Browsing by Subject "Cytosol"
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Item Influence of Cellular Localization on Activity of Hydroxysteroid Dehydrogenases(2011-12-14) Wooding, Kerry Michael; Auchus, Richard J.Hydroxysteroid dehydrogenases (HSDs) catalyze the interconversion of inactive steroids and active hormones. HSDs use nicotinamide cofactors in the cytosol and endoplasmic reticulum (ER) lumen to either reduce or oxidize their steroid substrates. Our lab has extensively studied the 17beta-HSDs types 1, 2 and 3 of the short-chain oxidoreductase family, particularly human 17beta-HSD1, which favors estrone reduction to estradiol. Rat AKR1C9 has also been thoroughly studied as a model HSD of the aldo-keto reductase family; AKR1C9 catalyzes the reduction of dihydrotestosterone to androstanediol. These two enzymes provide a basis for comparative studies with 11beta-HSD1, which catalyzes the reduction of cortisone to cortisol. Most mammalian cells supplied with adequate glucose and oxygen maintain cytoplasmic high nicotinamide concentration gradients, [NADPH] >> [NADP+] and [NAD+] >> [NADH], and in the strongly oxidizing environment of the ER lumen, both these gradients are shifted to more oxidized cofactor. Whereas 17betaHSD types 1, 2, 3 and AKR1C9 catalyze their respective reactions in a thermodynamically predictable manner based on cofactor gradients, 11beta-HSD1 does the opposite. 17beta-HSD1, 17beta-HSD3, and AKR1C9 favor reduction in the cytosol using NADPH, and 17beta-HSD2 favors oxidation in the ER lumen using NAD+. In contrast, 11beta-HSD1 reduces cortisone to cortisol in the highly oxidative ER lumen but requires hexose-6-phosphate-dehydrogenase (H6PD) to regenerate NADPH in the ER lumen. We hypothesize that H6PD directly channels NADPH to 11beta-HSD1 through specific interactions. To test this hypothesis, we have targeted 17beta-HSD1 and AKR1C9 to the ER lumen rather than the cytosol. Conversely, we have targeted 11beta-HSD1 to the cytoplasmic surface of the ER. In addition, we have engineered point mutations in 17beta-HSD1, AKR1C9, 11beta-HSD1 and H6PD, designed to attenuate the directional preferences by altering cofactor binding. Targeting and retaining 17beta-HSD1 in the ER lumen proved troublesome; regardless of the transfection conditions, ER-targeted 17beta-HSD1 was always detected in the cytosol. We conclude that either 17beta-HSD1’s activity or structure causes its translocation to the cytosol.Item Investigating the Roles of Vacuoles in Iron Trafficking in Saccharomyces cerevisiae(2013-11-27) Cockrell, Allison LeighTransition metals play essential roles in biological systems, but Fe can also be toxic to cells. In order to maintain this balance between necessity and toxicity mechanisms are employed for regulating and storing intracellular Fe. In Saccharomyces cerevisiae, vacuoles are responsible for sequestering, storing, and supplying Fe to the cytosol. Many of the proteins and regulatory pathways involved in Fe trafficking and storage in S. cerevisiae have been identified, but the forms of Fe which are involved in these processes have not been fully characterized. In these studies, biophysical and bioanalytical techniques were used to study intracellular Fe distributions in S. cerevisiae cells and organelles. Ultimately, Fe-containing species were biophysically characterized and absolute Fe concentrations in cells and organelles were quantified. The motivation for these studies stemmed from previous studies which revealed that the majority of the whole-cell Fe is a non-heme, high-spin (NHHS) form of Fe^(3+). This Fe is not localized to the mitochondria. The purpose of these studies was to determine if the vacuoles contained this NHHS Fe^(3+). A large-scale isolation procedure was developed to obtain purified vacuoles from S. cerevisiae and to investigate the Fe in these organelles. M?ssbauer and EPR analysis revealed that the primary form of Fe in vacuoles is a mononuclear, NHHS Fe^(3+) species. A second form of Fe was also observed as superparamagnetic ferric phosphate nanoparticles (NP). By investigating model compounds of Fe and polyphosphate we determined that a shift in vacuolar pH induces the conversion between NHHS Fe^(3+) and NP. These results showed that there are at least two forms of Fe in vacuoles, and that the ratio of these two forms is dependent upon the pH of these organelles. Biophysical analyses of whole cells also revealed the presence of low concentrations of a non-heme, high-spin Fe^(2+) species. The goal of these next projects was to determine if this NHHS Fe^(2+) species was localized to the cytosol. Genetic strains lacking or over-expressing the vacuolar Fe import protein Ccc1p were studied by M?ssbauer spectroscopy (?CCC1 and CCC1-up, respectively). ?CCC1 cells showed low vacuolar Fe (NHHS Fe3+ and NP), and increased NHHS Fe^(2+). We hypothesize that this NHHS Fe^(2+) is cytosolic Fe. We also propose that this NHHS Fe^(2+) is involved in the regulating intracellular Fe levels. CCC1-up cells accumulated more Fe than wild-type (WT) cells, and showed elevated levels of vacuolar Fe (NHHS Fe^(3+) and NP). These cells also accumulated high levels of NHHS Fe^(2+). The CCC1-up cells exhibited an adenine deficient phenotype, where the cells developed a red color during growth. With excess adenine the levels of NHHS Fe^(2+) declined, which indicated that this Fe accumulation was related to adenine deficiency. We conclude that adenine deficiency leads to the accumulation of a sequestered (possibly vacuolar) form of NHHS Fe^(2+). Overall, we have identified two separate pools of NHHS Fe^(2+) in ?CCC1 and CCC1-up cells. In ?CCC1 cells the NHHS Fe^(2+) pool is localized to the cytosol and is sensed by the cell. In CCC1-up cells the NHHS Fe^(2+) is sequestered from the Fe regulatory mechanism- possibly in the vacuoles. These data have helped us better understand the roles of vacuoles in Fe trafficking and the dynamics of vacuolar Fe trafficking.