Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Neuroscience

First Advisor

Harry Ischiropoulos

Second Advisor

Robert G. Kalb

Abstract

Nitric oxide (˙NO) is a critical contributor to glutamatergic neurotransmission in the central nervous system (CNS). Much of its influence is due to the ability of this molecule to regulate protein structure and function through its posttranslational modification of cysteine residues, a process known as S-nitrosylation. However, little is known about the extent of this modification and its associated functional effects in the brain under physiological conditions. We employed mass spectrometry (MS)-based methodologies to interrogate the S-nitrosocysteine proteome in wild-type (WT), neuronal nitric oxide synthase-deficient (nNOS-/-), and endothelial nitric oxide synthase-deficient (eNOS-/-) mouse brain. These approaches identified 269 sites from 136 proteins in the WT brain, with notable reductions in the number of sites detected in either eNOS-/- (50% of WT) or nNOS-/- brain (26% of WT). Gene ontology analysis revealed a cluster of S-nitrosylated proteins participating in the glutamate/glutamine cycle in wild-type and eNOS-/- mice that was underrepresented in nNOS-/- animals, suggesting a role for nNOS-derived ˙NO in the regulation of glutamate utilization in the CNS. Functional profiling of this pathway using 15N-glutamine based metabolomic analyses and enzymatic activity assays uncovered decreased conversion of glutamate to glutamine and increased glutamate oxidation in nNOS-/- mice relative to the other genotypes. Furthermore, site-directed mutagenesis of the rat sodium-dependent excitatory amino acid transporter 2 at Cys373 and Cys562 (Cys561 in mouse sequence), the two sites of S-nitrosylation observed in wild-type and eNOS-/- mice, revealed inhibition of glutamate transport through reversible S-nitrosylation. The selective, nNOS-dependent S-nitrosylation of proteins that govern glutamate transport and metabolism identifies a previously unknown function for ˙NO in glutamatergic neurotransmission.

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