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. ? ? ?
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