Cycles of Cdc53/Cullinl rubylation (a.k.a NEDDylation) protect ubiquitin-E3 SCF (Skpl-Cullinl-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. Cull 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 Cull 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 Cullinl rubylation/ derubylation ratio. We describe a mutant of the proteasome lid subunit, Rpnll that exhibits accumulated levels of Cullinl-Rubl conjugates, a characteristic phenotype of can mutants. By dissecting between distinct phenotypes of 7711 1 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 Cullinl rubylation in a reversible manner. Thus, the study adds the rubylation cascade to the list of cellular pathways regulated by redox homeostasis.

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.

Troxerutin, a flavonoid best known for its radioprotective and antioxidant properties is of considerable interest of study due to its broad pharmacological activities. The present study on troxerutin highlights its abilities to bind DNA and enhance cancer cell killing in response to radiation. Troxerutin showed strong binding with calf thymus DNA in vitro. Troxerutin-DNA interaction was confirmed by CD spectropolarimetry. The mode of binding of troxerutin to DNA was assessed by competing troxerutin with EtBr or DAPI, known DNA intercalator and a minor groove binder, respectively. DAPI fluorescence was drastically reduced with linear increase in troxerutin concentration suggesting possible binding of troxerutin to DNA minor groove. Further, computational studies of docking of troxerutin molecule on mammalian DNA also indicated possible troxerutin-DNA interaction at minor groove of DNA. Troxerutin was found to mainly localize in the nucleus of prostate cancer cells. It induced cytotoxicity in radioresistant (DU145) and sensitive (PC3) prostate cancer cells. When troxerutin pre-treated DU145 and PC3 cells were exposed to γ-radiation, cytotoxicity as estimated by MTT assay, was found to be further enhanced. In addition, the % subG1 population detected by propidium iodide staining also showed similar response when combined with radiation. A similar trend was observed in terms of ROS generation and DNA damage in DU145 cells when troxerutin and radiation were combined. DNA binding at minor groove by troxerutin may have contributed to strand breaks leading to increased radiation induced cell death.

Background: Free radicals are known to cause severe damage to most of the biomolecules in the human system and are responsible for various illnesses including neurodegenerative, cardiovascular and autoimmune disorders. Antioxidants can reduce effects of free radicals and are given to patients suffering from such diseases. There are certain natural antioxidants like flavonoids which possess free radical scavenging activities. The flavonoid quercetin is one such compound among naturally occurring antioxidants. The present study has been designed to determine the antioxidant activity in the synthetic antidepressant drug imipramine which is structurally similar to quercetin. Method: Specific standard procedures like ferric ion reducing capacity by FRAP assay, phosphomolybdenum assay and cupric ion reducing (CUPRAC) assay were carried out keeping ascorbic acid as the known standard. Results: Ferric ion reducing property of imipramine by FRAP assay revealed that reducing power of imipramine augmented with increasing amounts of the drug. In the phosphomolybdenum assay antioxidant capacity of imipramine increased in a dose dependent manner. In both these studies imipramine showed greater antioxidant action than ascorbic acid. In CUPRAC assay as the amount of imipramine was increased there was a definite elevation in antioxidant activity; however, it was comparatively less active than ascorbic acid. Conclusion: The highly potent antioxidant property in the antidepressant synthetic compound imipramine may be recognized by physicians involved in treatment of psychosis since patients receiving this drug regularly will certainly be in an advantageous position. The parent structure of imipramine can be modified further to potentiate antioxidant property of the drug.

Troxerutin (TRX) is a flavonoid present in tea, coffee, cereal grains, various fruits and vegetables have been reported to exhibit radioprotective, antithrombotic, nephro and hepato-protective effects. A systematic study was undertaken to evaluate its free radical scavenging ability and anti-apoptotic activity in cell-free and cellular systems. TRX scavenged superoxide, nitric oxide and also other model stable radicals like 1,1-diphenyl-2-picryl-hydazyl, and 2,2'-azinobis3-ethylbenzothiazoline-6-sulfonic acid. It reacted with hydroxyl radicals, carbonate and thiocyanate anions, as evaluated by pulse radiolysis and stopped flow techniques. TRX protected different cell types (epithelial cells, fibroblasts and lymphocytes) against peroxyl radical-induced apoptosis, necrosis and mitotic death. It scavenged intracellular basal and inducible ROS levels and also restored depletion of intracellular GSH levels, suggesting that free radical scavenging ability may be responsible for the observed cytoprotection of different cell types. TRX may find application as an adjuvant in treating various diseases attributed to oxidative stress.

Introduction: Flavonoid Quercetin is a major constituent of fruits and vegetables which naturally exists in the form of its glycosides, foremost as C3 glycosides. Three Quercetin glycosides namely quercitrin, quercetin 3-β-D-glucoside and quercetin 3-O-(6”-O-malonyl)-β-D-glucoside, differing at their C3 glycosylation were explored for their antioxidant properties and were compared to the parent quercetin. Methods: Free radical scavenging ability of quercetin glycosides was assessed by UV-VIS spectrophotometry, pulse radiolysis and cyclic voltammetry. Their protective effects in Fenton radical induced DNA strand breaks and mitigating oxidative stress in subcellular organelles such as mitochondria were also examined. Results: Unlike earlier reports we found that C3 glycosides of quercetin exhibited better free radical scavenging in cell free environment. These glycosides effectively protected pBR322 DNA against Fenton radical induced-DNA strand breaks and this could be attributed to their interaction with 2 deoxy guanosine base transient. However, amongst these glycosides, Quercetin 3-O-(6”-O-malonyl)-β-D-glucoside which exhibited excellent antioxidant ability in cell-free environment, could not effectively protect mitochondria from lipid peroxidation but conferred protection against protein sulphydryl depletion at lower concentrations. Conclusion: All quercetin glycosides exhibited excellent antioxidant activities in cell free environment but did not exert comparable protective effects in biological systems. Varied responses of these derivatives can be attributed to their C3 glycosylation and the derivatizations at C3 sugars.