 6 delivery attenuates TNF-alpha-and glutamate-induced retinal ganglion cell death by limiting ROS levels and maintaining Ca2+ homeostasis.

DOI：
10.1016/j.brainres.2008.07.076
发表时间：
2008-10-03
期刊：
Brain research
影响因子：
2.9
作者：
Fatma N;Kubo E;Sen M;Agarwal N;Thoreson WB;Camras CB;Singh DP
通讯作者：
Singh DP

Dynamic and differential regulation in the microRNA expression in the developing and mature cataractous rat lens.

DOI：
10.1111/jcmm.12094
发表时间：
2013-09
期刊：
Journal of cellular and molecular medicine
影响因子：
5.3
作者：
Kubo E;Hasanova N;Sasaki H;Singh DP
通讯作者：
Singh DP

Lens epithelium-derived growth factor deSumoylation by Sumo-specific protease-1 regulates its transcriptional activation of small heat shock protein and the cellular response.

DOI：
10.1111/j.1742-4658.2012.08686.x
发表时间：
2012-09
期刊：
The FEBS journal
影响因子：
0
作者：
Ishihara K;Fatma N;Bhargavan B;Chhunchha B;Kubo E;Dey S;Takamura Y;Kumar A;Singh DP
通讯作者：
Singh DP

Neuroprotective effect of peroxiredoxin 6 against hypoxia-induced retinal ganglion cell damage.