Gene Regulation by and of LEDGF

LEDGF 的基因调控

• 批准号: 7735580
• 负责人: DHIRENDRA P SINGH
• 金额: $ 36.38万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2012-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7735580
• 关键词: Acquired Immunodeficiency Syndrome; Acute; Affect; Affinity; Age; 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; Critiques; Crystallins; Cytoprotection; DNA Binding; DNA Binding Domain; DNA-Protein Interaction; Data; Disease; Drug Formulations; Electrophoretic Mobility Shift Assay; Elements; Epithelial Cells; Evaluation; 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; Therapeutic; Time; Transactivation; Transfection; Ubiquitin; Work; age related; cell type; combat; experience; expression vector; gel mobility shift assay; gene interaction; heat-shock factor 1; innovation; 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

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(申请人提供)：环境压力是老年性白内障(ARC)的主要原因。急性或慢性应激可引起多种细胞反应，包括激活热休克或应激蛋白反应。晶状体上皮细胞衍生生长因子(LEDGF)是一种应激诱导的转录因子，具有促存活、细胞保护功能。总体而言，LEDGF通过反式激活与压力相关的基因(如热休克蛋白基因)来实现这一功能，对于这些基因，LEDGF与这些基因启动子中存在的热休克元件(HSE)和应激元件(StrE)结合。LEDGF在细胞压力下发挥细胞保护作用的具体分子机制(S)尚不清楚，但可能存在多种可能性，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，以及LEDGF基因表达水平。我们的初步数据显示：(A)LEDGF在应激过程中与三聚体HSF1结合，协同反式激活热休克基因--这种相互作用导致细胞保护；(B)LEDGF和HSF1的相互作用对于细胞在应激状态下的生存至关重要，因为HSF1缺失的细胞尽管显示出更高的LEDGF表达，但比野生型细胞更容易因应激诱导死亡；以及(C)转录因子LEDGF的SUMO-1修饰和转录蛋白的总和作用导致基因调制；这是细胞信号传递中的一个机制。这些数据表明LEDGF和HSF1之间存在分子相互作用。更具体地说，这两种转录蛋白是如何以及何时相互作用的，LEDGF的表达是如何上调的，以及它的总和如何影响热休克基因的转录，仍有待了解。因此，我们推测，在应激和/或正常生理条件下，LEDGF是一种多结构域蛋白，通过多种作用方式发挥其细胞保护作用。通过DNA结合活性、通过苏莫化和蛋白质-蛋白质相互作用进行的蛋白质修饰，以及其表达水平。本研究的具体目标如下：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影响；2)定义和表征LEDGF和HSF1的相互作用结构域，并确定它们在应激过程中相互作用的潜在机制(S)；3)证明LEDGF和HSF1相互作用和/或它们的总和作用在热休克基因Hsp27或1B晶体蛋白的反式激活中的作用；以及4)证明LEDGF和HSF1相互作用对于应激诱导的细胞保护是必要的。这些研究将揭示有关晶状体上皮细胞在细胞应激下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晶状体功能损害，以及其他一般疾病，如癌症和艾滋病。克隆的LEDGF是一种新的生长和生存因子，具有转录因子的作用。尽管已知LEDGF的调控功能，但对LEDGF结构域的分子结构-功能关系的研究还很有限。目前GRANT中提出的研究将揭示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，如何调控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LEDGF的表达水平如何在面临应激的细胞中进行调控提供重要的见解。因此，实现这些目标将提供机会来操纵这种分子来治疗和控制各种与年龄相关的疾病，包括白内障以及其他疾病，如癌症、艾滋病和自身免疫性疾病。