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Identification of tyrosine-nitrated proteins in HT22 hippocampal cells during glutamate-induced oxidative stress.

ABSTRACT: OBJECTIVES:Nitration of tyrosine residues in protein is a post-translational modification, which occurs under oxidative stress, and is associated with several neurodegenerative diseases. To understand the role of nitrated proteins in oxidative stress-induced cell death, we identified nitrated proteins and checked correlation of their nitration in glutamate-induced HT22 cell death. MATERIALS AND METHODS:Nitrated proteins were detected by western blotting using an anti-nitrotyrosine antibody, extracted from matching reference 2-dimensional electrophoresis gels, and identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS:Glutamate treatment induced apoptosis in HT22 cells, while reactive oxygen species (ROS) inhibitor or neuronal nitric oxide synthase (nNOS) inhibitor blocked glutamate-induced HT22 cell death. Nitration levels of 13 proteins were increased after glutamate stimulation; six of them were involved in regulation of energy production and two were related to apoptosis. The other nitrated proteins were associated with calcium signal modulation, ER dysfunction, or were of unknown function. CONCLUSIONS:The 13 tyrosine-nitrated proteins were detected in these glutamate-treated HT22 cells. Results demonstrated that cell death, ROS accumulation and nNOS expression were related to nitration of protein tyrosine in the glutamate-stimulated cells.

SUBMITTER: Yoon SW

PROVIDER: S-EPMC6496341 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Identification of tyrosine-nitrated proteins in HT22 hippocampal cells during glutamate-induced oxidative stress.

Yoon S-W SW Kang S S Ryu S-E SE Poo H H

Cell proliferation 20101201 6

<h4>Objectives</h4>Nitration of tyrosine residues in protein is a post-translational modification, which occurs under oxidative stress, and is associated with several neurodegenerative diseases. To understand the role of nitrated proteins in oxidative stress-induced cell death, we identified nitrated proteins and checked correlation of their nitration in glutamate-induced HT22 cell death.<h4>Materials and methods</h4>Nitrated proteins were detected by western blotting using an anti-nitrotyrosine ...[more]

PMID: 21039997

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Project description:Protein kinase A (PKA) is a ser/thr kinase that is critical for maintaining essential neuronal functions including mitochondrial homeostasis, bioenergetics, neuronal development, and neurotransmission. The endogenous pool of PKA is targeted to the mitochondrion by forming a complex with the mitochondrial scaffold A-kinase anchoring protein 121 (AKAP121). Enhanced PKA signaling via AKAP121 leads to PKA-mediated phosphorylation of the fission modulator Drp1, leading to enhanced mitochondrial networks and thereby blocking apoptosis against different toxic insults. In this study, we show for the first time that AKAP121/PKA confers neuroprotection in an in vitro model of oxidative stress induced by exposure to excess glutamate. Unexpectedly, treating mouse hippocampal progenitor neuronal HT22 cells with an acute dose or chronic exposure of glutamate robustly elevates PKA signaling, a beneficial compensatory response that is phenocopied in HT22 cells conditioned to thrive in the presence of excess glutamate but not in parental HT22 cells. Secondly, redirecting the endogenous pool of PKA by transiently transfecting AKAP121 or transfecting a constitutively active mutant of PKA targeted to the mitochondrion (OMM-PKA) or of an isoform of AKAP121 that lacks the KH and Tudor domains (S-AKAP84) are sufficient to significantly block cell death induced by glutamate toxicity but not in an oxygen deprivation/reperfusion model. Conversely, transient transfection of HT22 neuronal cells with a PKA-binding-deficient mutant of AKAP121 is unable to protect against oxidative stress induced by glutamate toxicity suggesting that the catalytic activity of PKA is required for AKAP121's protective effects. Mechanistically, AKAP121 promotes neuroprotection by enhancing PKA-mediated phosphorylation of Drp1 to increase mitochondrial fusion, elevates ATP levels, and elicits an increase in the levels of antioxidants GSH and superoxide dismutase 2 leading to a reduction in the level of mitochondrial superoxide. Overall, our data supports AKAP121/PKA as a new molecular target that confers neuroprotection against glutamate toxicity by phosphorylating Drp1, to stabilize mitochondrial networks and mitochondrial function and to elicit antioxidant responses.

Project description:Significance"Nitroproteomic" is under active development, as 3-nitrotyrosine in proteins constitutes a footprint left by the reactions of nitric oxide-derived oxidants that are usually associated to oxidative stress conditions. Moreover, protein tyrosine nitration can cause structural and functional changes, which may be of pathophysiological relevance for human disease conditions. Biological protein tyrosine nitration is a free radical process involving the intermediacy of tyrosyl radicals; in spite of being a nonenzymatic process, nitration is selectively directed toward a limited subset of tyrosine residues. Precise identification and quantitation of 3-nitrotyrosine in proteins has represented a "tour de force" for researchers. Recent Advances: A small number of proteins are preferential targets of nitration (usually less than 100 proteins per proteome), contrasting with the large number of proteins modified by other post-translational modifications such as phosphorylation, acetylation, and, notably, S-nitrosation. Proteomic approaches have revealed key features of tyrosine nitration both in vivo and in vitro, including selectivity, site specificity, and effects in protein structure and function.Critical issuesIdentification of 3-nitrotyrosine-containing proteins and mapping nitrated residues is challenging, due to low abundance of this oxidative modification in biological samples and its unfriendly behavior in mass spectrometry (MS)-based technologies, that is, MALDI, electrospray ionization, and collision-induced dissociation.Future directionsThe use of (i) classical two-dimensional electrophoresis with immunochemical detection of nitrated proteins followed by protein ID by regular MS/MS in combination with (ii) immuno-enrichment of tyrosine-nitrated peptides and (iii) identification of nitrated peptides by a MIDAS™ experiment is arising as a potent methodology to unambiguously map and quantitate tyrosine-nitrated proteins in vivo. Antioxid. Redox Signal. 26, 313-328.

Dataset Information

Identification of tyrosine-nitrated proteins in HT22 hippocampal cells during glutamate-induced oxidative stress.

Publications

Identification of tyrosine-nitrated proteins in HT22 hippocampal cells during glutamate-induced oxidative stress.

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