Abstract
Aberrant Sumoylation of protein(s) in response to oxidative stress or during aging is known to be involved in etiopathogenesis of many diseases. Upon oxidative stress, Peroxiredoxin (Prdx) 6 is aberrantly Sumoylated by Sumo1, resulting in loss of functions and cell death. We identified lysines (K) 122 and 142 as the major Sumo1 conjugation sites in Prdx6. Intriguingly, the mutant Prdx6 K122/142 R (arginine) gained protective efficacy, increasing in abundance and promoting glutathione (GSH) peroxidase and acidic calcium-independent phospholipase A2 (aiPLA2) activities. Using lens epithelial cells derived from targeted inactivation of Prdx6−/− gene and relative enzymatic and stability assays, we discovered dramatic increases in GSH-peroxidase (30%) and aiPLA2 (37%) activities and stability in the K122/142 R mutant, suggesting Sumo1 destabilized Prdx6 integrity. Prdx6−/−LECs with EGFP-Sumo1 transduced or co-expressed with mutant TAT-HA-Prdx6K122/142 R or pGFP-Prdx6K122/142 R were highly resistant to oxidative stress, demonstrating mutant protein escaped and interrupted the Prdx6 aberrant Sumoylation-mediated cell death pathway. Mutational analysis of functional sites showed that both peroxidase and PLA2 active sites were necessary for mutant Prdx6 function, and that Prdx6 phosphorylation (at T177 residue) was essential for optimum PLA2 activity. Our work reveals the involvement of oxidative stress-induced aberrant Sumoylation in dysregulation of Prdx6 function. Mutant Prdx6 at its Sumo1 sites escapes and abates this adverse process by maintaining its integrity and gaining function. We propose that the K122/142R mutant of Prdx6 in the form of a TAT-fusion protein may be an easily applicable intervention for pathobiology of cells related to aberrant Sumoylation signaling in aging or oxidative stress.
Highlights
Maintaining cellular integrity in the face of diverse causes and effects of oxidative stress is a challenge for cells
We propose that the K122/142R mutant of Prdx[6] in the form of a TAT-fusion protein may be an applicable intervention for pathobiology of cells related to aberrant Sumoylation signaling in aging or oxidative stress
We found that cellular abundance of mutants K122R or K142R or K122/142 R proteins significantly higher than GFP-Prdx6WT protein at 20 μg/ml and 40 μg/ml (Figure 6a), suggesting that it is likely that Sumoylation mediates Prdx[6] degradation
Summary
Maintaining cellular integrity in the face of diverse causes and effects of oxidative stress is a challenge for cells. Enhanced cytoprotection of mutant Prdx6K122/142R B Chhunchha et al been identified, including Prdx6.8,16 Sumoylation occurs predominantly at a core consensus motif in substrate proteins (Ψ-K-X-[D/E], where Ψ is any large hydrophobic residue (I, V or L), K is target lysine, X is any residue and D/E is aspartate or glutamate).[17] Recently, an extended consensus motif for Sumo binding was found[18,19] and was termed a nonconsensus motif for Sumo binding.[8] aberrant Sumoylation signaling has been shown to be a cause of initiation and progression of various diseases including cancer, heart failure, diabetes and pathogenic inflammations caused by infectious agents.[20,21] ROS can modulate the process of Sumoylation by affecting the activation of conjugation and deconjugation enzymes.[10,22] During oxidative stress, Sumoylation levels have been found to be altered in several proteins, such as HIPK2,23 TP53INP1,24 Prdx[6] and LEDGF.[8,25] the crosstalk between Sumoylation and other post-translational modifications including ubiquitination has been well documented.[26,27] Sumoylation and ubiquitination can act either cooperatively or independently and thereby determine the fate of proteins and the future of cell integrity.[28,29]. Discovery of a protective mutant of Prdx[6] should provide a foundation for useful strategies for configuring proteins to enhance their protective efficacy and stability
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