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Cycles of Cdc53/Cullin1 rubylation (a.k.a NEDDylation) protect ubiquitin-E3 SCF (Skp1-Cullin1-F-box protein) complexes from self-destruction and play an important role in mediating the ubiquitination of key protein substrates involved in cell cycle progression, development, and survival. Cul1 rubylation is balanced by the COP9 signalosome (CSN), a multi-subunit derubylase that shows 1:1 paralogy to the 26S proteasome lid. The turnover of SCF substrates and their relevance to various diseases is well studied, yet, the extent by which environmental perturbations influence Cul1 rubylation/derubylation cycles per se is still unclear. In this study, we show that the level of cellular oxidation serves as a molecular switch, determining Cullin1 rubylation/derubylation ratio. We describe a mutant of the proteasome lid subunit, Rpn11 that exhibits accumulated levels of Cullin1-Rub1 conjugates, a characteristic phenotype of csn mutants. By dissecting between distinct phenotypes of rpn11 mutants, proteasome and mitochondria dysfunction, we were able to recognize the high reactive oxygen species (ROS) production during the transition of cells into mitochondrial respiration, as a checkpoint of Cullin1 rubylation in a reversible manner. Thus, the study adds the rubylation cascade to the list of cellular pathways regulated by redox homeostasis.

Introduction: Fenugreek is known to possess anti-diabetic as well as antioxidant properties known to increase with germination. Previous study from our laboratory has reported vitexin and isovitexin as major antioxidant compounds from germinated fenugreek seeds. The present study aimed to comprehensively assesses and compare their antioxidant activity. Methods: The antioxidant ability of these compounds against various radicals was checked using standard in vitro radical scavenging assays and pulse radiolysis. Protection conferred to mitochondria against oxidative damage was assessed by measuring the levels of lipid peroxides and protein sulphydryls. Protection from hydrogen peroxide induced cytotoxicity in HepG2 cells was checked by MTT assay and by measuring intracellular ROS. Their ability to modulate intracellular antioxidant status was checked both at activity and RNA level. Result: Vitexin and isovitexin exhibited differential antioxidant activity against various radicals. Vitexin was a better nitric oxide scavenger while isovitexin scavenged superoxide radicals more efficiently. Pulse radiolysis studies revealed good antioxidant activity of both compounds against short-lived radicals and affected the formation and decay of the 2-dG transient species to the same extent. In oxidatively damaged mitochondria, lipid peroxidation was inhibited significantly by isovitexin whereas vitexin prevented decrease in protein sulphydryl content more effectively. These compounds protected HepG2 cells to the same extent against hydrogen peroxide induced oxidative insult by reducing intracellular ROS and modulating the levels of antioxidant enzymes. Conclusion: The present study clearly demonstrates differential antioxidant potential of both vitexin and isovitexin and their role in mitigating oxidative stress induced damage and maintaining cellular redox homeostasis.

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