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D. P. Singh, E. Kubo, K. Ishihara, Y. Takamura, N. Fatma; SUMO-Specific Protease SENP1 Regulates LEDGF-Dependent Transcription of Small Heat Shock Proteins. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4345.
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© ARVO (1962-2015); The Authors (2016-present)
Lens epithelium derived-growth factor (LEDGF) is a transcriptional protein that plays role in regulating cell survival and apoptosis by regulating small heat shock proteins (sHsp) gene transcription. We have reported earlier that LEDGF is substrate for Small Ubiquitin-like MOdifier protein (SUMO)1. Herein, we investigated that LEDGF-mediated sHsps transcription is markedly enhanced by SENP1 and the LEDGF-dependent transcription and enhanced cell survival is mediated through its desumoylation and increased expression.
Sumoplot was used to spot out SUMO1 motif, and deletion mutants of LEDGF linked to GST or GFP vectors were prepared. Site-directed-mutagenesis was done to create point mutation (lysine [K] to arginine [R]) in LEDGF. Constructs of GFP or GST-SUMO1 and Flag-SENP1, were prepared for in vivo and in vitro sumoylation and desumoylation assays, respectively. Gel-mobility and transactivation experiments with Hsp27/B-crystallin promoter and LEDGF promoters monitored the effects of SENP1 on LEDGF activity. LEDGF SiRNA was used to specify the results. Real time PCR, Western blot and immunocytochemistry were done to monitor mRNA, protein expression and localization of SENP1 in LECs facing heat/oxidative stress and MTS assay for cell survival.
Deletion mutants with point mutation disclosed that LKID positioned at 364aa of the LEDGF was SUMO1 modification site. SENP1enhances markedly the LEDGF-dependent activation of sHsp gene transcription in dose dependent fashion and SENP1-induced LEDGF transactivational activity occurred dependently of LEDGF desumoylation, and this is accompanied by an increase of LEDGF DNA binding activity and cell survival. LEDGF’s transactivation effect on Hsps could be reversed by RNA interference depletion of LEDGF validated specificity. Cells over-expressed with LEDGF-K364R, mutation of SUMO1 site showed increased cellular survival. While LECs with GFP-SUMO1, showed reduced Hsps transcriptional activity with decrease cell survival. LEDGF, SUMO1 and SENP1 were colocalized in nuclei of cells. Interestingly, LECs bearing SENP1 displayed increased endogenous LEDGF.
Together these findings point to a central role of SENP1 in the regulation and regulatory functions of LEDGF in sumoylation/ desumoylation-dependent transcriptional control of sHsps, and may be essential for tuning lens cell physiology in maintaining eye lens homeostasis.
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