Two cytosolic sources of NADPH, the hexose monophosphate pathway and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2), have been found to be essential in preventing the accumulation of deleterious oxidative byproducts of endogenous metabolic pathways including peroxisomal beta-oxidation and mitochondrial respiration. Loss of glucose-6-phosphate dehydrogenase (ZWF1, the first enzyme in the hexose monophosphate pathway) and of IDP2 results in a rapid loss in viability of yeast cells transferred to medium with oleate or acetate as the carbon source. In contrast, loss of major cellular peroxidative enzymes has no effect on growth with these carbon sources. The lethality resulting from endogenous metabolic byproducts, and the specific requirement for cytosolic sources of NADPH to protect from this lethality, are novel observations in the area of cellular oxidative stress. Further investigation of these phenomena is proposed because oxidative damage to cellular macromolecules has been associated with numerous degenerative diseases and with the process of aging. Primary aims of this proposal include the following: (A) Proteins and DNA from strains lacking cytosolic sources of NADPH will be examined to identify specific cellular and organellar macromolecular targets of deleterious oxidants generated by normal metabolic pathways. (B) We will assess the importance of cytosolic sources and of levels of NADPH as determinants of longevity, using yeast strains lacking or overexpressing these key antioxidant enzymes for life-span analysis. (C) We will initiate biochemical analyses of mammalian IDP2, and examine the physiological relevance of co-localization of this enzyme in the cytosol and in peroxisomes using both yeast and mouse models. In addition, we will continue to examine the structural basis for the unique physiological functions of yeast IDP2. (D) Finally, we will assess global changes in pools of central metabolites and reducing equivalents to directly test fundamental hypotheses related to loss or replacement of cytosolic sources of NADPH.

Agency
National Institute of Health (NIH)
Institute
National Institute on Aging (NIA)
Type
Research Project (R01)
Project #
5R01AG017477-07
Application #
7198112
Study Section
Special Emphasis Panel (ZRG1-CMAD (01))
Program Officer
Finkelstein, David B
Project Start
2000-03-01
Project End
2009-02-28
Budget Start
2007-03-01
Budget End
2008-02-29
Support Year
7
Fiscal Year
2007
Total Cost
$270,635
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Biochemistry
Type
Other Domestic Higher Education
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
Lu, Qian; McAlister-Henn, Lee (2010) Peroxisomal localization and function of NADP+ -specific isocitrate dehydrogenases in yeast. Arch Biochem Biophys 493:125-34
Garcia, Joshua A; Minard, Karyl I; Lin, An-Ping et al. (2009) Disulfide bond formation in yeast NAD+-specific isocitrate dehydrogenase. Biochemistry 48:8869-78
Minard, Karyl I; McAlister-Henn, L (2009) Redox responses in yeast to acetate as the carbon source. Arch Biochem Biophys 483:136-43
Lu, Qian; Minard, Karyl I; McAlister-Henn, Lee (2008) Dual compartmental localization and function of mammalian NADP+-specific isocitrate dehydrogenase in yeast. Arch Biochem Biophys 472:17-25
Lin, An-Ping; Hakala, Kevin W; Weintraub, Susan T et al. (2008) Suppression of metabolic defects of yeast isocitrate dehydrogenase and aconitase mutants by loss of citrate synthase. Arch Biochem Biophys 474:205-12
Minard, Karyl I; Carroll, Christopher A; Weintraub, Susan T et al. (2007) Changes in disulfide bond content of proteins in a yeast strain lacking major sources of NADPH. Free Radic Biol Med 42:106-17
Minard, Karyl I; McAlister-Henn, Lee (2005) Sources of NADPH in yeast vary with carbon source. J Biol Chem 280:39890-6
Contreras-Shannon, Veronica; Lin, An-Ping; McCammon, Mark T et al. (2005) Kinetic properties and metabolic contributions of yeast mitochondrial and cytosolic NADP+-specific isocitrate dehydrogenases. J Biol Chem 280:4469-75
Contreras-Shannon, Veronica; McAlister-Henn, Lee (2004) Influence of compartmental localization on the function of yeast NADP+-specific isocitrate dehydrogenases. Arch Biochem Biophys 423:235-46
McCammon, Mark T; McAlister-Henn, Lee (2003) Multiple cellular consequences of isocitrate dehydrogenase isozyme dysfunction. Arch Biochem Biophys 419:222-33

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