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  • Filipin III We further demonstrated that in the presence of


    We further demonstrated that in the presence of the proteasomal inhibitors, Dp44mT was unable to increase the levels of the top and middle NDRG1 bands, as well as is phosphorylated forms (Figs. 5A, 6A). These observations demonstrated the role of the active proteasome in the ability of Dp44mT to induce NDRG1 Filipin III and phosphorylation. Considering this, proteasomal inhibitors could be potentially inhibiting upstream kinases of NDRG1 that could control its phosphorylation and also potentially its activation. Previous investigations by others have reported the phosphorylation of NDRG1 can be mediated through both GSK3β and SGK1 activity [16]. Initial studies to determine their roles in NDRG1 processing demonstrated that specific inhibitors for these kinases were able to markedly reduce the level of the top NDRG1 and p-NDRG1 bands at 47-kDa (Fig. 7, Fig. 8). To assess whether this observation was due to the ability of these inhibitors to decrease kinase activity, or due to alternate mechanisms, more detailed mechanistic dissection was performed. These studies demonstrated that GSK3β inhibition is involved in a kinase-independent decrease in NDRG1 levels (Fig. 11B). Previous reports have demonstrated that GSK3β inhibition can result in the activation of a lysosomal pathway [61], that could potentially be responsible for the decrease in the NDRG1 top band observed under these conditions. In contrast, the effects of SGK1 inhibition on the top NDRG1 band were demonstrated to be mediated through its ability to inhibit NDRG1 phosphorylation (Fig. 11B).
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    Acknowledgments S.S. appreciates a Young Investigator PdCCRS Grant jointly funded by Cancer Australia and the Cure Cancer Australia Foundation. S.S. also thanks AMP Foundation for the AMP Tomorrow Fund grant. K. C. P. is a grateful recipient of Research Training Program Stipend Scholarship from the University of Sydney. ZK appreciates fellowships from the National Health and Medical Research Council of Australia and the Cancer Institute NSW as well as grant funding from the Avner Pancreatic Cancer Foundation and a PdCCRS grant jointly funded by Cancer Australia and the Cure Cancer Australia Foundation. D. R. R. appreciates a Senior Principal Research Fellowship and Project Grant funding from the National Health and Medical Research Council of Australia.
    Introduction Methylglyoxal (MG) belongs to a heterogeneous group of low-molecular weight dicarbonyls derived from metabolic processes and, in particular, from glycolysis. Dicarbonyl species are highly reactive and promote posttranslational modification of proteins by glycation, a non-enzymatic reaction with free amino groups of proteins, lipids, and nucleic acids [1]. The early stage of this process involves a complex series of reactions, often referred to collectively as the Maillard reaction, leading to formation of intermediates that are initially reversible but ultimately form stable end-stage adducts called advanced glycation end-products (AGEs) [2]. MG is known to react primarily with arginine residues to form hydroimidazolones and argpyrimidine [3,4], here referred to as MG-AGEs. Detoxification of MG mainly occurs via glyoxalase 1 (GLO1) and glyoxalase 2 (GLO2), with GLO1 catalysing the glutathione (GSH)-dependent formation of S-d-lactoylglutathione from MG, and GLO2 hydrolysing S-d-lactoylglutathione to d-lactate and GSH [5]. MG accumulation may arise from increased glycolytic metabolism and/or reduced glyoxalase-mediated removal. The latter may occur as a consequence of reduced GSH availability (e.g. oxidative stress) and/or decreased glyoxalase expression (e.g. aging) [5]. The build-up of MG affects mitochondrial proteins and increases AGEs, which can also activate receptor-mediated pro-oxidant signalling pathways, thus generating a vicious cycle. Therefore one of the main aspects of the glycative burden is oxidative stress [5]. Both MG and protein glycation (dicarbonyl stress) have been attracting considerable attention because of their role in the chronic side effects in diabetic patients with enduring hyperglycaemia and in the pathogenesis of numerous diseases associated with altered redox homeostasis [5]. About ten years ago, Diamanti-Kandarakis' research group proposed the involvement of AGEs in ovarian dysfunctions based on the observation of increased levels of AGEs in the serum and ovary of women affected by polycystic ovary syndrome (PCOS) [[6], [7], [8]]. Specific serum levels of AGEs were established to indicate diminished fertility, and AGEs were shown to correlate positively with altered glucose metabolism, age, and factors related to obesity, dyslipidaemia, hyperglycaemia, and insulin resistance in patients undergoing in vitro fertilization (IVF) [9]. In addition, elevated MG and MG-AGEs were reported in ovaries of reproductively aged mice [[10], [11], [12]]. A preliminary analysis of proteome revealed increased glycation of specific polypeptides in concomitance with decreased activity and expression of GLO1 [10]. Further evidence for the role of AGEs in ovarian aging was provided by measurements in follicular fluid and serum of soluble RAGE (s-RAGE), a circulating isoform of RAGE that can neutralize the ligand-mediated damage [13,14]. The crucial role of glyoxalases in the ovary has been confirmed by the finding that dietary glycotoxins and hyperandrogenic status decrease GLO1 activity in rat ovaries, possibly contributing to increased AGE accumulation in granulosa cells [15].