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S. Plafker, K. Plafker, S. Mirza, M. Barneche; The Ubiquitin Conjugating Enzyme, UbcM2, Is a Novel Regulator of the Anti-Oxidant Transcription Factor, Nrf2, in RPE Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4186.
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Oxidative stress is widely held to be a root cause of age-related macular degeneration (AMD). Compelling experimental evidence in support of this comes from recent work by Hollyfield and colleagues showing that major hallmarks of dry AMD can be reconstituted in mice immunized with carboxyethylpyrrole (CEP)-derivatized albumin. CEP is a unique oxidation product of docosahexaenoic acid and it is adducted to proteins in drusen deposits and in the plasma of AMD patients. Corroborating clinical evidence comes from the Age-Related Eye Disease Study in which high doses of anti-oxidants and zinc slowed wet AMD progression and vision loss. Retinal pigment epithelial (RPE) cells are particularly sensitive to oxidative damage. They utilize a host of defense mechanisms to counter the harmful effects of reactive oxygen species (ROS) and the transcription factor, Nrf2, is a central coordinator of these defenses. Nrf2 induces the expression of enzymes that re-establish redox homeostasis by neutralizing intracellular ROS. Yet, we still do not understand the molecular mechanisms by which Nrf2 stability and function are regulated in RPE cells.
A combination of recombinant pulldowns, siRNA studies and transient transfection over-expression studies were employed.
We have discovered a novel mechanism of Nrf2 regulation. We find that UbcM2, a ubiquitin conjugating enzyme present in RPE cells, binds to and stabilizes Nrf2 in a redox-dependent fashion. In vitro, recombinant UbcM2 and Nrf2 bind directly to each other and this interaction is induced by alkylation of UbcM2 on a unique cysteine residue. In vivo, UbcM2 knockdown in human RPE cells prevents Nrf2 stabilization in response to oxidative stress. Conversely, UbcM2 over-expression stabilizes Nrf2.
These findings support a model in which UbcM2 binds to and protects Nrf2 from degradation in response to an oxidative insult. This work represents a new paradigm by which components of the ubiquitin proteolytic system protect vulnerable cells of the retina from the deleterious effects of oxidative stress. We anticipate that this avenue of investigation will ultimately provide a molecular rationale for the development of UbcM2-based ocular gene therapy for the treatment of AMD.
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