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S. Plafker, K. S. Plafker, L. Nguyen, M. Barneche, S. Mirza; Regulation of the Master Anti-Oxidant Transcription Factor, Nrf2, Through a Redox-Sensing Cysteine in the Ubiquitin Conjugating Enzyme, UbcM2. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3502.
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Age-related macular degeneration (AMD) is a leading cause of vision impairment and loss in the elderly. Clinical studies and animal models support that chronic oxidative stress, which arises from an imbalance in the production and elimination of reactive oxygen species (ROS), is a major etiological factor of AMD. Our long-term goal is to develop strategies that protect the retina from the damaging effects of ROS and AMD development. Our work focuses on the retinal pigment epithelium (RPE). This cell layer is highly susceptible to ROS damage and atrophy of the RPE has been implicated as a precipitating event for AMD. The RPE employs a host of defense mechanisms to counter chronic oxidative stress and the transcription factor, Nrf2, is a central coordinator of these defenses. Nrf2 induces the expression of enzymes that neutralize intracellular ROS. Yet, a complete molecular understanding of how Nrf2 stability and function are regulated in RPE cells remains to be elucidated.
A combination of recombinant pulldowns, co-immunoprecipitations, transcription assays, siRNA studies and mutant over-expression studies were used.
We have discovered a novel mechanism of Nrf2 regulation. We find that UbcM2, a ubiquitin conjugating enzyme present in RPE cells, stabilizes and induces the transcriptional activity of Nrf2 in a redox-dependent fashion. The mechanism underlying this effect resides in a unique cysteine residue of UbcM2. To mimic oxidation of this cysteine, we changed the cysteine to a phenylalanine. This mutant enzyme constitutively activates Nrf2 and is currently being explored for its capacity to amplify the endogenous anti-oxidant system in a mouse model of AMD. Complementary siRNA studies revealed that UbcM2 knockdown in RPE cells prevents Nrf2 stabilization in response to oxidative stress.
These findings support a model in which oxidation of a redox-sensing cysteine in UbcM2 results in the enhanced activity of Nrf2. 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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