Regulation of the Glutamate/glutamine Cycle by Nitric Oxide in the Central Nervous System

Loading...
Thumbnail Image
Degree type
Doctor of Philosophy (PhD)
Graduate group
Neuroscience
Discipline
Subject
Glutamate
Nitric Oxide
Proteomics
S-Nitrosylation
Biochemistry
Biology
Neuroscience and Neurobiology
Funder
Grant number
License
Copyright date
2016-11-29T00:00:00-08:00
Distributor
Related resources
Contributor
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.

Advisor
Harry Ischiropoulos
Robert G. Kalb
Date of degree
2015-01-01
Date Range for Data Collection (Start Date)
Date Range for Data Collection (End Date)
Digital Object Identifier
Series name and number
Volume number
Issue number
Publisher
Publisher DOI
Journal Issue
Comments
Recommended citation