Manganese is an essential transition metal that, among other functions, can act independently of proteins to either defend against or promote oxidative stress and disease. The majority of cellular manganese exists as low molecular-weight Mn 2+ complexes, and the balance between opposing “essential” and “toxic” roles is thought to be governed by the nature of the ligands coordinating Mn 2+ . Until now, it has been impossible to determine manganese speciation within intact, viable cells, but we here report that this speciation can be probed through measurements of 1 H and 31 P electron-nuclear double resonance (ENDOR) signal intensities for intracellular Mn 2+ . Application of this approach to yeast ( Saccharomyces cerevisiae ) cells, and two pairs of yeast mutants genetically engineered to enhance or suppress the accumulation of manganese or phosphates, supports an in vivo role for the orthophosphate complex of Mn 2+ in resistance to oxidative stress, thereby corroborating in vitro studies that demonstrated superoxide dismutase activity for this species.
Among the phenotypes of Saccharomyces cerevisiae mutants lacking CuZn-superoxide dismutase (Sod1p) is an aerobic lysine auxotrophy; in the current work we show an additional leaky auxotrophy for leucine. The lysine and leucine biosynthetic pathways each contain a 4Fe-4S cluster enzyme homologous to aconitase and likely to be superoxide-sensitive, homoaconitase (Lys4p) and isopropylmalate dehydratase (Leu1p), respectively. We present evidence that direct aerobic inactivation of these enzymes in sod1⌬ yeast results in the auxotrophies. Located in the cytosol and intermembrane space of the mitochondria, Sod1p likely provides direct protection of the cytosolic enzyme Leu1p. Surprisingly, Lys4p does not share a compartment with Sod1p but is located in the mitochondrial matrix. The activity of a second matrix protein, the tricarboxylic acid cycle enzyme aconitase, was similarly lowered in sod1⌬ mutants. We measured only slight changes in total mitochondrial iron and found no detectable difference in mitochondrial "free" (EPRdetectable) iron making it unlikely that a gross defect in mitochondrial iron metabolism is the cause of the decreased enzyme activities. Thus, we conclude that when Sod1p is absent a lysine auxotrophy is induced because Lys4p is inactivated in the matrix by superoxide that originates in the intermembrane space and diffuses across the inner membrane.The antioxidant enzyme copper/zinc-superoxide dismutase 1 plays an integral role in the protection of many organisms from the oxidative aerobic environment.CuZn-SOD is localized to the cytosol, nucleus, and the intermembrane space (IMS) of the mitochondria, suggesting that it exerts its protective effect in multiple compartments (1). Another SOD that contains manganese (Mn-SOD or Sod2p) is located in the matrix of mitochondria. Saccharomyces cerevisiae lacking CuZn-SOD (sod1⌬) have distinct and well established aerobic phenotypes including diminished growth, auxotrophies for lysine and either methionine or cysteine, decreased ability to grow on nonfermentable carbon sources (2, 3), increased "free" (EPR-detectable) iron (4), hypersensitivity to millimolar concentrations of zinc (5), and exquisite sensitivity to redox-cycling drugs such as the herbicide paraquat (6, 7). All of these phenotypes (except zinc sensitivity) are also observed in mutants lacking the copper chaperone for CuZn-SOD 2 (lys7⌬ or ccs1⌬) and thus contain a form of the Sod1p polypeptide that is inactive because of a lack of copper in the active site (5, 8). Mutants lacking Sod2p have a less dramatic phenotype; they are sensitive to redox cycling drugs and grow poorly on nonfermentable carbon sources but show no aerobic auxotrophies (2).Many cellular components are susceptible to oxidative damage, including proteins, lipids, and DNA (9, 10). However, targets of superoxide-specific damage are much more limited. A particular type of protein prosthetic group, solvent-exposed 4Fe-4S clusters occurring in nonelectron transfer proteins, have been shown to be specifically damaged by superoxid...
Yeast lacking copper-zinc superoxide dismutase (sod1Δ ) have a number of oxygen-dependent defects, including auxotrophies for lysine and methionine and sensitivity to oxygen. Here we report additional defects in metabolic regulation. Under standard growth conditions with glucose as the carbon source, yeast undergo glucose repression where mitochondrial respiration is de-emphasized, energy is mainly derived from glycolysis, and ethanol is produced. When glucose is depleted, the diauxic shift is activated, in which mitochondrial respiration is re-emphasized and stress resistance increases. We find that both of these programs are adversely affected by the lack of Sod1p. Key events in the diauxic shift do not occur and sod1Δ cells do not utilize ethanol and stop growing. The ability to shift to growth on ethanol is gradually lost as time in culture increases. In early stages of culture, sod1Δ cells consume more oxygen and have more mitochondrial mass than wild-type cells, indicating that glucose repression is not fully activated. These changes are at least partially dependent on the activity of the Hap2,3,4,5 complex, as indicated by CYC1-lacZ reporter assays. These changes may indicate a role for superoxide in metabolic signaling and regulation and/or a role for glucose derepression in defense against oxidative stress.
Oxidative stress, caused by free radicals within the body, has been associated with the process of aging and many human diseases. As free radicals, in particular superoxide, are difficult to measure, an alternative indirect method for measuring oxidative stress levels has been successfully used in E. coli and yeast. This method is based on a proposed connection between elevated superoxide levels and release of iron from solvent exposed [4Fe-4S] enzyme clusters, which eventually leads to an increase in hydroxyl radical production. In past studies using bacteria and yeast, a positive correlation was found between superoxide production or oxidative stress due to superoxide within the organism and EPR (electron paramagnetic resonance) detectable "free" iron levels. In the present study, we have developed a reliable and an efficient method for measuring "free" iron levels in C. elegans using low temperature Fe(III) EPR at g = 4.3. This method utilizes synchronized worm cultures grown on plates, which are homogenized and treated with desferrioxamine, an Fe(III) chelator, prior to packing the EPR tube. Homogenization was found not to alter "free" iron levels, while desferrioxamine treatment significantly raised these levels, indicating presence of both Fe(II) and Fe(III) in the "free" iron pool. The correlation between free radical levels and the observed "free" iron levels was examined by using heat stress and paraquat treatment. The intensity of the Fe(III) EPR signal and thus, the concentration of the "free" iron pool, varied with the treatments that altered radical levels without changing the total iron levels. This study provides the groundwork needed to uncover the correlation between oxidative stress, "free" iron levels, and longevity in C. elegans.
IntroductionTens of millions of people worldwide will develop Alzheimer's disease (AD), and only by intervening early in the preclinical disease can we make a fundamental difference to the rates of late-stage disease where clinical symptoms and societal burden manifest. However, collectively utilizing data, samples, and knowledge amassed by large-scale projects such as the Innovative Medicines Initiative (IMI)-funded European Prevention of Alzheimer's Dementia (EPAD) program will enable the research community to learn, adapt, and implement change.MethodIn the current article, we define and discuss the substantial assets of the EPAD project for the scientific community, patient population, and industry, describe the EPAD structure with a focus on how the public and private sector interacted and collaborated within the project, reflect how IMI specifically supported the achievements of the above, and conclude with a view for future.ResultsThe EPAD project was a €64-million investment to facilitate secondary prevention of AD dementia research. The project recruited over 2,000 research participants into the EPAD longitudinal cohort study (LCS) and included over 400 researchers from 39 partners. The EPAD LCS data and biobank are freely available and easily accessible via the Alzheimer's Disease Data Initiative's (ADDI) AD Workbench platform and the University of Edinburgh's Sample Access Committee. The trial delivery network established within the EPAD program is being incorporated into the truly global offering from the Global Alzheimer's Platform (GAP) for trial delivery, and the almost 100 early-career researchers who were part of the EPAD Academy will take forward their experience and learning from EPAD to the next stage of their careers.DiscussionThrough GAP, IMI-Neuronet, and follow-on funding from the Alzheimer's Association for the data and sample access systems, the EPAD assets will be maintained and, as and when sponsors seek a new platform trial to be established, the learnings from EPAD will ensure that this can be developed to be even more successful than this first pan-European attempt.
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