Del uso de las zonas verdes

Anupama Shanmuganathan, Amrita Nargund, Simon V. Avery and John E. Houghton

Several mechanisms are reportedly used by cells to counter the effects of the toxic metal, cadmium, including a sulfur metabolism response that stimulates glutathione production. (M.

Fauchon et al., (2002) Mol. Cell 9: 713-723). Here, we show that cadmium also provokes a

carbon-specific metabolic response, which is required for Cd resistance. Using immuno- detection of oxidized proteins, we demonstrated that cadmium (which is redox inactive) induces a transient, targeted oxidation of glycolytically associated enzymes in yeast, similar to that reported previously in cells exposed to redox-active metals. An observed decrease in glycolytic enzyme activity, at sub-lethal Cd concentrations, contrasted with elevated expression and activity of the key pentose phosphate pathway (PPP) enzyme, glucose-6-phosphate dehydrogenase (Zwf1p). The results predicted a response to Cd involving enhanced flow of carbon through the PPP, which was substantiated by a significant increase in cellular reducing power (in the form of NADPH) during Cd exposure. The increased reducing power was further reflected in a concomitant increase in the cellular levels of reduced glutathione (GSH), a major defense molecule against Cd. These cadmium-induced increases in NADPH and GSH were both blocked in PPP mutants, which exhibited a marked sensitivity to Cd. The results reveal that a re-direction

of carbon metabolism occurs in response to cellular Cd exposure, providing a mechanism additional to the sulfur response for enabling GSH dependent Cd resistance.

Introduction

The widespread industrial use of cadmium (Cd) has contributed significantly to its anthropogenic introduction and persistence in the environment. With little to no known biological function (Lane et al., 2005), cadmium can be rapidly absorbed from the blood in

mammals, and thereafter accumulates in metallothionein-rich organs, thereby increasing its accrual and distribution through the food chain. As an established genotoxin and carcinogen, cadmium’s relatively long biological half-life (greater than 30 years in some instances) further heightens its toxic threat. Even though exposure to this transition metal has been directly associated with hyper mutability in yeast (Jin et al., 2003), the variety of effects that cadmium

elicits within different organisms at different concentrations has made it difficult to identify specific molecular mechanisms of its toxicity (Filipic et al., 2006). Unlike many other

carcinogenic metals, cadmium is redox inactive, in that it is unable to catalyze directly the Fenton and Harber-Weiss reactions, which produce potent reactive oxygen species (ROS). Nonetheless, cadmium is still considered an effective prooxidant in cells, presumably through its ability to produce ROS indirectly, either by displacing redox-active metals such as copper and

iron from the active sites of enzymes (Stohs and Bagchi et al., 1995) or by selective depletion of

the cells’ anti-oxidant defences (Fortuniak et al., 1996 and Stohs et al., 2001). It is this role of

cadmium, acting as an oxidative stressor in the model system Saccharomyces cerevisiae, that

provides a basis for the response reported in this work. The yeast S. cerevisiae has been used

Transcription factors such as Yap1p and Met4p mediate a series of transcriptional responses to Cd, which involve upregulation of the GSH1 gene among others (Lee et al., 1999 and Dormer et

al., 2000). Gsh1p catalyzes the rate limiting step in the biosynthesis of glutathione, a thiol

metabolite that is essential for Cd detoxification (Li et al., 1997 and Vido et al., 2001). Another

outcome of Cd-induced transcriptional reprogramming that enables glutathione-mediated Cd resistance is the sulfur sparing response (Fauchon et al., 2002). This response to Cd involves a

decrease in the production of abundant sulfur-rich proteins, allowing a greater proportion of cellular sulfur to be directed towards glutathione biosynthesis. Certain aspects of the above consequences of cadmium exposure overlap with responses reported for other particular metals or prooxidants (Lee et al., 1999 and Thorsen et al., 2007). Such overlaps in the stress response

pathways of cells are common, whereas other effects appear to be restricted to particular subclasses of stressors. Thus, in studying the effects of redox-active agents (H2O2, copper and chromium), we and others have shown that a discrete subset of glycolytic enzymes within the cell become oxidized (Cabiscol et al., 2000; Costa et al., 2002; Shanmuganathan et al., 2004 and

Sumner et al., 2005). It has been suggested, but not shown, that such a targeted oxidation of

glycolytically associated enzymes could redirect the flow of carbon equivalents away from glycolysis and into the pentose phosphate pathway (PPP) (Costa et al., 2002; Shanmuganathan et

al., 2004; Ravichandran et al., 1994 and Shenton and Grant, 2003), giving rise to elevated levels

of cellular reducing power in the form of NADPH. In turn, it has been proposed that this could bolster cellular defenses against the immediate effects of redox-active stressors by, among other things, recycling cellular antioxidants such as reduced glutathione. Given the relatively rapid rate of targeted protein oxidation (within minutes) observed following exposure to redox-active metals (Shanmuganathan et al., 2004 and Sumner et al., 2005), if the association between protein

oxidation and cellular reducing power holds true, there could be a similarly rapid elevation in the levels of the necessary reducing power, NADPH. As described above, a large component of the extensive transcriptional reprogramming that occurs in Cd-stressed cells is directed towards increasing the availability of glutathione. With this in mind, here we hypothesize that the availability of any further mechanisms for increasing glutathione capacity (such as the possible reshuffling of carbon equivalents through the PPP) will have been assimilated into the cadmium stress response during evolution. Although a targeted PPP response has only previously been suggested for redox-active metals (see above), and other key responses to chemically disparate metals can be distinct (Lee et al., 1999; Gross et al., 2000; van Bakel et al., 2000 and Holland et

al., 2007), here we show that redox-inactive cadmium elicits a pattern of protein oxidation in the

cell comparable to that observed previously for copper and chromium (Shanmuganathan et al.,

2004 and Sumner et al., 2005). Moreover, we show that this focused protein oxidation comprises

part of a rapid and coordinated response that is specific to cadmium. This response serves to enhance reduced-glutathione availability and, consequently, Cd resistance.

Materials and Methods