PhD

dissertationMetal ions can interact with specific biological molecules and activate or regulate the function of these molecules by causing a change in molecular structure. Metallothioneins (MTs) are small, cysteine rich proteins that bind metal ions tenaciously. Metal ions are chelated within polymetallic clusters and undergo facile metal exchange reactions. MTs appear to function in metal ion regulation and detoxification. Metallothionein-3 (MT-3) is a brain specific member of the metallothionein family of metal binding proteins, and its absence has been implicated in the development of Alzheimer's disease (AD). The growth inhibitory activity of MT-3 appears to result from a distinct amino acid sequence and not unusual metal-binding properties compared with MT-1 or MT-2. The transcription factor Macl from S. cerevisiae regulates the expression of the high affinity copper uptake system in a copper dependent manner. When cells are exposed to copper concentration above starvation levels expression of genes in this uptake system is inhibited. Macl contains two cysteine rich motifs in the C-terminus which resemble Cu(I) binding cysteinyl sequences found in MTs. Macl was found to directly bind Cu(I) ions and a copper dependent intramolecular interaction between the N-terminal DNA binding domain and the C-terminal copper binding activation domain was identified. This interaction may result in the masking of the DNA binding domain in a copper dependent manner abolishing expression of Macl regulated genes. In yeast, MT genes are transcriptionally induced by copper, but not by other metal ions. The primary copper buffering protein in S. cerevisiae is a M

Genetic Analysis of Peroxisomal Genes Required for Longevity in a Yeast Model of Citrin Deficiency

Citrin is a liver-specific mitochondrial aspartate–glutamate carrier encoded by SLC25A13. Citrin deficiency caused by SLC25A13 mutation results in carbohydrate toxicity, citrullinemia type II, and fatty liver diseases, the mechanisms of some of which remain unknown. Citrin shows a functional homolog in yeast aspartate-glutamate carrier (Agc1p) and agc1Δ yeasts are used as a model organism of citrin deficiency. Here, we found that agc1Δ yeasts decreased fat utilization, impaired NADH balance in peroxisomes, and decreased chronological lifespan. The activation of GPD1-mediated NAD+ regeneration in peroxisomes by GPD1 over-expression or activation of the malate–oxaloacetate NADH peroxisomal shuttle, by increasing flux in this NADH shuttle and over-expression of MDH3, resulted in lifespan extension of agc1Δ yeasts. In addition, over-expression of PEX34 restored longevity of agc1Δ yeasts as well as wild-type cells. The effect of PEX34-mediated longevity required the presence of the GPD1-mediated NADH peroxisomal shuttle, which was independent of the presence of the peroxisomal malate–oxaloacetate NADH shuttle and PEX34-induced peroxisome proliferation. These data confirm that impaired NAD+ regeneration in peroxisomes is a key defect in the yeast model of citrin deficiency, and enhancing peroxisome function or inducing NAD+ regeneration in peroxisomes is suggested for further study in patients’ hepatocytes

Possible Role of the Ca²⁺/Mn²⁺ P-Type ATPase Pmr1p on Artemisinin Toxicity through an Induction of Intracellular Oxidative Stress

Artemisinins are widely used to treat Plasmodium infections due to their high clinical efficacy; however, the antimalarial mechanism of artemisinin remains unresolved. Mutations in P. falciparum ATPase6 (PfATP6), a sarcoplasmic endoplasmic reticulum Ca2+-transporting ATPase, are associated with increased tolerance to artemisinin. We utilized Saccharomyces cerevisiae as a model to examine the involvement of Pmr1p, a functional homolog of PfATP6, on the toxicity of artemisinin. Our analysis demonstrated that cells lacking Pmr1p are less susceptible to growth inhibition from artemisinin and its derivatives. No association between sensitivity to artemisinin and altered trafficking of the drug efflux pump Pdr5p, calcium homeostasis, or protein glycosylation was found in pmr1∆ yeast. Basal ROS levels are elevated in pmr1∆ yeast and artemisinin exposure does not enhance ROS accumulation. This is in contrast to WT cells that exhibit a significant increase in ROS production following treatment with artemisinin. Yeast deleted for PMR1 are known to accumulate excess manganese ions that can function as ROS-scavenging molecules, but no correlation between manganese content and artemisinin resistance was observed. We propose that loss of function mutations in Pmr1p in yeast cells and PfATP6 in P. falciparum are protective against artemisinin toxicity due to reduced intracellular oxidative damage

The interaction of mitochondrial iron with superoxide dismutase

Superoxide dismutase 2 (SOD2) is one of the rare mitochondrial enzymes evolved to use manganese as a cofactor over the more abundant element iron. Although mitochondrial iron does not normally bind SOD2, iron will misincorporate into Saccharomyces cerevisiae Sod2p when cells are starved for manganese or when mitochondrial iron homeostasis is disrupted by mutations in yeast grx5, ssq1, and mtm1. We report here that such changes in mitochondrial manganese and iron similarly affect cofactor selection in a heterologously expressed Escherichia coli Mn-SOD, but not a highly homologous Fe-SOD. By x-ray absorption near edge structure and extended x-ray absorption fine structure analyses of isolated mitochondria, we find that misincorporation of iron into yeast Sod2p does not correlate with significant changes in the average oxidation state or coordination chemistry of bulk mitochondrial iron. Instead, small changes in mitochondrial iron are likely to promote iron-SOD2 interactions. Iron binds Sod2p in yeast mutants blocking late stages of iron-sulfur cluster biogenesis (grx5, ssq1, and atm1), but not in mutants defective in the upstream Isu proteins that serve as scaffolds for iron-sulfur biosynthesis. In fact, we observed a requirement for the Isu proteins in iron inactivation of yeast Sod2p. Sod2p activity was restored in mtm1 and grx5 mutants by depleting cells of Isu proteins or using a dominant negative Isu1p predicted to stabilize iron binding to Isu1p. In all cases where disruptions in iron homeostasis inactivated Sod2p, we observed an increase in mitochondrial Isu proteins. These studies indicate that the Isu proteins and the iron-sulfur pathway can donate iron to Sod2p

The Interaction of Mitochondrial Iron with Manganese Superoxide Dismutase*

Superoxide dismutase 2 (SOD2) is one of the rare mitochondrial enzymes evolved to use manganese as a cofactor over the more abundant element iron. Although mitochondrial iron does not normally bind SOD2, iron will misincorporate into Saccharomyces cerevisiae Sod2p when cells are starved for manganese or when mitochondrial iron homeostasis is disrupted by mutations in yeast grx5, ssq1, and mtm1. We report here that such changes in mitochondrial manganese and iron similarly affect cofactor selection in a heterologously expressed Escherichia coli Mn-SOD, but not a highly homologous Fe-SOD. By x-ray absorption near edge structure and extended x-ray absorption fine structure analyses of isolated mitochondria, we find that misincorporation of iron into yeast Sod2p does not correlate with significant changes in the average oxidation state or coordination chemistry of bulk mitochondrial iron. Instead, small changes in mitochondrial iron are likely to promote iron-SOD2 interactions. Iron binds Sod2p in yeast mutants blocking late stages of iron-sulfur cluster biogenesis (grx5, ssq1, and atm1), but not in mutants defective in the upstream Isu proteins that serve as scaffolds for iron-sulfur biosynthesis. In fact, we observed a requirement for the Isu proteins in iron inactivation of yeast Sod2p. Sod2p activity was restored in mtm1 and grx5 mutants by depleting cells of Isu proteins or using a dominant negative Isu1p predicted to stabilize iron binding to Isu1p. In all cases where disruptions in iron homeostasis inactivated Sod2p, we observed an increase in mitochondrial Isu proteins. These studies indicate that the Isu proteins and the iron-sulfur pathway can donate iron to Sod2p

Chemical-Genetic Interactions of Bacopa monnieri Constituents in Cells Deficient for the DNA Repair Endonuclease RAD1 Appear Linked to Vacuolar Disruption

Colorectal cancer is a common cancer worldwide and reduced expression of the DNA repair endonuclease XPF (xeroderma pigmentosum complementation group F) is associated with colorectal cancer. Bacopa monnieri extracts were previously found to exhibit chemical-genetic synthetic lethal effects in a Saccharomyces cerevisiae model of colorectal cancer lacking Rad1p, a structural and functional homologue of human XPF. However, the mechanisms for B. monnieri extracts to limit proliferation and promote an apoptosis-like event in RAD1 deleted yeast was not elucidated. Our current analysis has revealed that B. monnieri extracts have the capacity to promote mutations in rad1∆ cells. In addition, the effects of B. monnieri extracts on rad1∆ yeast is linked to disruption of the vacuole, similar to the mammalian lysosome. The absence of RAD1 in yeast sensitizes cells to the effects of vacuole disruption and the release of proteases. The combined effect of increased DNA mutations and release of vacuolar contents appears to induce an apoptosis-like event that is dependent on the meta-caspase Yca1p. The toxicity of B. monnieri extracts is linked to sterol content, suggesting saponins may be involved in limiting the proliferation of yeast cells. Analysis of major constituents from B. monnieri identified a chemical-genetic interaction between bacopasaponin C and rad1∆ yeast. Bacopasaponin C may have potential as a drug candidate or serve as a model for the development of analogs for the treatment of colorectal cancer