May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
SUMO-1 Conjugation Motif of C-terminus LEDGF Is Essential for Its Interaction with HSF1 and Modulation of Small Heat Shock Protein Genes Transcription During Stress
Author Affiliations & Notes
  • D. P. Singh
    Ophthalmology and Visual Sciences, Univ of Nebraska Medical Ctr, Omaha, Nebraska
  • E. Kubo
    Ophthalmology, University of Fukui, Fukui, Japan
  • K. Ishihara
    Ophthalmology and Visual Sciences, Univ of Nebraska Medical Ctr, Omaha, Nebraska
  • Y. Takamura
    Ophthalmology and Visual Sciences, Univ of Nebraska Medical Ctr, Omaha, Nebraska
  • N. Fatma
    Ophthalmology and Visual Sciences, Univ of Nebraska Medical Ctr, Omaha, Nebraska
  • Footnotes
    Commercial Relationships  D.P. Singh, None; E. Kubo, None; K. Ishihara, None; Y. Takamura, None; N. Fatma, None.
  • Footnotes
    Support  NIH Grant EY13394, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2276. doi:https://doi.org/
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      D. P. Singh, E. Kubo, K. Ishihara, Y. Takamura, N. Fatma; SUMO-1 Conjugation Motif of C-terminus LEDGF Is Essential for Its Interaction with HSF1 and Modulation of Small Heat Shock Protein Genes Transcription During Stress. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2276. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : The stress inducible transcriptional protein LEDGF provides cytoprotection by activating small heat shock proteins (sHsp) gene transcription. The constitutive versus stress-inducible transcriptional activities of LEDGF can be regulated by its posttranslational modifications. Herein, we investigated that the SUMO (small ubiquitin-like modifier)-1 conjugation motif of C-terminus LEDGF is essential for its interaction with HSF1, and sumoylation, a dynamic and reversible process, is crucial in LEDGF-dependent transcription of sHsp.

Methods: : Web based analysis (Sumoplot) was used to spot out SUMO1 motif, and predefined deletion constructs of LEDGF linked to GST or GFP vectors were prepared. To ascertain Sumo-1 motif, site-directed-mutagenesis was done to create point mutation (lysine [K] to arginine [R]). Constructs of GFP or GST-SUMO1 and Flag-SENP1, a nuclear protease, were prepared for in vivo and in vitro sumoylation and desumoylation assays, respectively. Gel-mobility and transactivation experiments with Hsp27/αB-crystallin promoter monitored the effects of SUMO1 modification of LEDGF activity. Cellular extract isolated from heat stressed or unstressed hsf-/- and hsf+/+ lens epithelial cells (LECs) or cos-7 cells or hLECs were used for pull-down assay, immunoprecipitation, immunohistochemistry and Western analysis, using antibodies specific to SUMO1, LEDGF and HSF1.

Results: : Deletion derivative consisting of N-terminal 250 amino acids (aa) with five putative SUMO1 motifs were not sumoylated. LKID (lysine [K] 364), positioned at 364aa of the C-terminal LEDGF, was modified by SUMO1; conversely, mutation of K364 to R abolished LEDGF-SUMO1 conjugation. LEDGF desumoylation by SENP1 or mutation of SUMO1 motif significantly increased transactivation activity of LEDGF, and increased LEDGF-DNA binding activity. LEDGF and SUMO1 were present in nucleus and colocalized with HSF1 in nuclei during heat stress. Only wild type LEDGF, not its mutant form (K364R), interacted with HSF1. Transactivation assay using hsf1-/- LECs disclosed that interaction of LEDGF and HSF1 was essential for elevated activation of hsp27/αB-crystallin gene in cells facing stress.

Conclusions: : These findings provide novel insights into regulation and regulatory functions of LEDGF in sumoylation-dependent transcriptional control that may be essential for modifying the physiology of cells to maintain cellular homeostasis in cellular microenvironment.

Keywords: transcription factors • transcription • protein modifications-post translational 
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