Abstract
Purpose: :
LEDGF plays a role in regulating the transcription of heat shock genes and in doing so helps to provide cytoprotection. The stress–inducible versus constitutive activities of LEDGF are critical for expression of heat shock genes. Herein, we investigated the influence of posttranslational modification, SUMO (small ubiquitin–like modifier)–1 conjugation of LEDGF, and its interaction to HSF1 in activating heat shock gene expression and cellular survival.
Methods: :
Based on bioinformatics analysis (Sumoplot) of Sumo–1 motif of LEDGF, deletion constructs of LEDGF linked to GST/GFP vectors were prepared. Point mutation (lysine (K) to arginine (R)) was performed to ascertain Sumo–1 motif. Sumoylation assays were conducted to demonstrate the SUMO–1 conjugation of LEDGF. EMSA and transactivation experiment with Hsp27/αB–crystallin promoter were done to monitor the effect of SUMO–1 modification of LEDGF to its DNA–binding and transactivation activities. GFP/GST–SUMO–1 proteins were expressed to accomplish the experiments. Cell–extract isolated from heat treated hsf–/– and hsf+/+ cells or cos–7 cell were used for Pull–down assay and Western analysis with specific antibodies
Results: :
Inspection of the sequence of LEDGF revealed the presence of a sumoylation motif LKID, around lysine 364 (K364) near the C–terminal helix–turn–helix domain. Sumoylation reaction as well as DNA transfection experiments revealed that the SUMO–1 conjugation occurs at lysine 364(K364) of LEDGF. LEDGF sumoylation enhances the transactivation capability of LEDGF, and this is accompanied by an increase of LEDGF DNA binding activity. Mutation of K364 to R364 reduces its DNA binding activity and transactivation potential. Transfection assay revealed that only wild type LEDGF, but not its mutant form (K364R) could efficiently enhance Hsp27 and αB–crystallin genes transcription and rescue the lens epithelial cells from heat stress. Pull down and transactivation experiments using heat shock factor (HSF1) depleted cells disclosed the interaction of LEDGF and HSF1, is essential for rapid activation of heat shock genes for cellular protection against stress.
Conclusions: :
Our data together point a new information concerning the molecular mechanism of LEDGF’s regulatory function in cells and cells facing stress, and may provide a strategy for manipulation of cellular heat shock protein expression, vitally important proteins in maintaining lens homeostasis.
Keywords: stress response • gene/expression • transcription