Efficient intracellular electron transfer is essential to organisms that use oxygen. An exquisite balance between oxidized and reduced enzymes and cofactors, termed redox homeostasis, must be carefully regulated to maintain cellular function. Loss of redox balance underlies molecular changes associated with aging and age related diseases. Consequences of acute or chronic disruption in redox homeostasis include neurodegenerative diseases, cancer, diabetes mellitus, atherosclerosis, and rheumatoid arthritis. To investigate the specific molecular and chemical events that govern redox balance, structural and biochemical studies of enzymes that preserve cellular redox potential will be examined. In particular, the proposed research will consider the enzymatic pathways responsible for the maintenance of reduced thioredoxin and glutathione pools by addressing the following specific aims: (i) Characterize the electron transfer cascade catalyzed by mitochondrial thioredoxin reductase at the molecular level. The goal of this aim is to use structural analysis and biochemical characterizations to examine the mechanistic details of the thioredoxin system as it pertains to mitochondrial redox homeostasis. We will determine the crystal structures of mitochondrial thioredoxin and glutaredoxin alone and in complex with mitochondrial thioredoxin reductase. (ii) investigate the structural and mechanistic features of essential glutathione homeostasis enzymes. The goal of this aim is to gain insight into the regulation of glutathione levels. We will examine the molecular details of allosteric regulation of glutamate cysteine ligase, which catalyzes the committed step of glutathione bisoynthesis. We will also examine the auto-activation mechanism of g-glutamyl transpeptidase, an ectoenzyme required for glutathione salvage, and the effects of self-processing on catalytic activity. Structural characterizations of these key thioredoxin and glutathione systems will provide new insights into mechanisms of cellular redox homeostasis. Understanding the crucial details may translate to new therapeutic targets in the array of difficult health problems caused by oxidative damage. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM077289-02
Application #
7196498
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Jones, Warren
Project Start
2006-04-01
Project End
2011-03-31
Budget Start
2007-04-01
Budget End
2008-03-31
Support Year
2
Fiscal Year
2007
Total Cost
$196,471
Indirect Cost
Name
University of Nebraska Lincoln
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
555456995
City
Lincoln
State
NE
Country
United States
Zip Code
68588
Ozer, Hatice K; Dlouhy, Adrienne C; Thornton, Jeremy D et al. (2015) Cytosolic Fe-S Cluster Protein Maturation and Iron Regulation Are Independent of the Mitochondrial Erv1/Mia40 Import System. J Biol Chem 290:27829-40
Liu, Yilin; Hyde, Annastasia S; Simpson, Melanie A et al. (2014) Emerging regulatory paradigms in glutathione metabolism. Adv Cancer Res 122:69-101
Hyde, Annastasia S; Thelen, Ashley M; Barycki, Joseph J et al. (2013) UDP-glucose dehydrogenase activity and optimal downstream cellular function require dynamic reorganization at the dimer-dimer subunit interfaces. J Biol Chem 288:35049-57
Hyde, Annastasia S; Farmer, Erin L; Easley, Katherine E et al. (2012) UDP-glucose dehydrogenase polymorphisms from patients with congenital heart valve defects disrupt enzyme stability and quaternary assembly. J Biol Chem 287:32708-16
Willis, Melanie Neely; Liu, Yilin; Biterova, Ekaterina I et al. (2011) Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits. Biochemistry 50:6508-17
Biterova, Ekaterina I; Barycki, Joseph J (2010) Structural basis for feedback and pharmacological inhibition of Saccharomyces cerevisiae glutamate cysteine ligase. J Biol Chem 285:14459-66
Biterova, Ekaterina I; Barycki, Joseph J (2009) Mechanistic details of glutathione biosynthesis revealed by crystal structures of Saccharomyces cerevisiae glutamate cysteine ligase. J Biol Chem 284:32700-8
Williams, Kristin; Cullati, Sierra; Sand, Aaron et al. (2009) Crystal structure of acivicin-inhibited gamma-glutamyltranspeptidase reveals critical roles for its C-terminus in autoprocessing and catalysis. Biochemistry 48:2459-67
Morrow, Amy L; Williams, Kristin; Sand, Aaron et al. (2007) Characterization of Helicobacter pylori gamma-glutamyltranspeptidase reveals the molecular basis for substrate specificity and a critical role for the tyrosine 433-containing loop in catalysis. Biochemistry 46:13407-14
Boanca, Gina; Sand, Aaron; Barycki, Joseph J (2006) Uncoupling the enzymatic and autoprocessing activities of Helicobacter pylori gamma-glutamyltranspeptidase. J Biol Chem 281:19029-37