April 2011
Volume 52, Issue 14
Free
ARVO Annual Meeting Abstract  |   April 2011
Characterization Of A Protective Ubiquitin Conjugating Enzyme In The Retina
Author Affiliations & Notes
  • Scott Plafker
    Cell Biology, Univ of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
  • Linda Nguyen
    Cell Biology, Univ of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
  • Kendra Plafker
    Cell Biology, Univ of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
  • Saima Mirza
    Cell Biology, Univ of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
  • Footnotes
    Commercial Relationships  Scott Plafker, None; Linda Nguyen, None; Kendra Plafker, None; Saima Mirza, None
  • Footnotes
    Support  NIH/NCRR grant P20RR024215; AHAF grant M2009095; OCASY grant HR08-076; Karl Kirchgessner Foundation for Vision Research Grant
Investigative Ophthalmology & Visual Science April 2011, Vol.52, 2318. doi:
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      Scott Plafker, Linda Nguyen, Kendra Plafker, Saima Mirza; Characterization Of A Protective Ubiquitin Conjugating Enzyme In The Retina. Invest. Ophthalmol. Vis. Sci. 2011;52(14):2318.

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Abstract

Purpose: : Chronic oxidative stress is a major pathogenic factor of age-related macular degeneration (AMD). Our goal is to develop strategies that protect the retina from the damaging effects of reactive oxygen species (ROS). Toward this end, we are identifying and characterizing proteins in the retina that confer protection against ROS-mediated insults. The current project focuses on a ubiquitin (Ub) conjugating enzyme with a putative role in protecting the retina from the accumulation of damaged proteins. This enzyme, UbcM2, is a functional homolog of a pair of yeast enzymes that mediate the degradation of oxidatively-damaged proteins.

Methods: : Western blotting, immunohistochemistry, and recombinant ubiquitylation assays were used.

Results: : We have discovered by western blotting that UbcM2 is enriched in the retina. Complementary immunohistochemical analyses revealed nuclear UbcM2 expression in the inner nuclear layer, ganglion cells, outer nuclear layer, and the retinal pigment epithelial (RPE). Recombinant ubiquitylation assays demonstrated that UbcM2 preferentially synthesizes lysine (K)63-linked polyUb chains. This configuration of polyUb can target proteins for proteasomal degradation and protein aggregates and dysfunctional organelles for autophagy-mediated degradation. We have also shown that the capacity of UbcM2 to synthesize these chains is disrupted by mutation of six non-adjacent residues. This dominant-negative enzyme provides a unique opportunity to dissect the consequences of disrupting UbcM2 function on retinal susceptibility to oxidative stress. Complementary studies to generate and study an RPE-specific UbcM2 knockout mouse are under way.

Conclusions: : These findings support a model in which UbcM2 protects susceptible cells of the retina from the accumulation of oxidatively-damaged proteins and organelles. It does so by tagging these toxic species with K63-linked polyUb chains and thus mediating their proteasomal and/or autophagic destruction. We anticipate that this work will ultimately provide a rationale for the development of therapeutics that specifically enhance UbcM2 activity to prevent AMD initiation and/or progression.

Keywords: age-related macular degeneration • oxidation/oxidative or free radical damage • retinal degenerations: cell biology 
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