Higuchi Biosciences Center - University of Kansas

Fall 2008 Science Talks
December 5, 2008

F1. Characterization of inter-domain electron transfer in Ncb5or, a redox enzyme involved in fatty acid desaturation
Bin Deng¹, Sudharsan Parthasarathy³, Ming Xu¹, David R. Benson^2,3, Hao Zhu^1,4,5
Departments of ¹Physical Therapy and Rehabilitation Science, ⁴Clinical Laboratory Sciences, and ⁵Biochemistry and Molecular Biology, University of Kansas Medical Center, Kansas City, KS 66160
Departments of ²Chemistry and ³Molecular Biosciences, University of Kansas, Lawrence, KS 66045

Microsomal cytochrome b5 (b5) and cytochrome b5 reductase (b5R) are singular redox proteins that donate electrons to stearoyl-CoA desaturase (SCD) in fatty acid desaturation. NADH cytochrome b5 oxidoreductase (Ncb5or) contains b5 and b5R domains, and a linkage domain which shares homology with the CS (CHORD-SGT1) family members, including HSP20 (heat shock protein 20, a putative partner of HSP90). Our recent studies show that Ncb5or functions as an alternative electron donor for SCD. Ncb5or knockout mice display lipoatrophy and diabetes as a result of impaired fatty acid desaturation. The purpose of this study is to characterize the properties and interactions of individual Ncb5or domains, and potential sites for functional modulation of inter-domain electron transfer. The b5, b5R and CS+b5R domains were generated individually from a bacterial expression system, purified to homogeneity and used for functional studies. The b5R and CS+b5R domains tend to aggregate under low salt conditions, but are stable under medium salt conditions. In contrast, the b5 domain is stable under all salt conditions. Each of the native b5, b5R and CS+b5R proteins formed a distinct band in native gel electrophoresis. While bands for b5R and CS+b5R were absent if the proteins had been heated to 60 ºC for 4 minutes prior to loading, the b5 band was not affected by the same treatment. Inter-domain electron transfer can be partially reconstituted by mixing b5 with b5R or CS+b5R in the presence of NADH. Compared to 100% heme reduction in native Ncb5or, only about 20% of heme iron was reduced with the mixture of b5 and b5R (or CS+b5R) proteins under medium salt conditions. Interestingly, under low salt conditions, electron transfer was greatly enhanced, resulting in ~ 80% heme reduction with the same mixture. The presence of the CS domain appeared to have no impact on the electron flow from b5R to b5 domains. We conclude that the CS linkage domain may help the proper docking between the b5 and b5R domains, but is not essential to this interaction.

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F2. How Do Reactive Metabolites Kill Cells? The Functional Proteomics of Xenobiotic Electrophiles
Robert Hanzlik, Yakov Koen, Jianwen Fang
Medicinal Chemistry, University of Kansas, KU-MedChem, 4048 Malott Hall, 1251 Wescoe Hall Drive, Lawrence, KS, 66045-7582

Background. Post-translational modification is a well-known mechanism for regulating protein activity. In parallel with this, the covalent binding of reactive metabolites of xenobiotic drugs and chemicals is often strongly associated with cytotoxicity. The detailed chemistry of protein adduct formation is well understood, but major unanswered questions are 1) which proteins become adducted, and 2) how does the cell recognize and react to their covalent modification by xenobiotic residues.

Methods. Rats were given a hepatotoxic dose of [C-14]-bromobenzene or [C-14]-thiobenzamide. After 4 hr microsomal and cytosolic proteins were resolved by 2D gel electrophoresis, and the proteins in over 250 radioactive spots were identified by in-gel digestion and peptide mass mapping. The >100 proteins identified were compiled with >100 others from literature reports (http://tpdb.medchem.ku.edu:8080/protein_database), and analyzed to illuminate possible mechanisms of toxicity.

Results. Covalent binding by reactive metabolites is selective, not random. Among >200 target proteins of 21 small molecules, evidence of commonality is modest; the most common protein target is "hit" by only 7 of 21 chemicals while only 8 proteins are hit by 4 or more of the 21 chemicals. The target proteins have diverse biological roles as enzymes of intermediary metabolism, xenobiotic metabolism, binding/carrier proteins, and proteins related to protein folding, heat shock and stress responses; no single protein or class plausibly accounts for cytotoxicity. For 21 target proteins common to 3 or more reactive metabolites, bioinformatic methods identified 165 partner proteins and 528 known interactions. Sorting the 186 proteins into Gene Ontology categories and KEGG pathways revealed highly significant enrichments in several categories of interest in relation to cytotoxicity.

Conclusion. Functional proteomics combined with bioinformatic analysis of protein interactions has implicated the potential involvement of several functional and signaling pathways in cytotoxicity caused by chemically reactive metabolites. (Supported by NIH GM21784).

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F3. Transgenic mice over-expressing Glud1 in neurons: Selective age-associated neuronal, dendritic and synaptic losses, stress-related genomic expression changes, and increased sensitivity to Aβ
R. Pal*, X. Bao, X. Wang, D. Hui, A. Agbas, and E. K. Michaelis
Dept. of Pharmacology and Toxicology and the Higuchi Biosciences Center, University of Kansas, Lawrence, KS 66047

During aging, there is increased accumulation of glutamate (Glu) in the extracellular environment of neurons. The effects of gradual accumulation of extracellular glutamate (Glu) in the central nervous system (CNS) occurring during aging or in age-associated neurodegenerative diseases are not as well characterized as those of acute neurotoxicity produced by Glu. We created a transgenic (Tg) mouse model of life-long excess Glu release in CNS by introducing the transgene of Glu dehydrogenase 1 (Glud1) under the control of the neuron-specific enolase promoter. We reported previously that increased levels of GLUD protein and activity in neurons of hemizygous Tg mice was associated with increases in depolarization-induced, in vivo release of Glu, age-related neuronal losses in select brain regions, and subtle changes in behavior. In the CA1 region of the hippocampus, a vulnerable brain region, there were age-associated losses of dendrites (labeled with anti-MAP2A) and synapses (labeled with anti-synaptophysin), in addition to neuronal losses. Thus, the Glud1 hemizygous Tg mouse may be a model of accelerated effects of chronic, moderate, lifelong excess of Glu formation in brain. To explore the response of neurons to such chronic Glu hyperactivity and associated stress, we conducted microarray expression analyses of the hippocampus from 8-mo old Tg and wt mice, i.e., prior to the onset of extensive neuronal damage. There was significant up-regulation of genes involved in oxidative stress, neuroinflammation, and cellular Ca²⁺ regulation in Tg mice, processes also associated with AD pathogenesis. Hippocampus CA1 neurons from Glud1 mice were also significantly more sensitive to Aβ oligomer-induced toxicity than those from wt mice. Hippocampus cells in Glud1 mice also showed upregulation of genes related to dendrite and axon formation/growth, including that of proline-rich tyrosine kinase (PTK2B). Thus, despite the loss of dendrites in Tg mice, there was a significant relative increase in PTK2B and phospho-PTK2B protein levels in CA1 of Tg mice (increased ratio of PTK2B or phospho-PTK2B to MAP2), a sign of neuronal attempts toward neurite restructuring in the presence of the stress of a hyperglutamatergic state.

Supported by grants NIA AG12993, NICHD HD02528, NIAAA AA11419 and AA04732.

xiaodong bao [mailto:xiaodong_bao@yahoo.com]

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