PML‐Nuclear Bodies Regulate the Stability of the Fusion Protein Dendra2‐Nrf2 in the Nucleus of Single Live Cells

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Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a basic leucine‐zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1‐Cullin3/RING box 1 E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2‐Nrf2 in single live cells. Using this approach, we show that the half‐life (t 1/2 ) of Dendra2‐Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG‐132 or induction of oxidative stress with tert ‐butylhydroquinone ( t BHQ) extended the half‐life of Dendra2‐Nrf2 by 6‐ and 28‐fold, respectively. By inhibiting nuclear export using leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML‐NBs. We further demonstrate that co‐expression of Dendra2‐Nrf2 and Crimson‐PML‐I lacking two PML‐I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2‐Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Altogether, these data suggest that functional PML‐NBs alter the rate of degradation of nuclear Nrf2, which underscores the importance of these structures in Nrf2‐mediated response to oxidative stress. Support or Funding Information HHS |National Institutes of Health (NIH):, SC1CA143985; HHS |National Institutes of Health (NIH):, SC1CA211030; HHS |National Institutes of Health (NIH):, UL1TR000445; HHS |National Institutes of Health (NIH):, U54MD007593; HHS |National Institutes of Health (NIH):, G12MD007586; HHS |National Institutes of Health (NIH):, U54CA163069; HHS |National Institutes of Health (NIH):, R24DA036420; HHS |National Institutes of Health (NIH):, S10RR0254970; HHS |National Institutes of Health (NIH):, 5R25GM059994; HHS |National Institutes of Health (NIH):, 5T32GM007628‐37 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

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