Silicates: Novel Modulators of Biological Stress Response?

and examine their potential role in redox activity. We show that Zinc fingers are commonly associated with a specific type of forbidden disulfide motif. Forbidden disulfides are a canonical set of disulfides with abnormal stereochemistry associated with redox-activity. We show that in around 50% of Zinc finger 2+ structures two of the Zn -ligating thiols are embedded in a secondary structure similar to an anti-parallel β-diagonal disulfidelike motif (aBDD), located on the β-hairpin structure known as a Zinc knuckle. Formation of a disulfide by thiols of this motif has recently been characterized in the molecular chaperone Hsp33 and also demonstrated in several other transcription factors (1). Although other forbidden disulfide motifs are occasionally present in Zinc fingers, none are as ubiquitous as this aBDD-like motif. We show that the presence of this motif and its position in the structure is characteristic of different types of Zinc fingers, suggesting a functional relationship. As Zinc fingers comprise more than 17% of the annotated domain content of the human 2+ genome, this motif is likely important in Zn signalling. 1. Ilbert M, Horst J, Ahrens S, Winter J, Graf PCF, Lilie H, Jakob U. The redox-switch domain of Hsp33 functions as dual stress sensor. Nat Struct Mol Biol 14: 556-563, 2007. doi: 10.1016/j.freeradbiomed.2010.10.358

350 Silicates: Novel Modulators of Biological Stress Response? Dhiraj Anil Vattem1, Sarah Neely1, Nick Swift1, Deana Townsend1, Leanna McMillin1, Brandon Jamison1, Vatsala Maitin1, C. Reed Richardson1, Ignacio Cisneros2, and Sandra Duesler2 1 2 Texas State University, Cisne Enterprises, Inc. Physiological relevance of silicates is not very well understood. Investigations on their biochemical and molecular properties relevant to health are non-existent. Silicates are ubiquitous and speculated to be a trace nutrient, important in structural and connective tissue development. Based on the known electrochemical properties of silicates, we hypothesized that silicates may modulate the redox environment in biological systems and effect critical stress response signaling pathways. We established a transgenic c. elegans library expressing GFP promoter fusions of 63 genes important in the IGFR/PI3K/AKT, ARE/NRF2, Sir2.1/AMPK, TGF-β, p38 MAPK, mTOR/HIF-1α, apoptosis/cell cycle and UPR signaling pathways. We characterized the effect of silicates on these highly conserved pathways that regulate growth, development, metabolism, lifespan, immune, environmental and antioxidant stress response pathways. Our results suggest that biological effects of silicates are structure dependent. NMR and IR spectroscopy revealed a putative formula of the most bioactive silicate to be Na8.2Si4.4H9.7O17.6. Silicate treatment (200nM) resulted in a significant increase in expression of insulin and insulin like growth factors but not in the DAF-16 (FKHR). Upregulation in NRF2 (SKN-1) and of genes (GSR-1, ELT2) positively regulated by SKN-1 were noted. The expression of SIR2.1 increased significantly and correlated to upregulation of antioxidant enzymes SOD-3, CTL-2, TRX and heat-shock protein expression. Increased life expectancy and protection from heavy metal toxicity and heat induced accumulation of Aβ42 was also noted. Interestingly, genes in the mTOR/HIF-α and p38-MAPK pathways were also upregulated upon treatment and expression of CKI-1 an inhibitor of cell cycle decreased significantly. EGL-9, that targets HIF-α for proteosomal degradation also increased in response to the treatment. There was no change in expression of genes involved in TGFβ signaling, mitochondrial electron transfer and oxdative CYP450 metabolism. We propose that redox buffering by certain silicates may be important in regulation of cellular functions, especially in stress response and related pathologies. doi: 10.1016/j.freeradbiomed.2010.10.359

351 Glucocorticoid Resistance in Lymphoma Cells  Mediated by Hydrogen Peroxide Removal and  Disregulation of MAPK Signaling  Margaret E. Tome1, Melba C. Jaramillo1, and Margaret M. Briehl1 1 University of Arizona The role of ROS in glucocorticoid-induced apoptosis signaling is still controversial. Glucocorticoids induce apoptosis in lymphoid cells via the intrinsic pathway in which the committed step is release of cytochrome c. Using WEHI7.2 murine thymic lymphoma cells, we found that glucocorticoid treatment increases ROS in general and H2O2 in particular during the signaling phase of apoptosis. The steady state H2O2 concentration increases from 20.5 pM to 55.5 pM 12h after the addition of glucocorticoids. Cells that overexpress catalase show a delay or lack of cyctochrome c release after dexamethasone treatment. These cells produce H2O2 in response to glucocorticoids; however, when the catalase levels are high enough to remove the H2O2 the cells do not undergo apoptosis. If the glucocorticoids are removed from the WEHI7.2 cells after the H2O2 increases, but prior to any measures of apoptosis (cytochrome c release, phosphatidyl serine exposure or caspase 3 activation) ~30% of the cells will form colonies. At the same timepoint, catalase-overexpressing cells do not have an increase in H2O2 and show 100% cloning efficiency. The glucocorticoid-sensitive cells lose pERK signaling while the catalase-overexpressing cells maintain pERK after glucocorticoid treatment. Treatment of the catalaseoverexpressing cells with a MEK inhibitor decreased pERK and partially sensitized the cells to glucocorticoids. These data suggest that: 1) H2O2 is a required signal for glucocorticoidinduced apoptosis; and 2) loss of pERK contributes to the apoptotic signaling triggered by increased H2O2 in WEHI7.2 cells. Lymphomas often arise at the sites of chronic inflammation where cells are exposed to increased ROS. Our data imply that MEK inhibitors could have clinical efficacy in overcoming glucocorticoid resistance particularly in lymphomas that occur at these sites. doi: 10.1016/j.freeradbiomed.2010.10.360

352 The Expression of Extracellular­superoxide  Dismutase in 3T3­L1 Adipocytes during  Endoplasmic Reticulum Stress  Tetsuro Kamiya1,2, Hirokazu Hara1, Naoki Inagaki2, and Tetsuo Adachi1 1 2 Gifu Pharmaceutical University, Gifu University Purpose: Obesity is closely linked to a variety of metabolic disorders. It is known that adipocytes suffer an endoplasmic reticulum (ER) stress during the adipocytes hypertrophy. Extracellular-superoxide dismutase (EC-SOD) is one of the antioxidative enzymes and protects the cells from oxidative stress in vascular systems. We investigated the regulation of EC-SOD and other SOD isozymes in 3T3-L1 adipocytes during ER stress induced by thapsigargin (Tg) or tunicamycin (Tu). Methods: 3T3-L1 preadipocytes were differentiated into mature adipocytes by the conventional methods. The expression of SOD isozymes was measured by RT-PCR. Protein phospholylation was detected by western blotting. Results and Discussion: Tg treatment decreased the expression of EC-SOD mRNA in a Tg dose- and time-dependent manner, whereas the expressions of other SOD isozymes were not affected. On the other hand, Tu did not have the capacity to reduce all SOD isozymes. Tg treatment activated eukaryotic initiation factor 2 (eIF2) α. Moreover, pretreatment with salubrinal, an inhibitor of dephospholylation of eIF2α, significantly accelerated Tg-triggered EC-SOD reduction. From these results, we speculated that the expression of EC-SOD was regulated by the eIF2α-derived signaling in 3T3-L1 adipocytes during ER

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