研究概要 |
The accumulation of glutamate, which occurs immediately after ischemia, results in excessive stimulation of glutamate receptors (NMDA, AMPA receptors) leading to neurotoxicity. Thus, suppressing glutamate receptor function has gained significant interest towards the therapeutic treatment of ischemic stroke. However, clinical application of glutamate receptor antagonists in stroke treatment has failed since these treatments suppress postsynaptic glutamate response that is needed for normal brain function. Therefore, alternative targets/pathways for therapeutic treatment of ischemic stroke must be sought. In the past year, we used GST-fusion proteins encoding fragments of GluR2 subunit of AMPA receptor to affinity pull down GAPDH. By repeating the GST-pull down experiment, we have successfully delineated the region (Y142-K172) of GluR2 involved in the interaction with GAPDH. The presence of peptide encoding Y142-K172 region of GluR2 is able to competitively interfere with the protein complex. Moreover, we have confirmed that AMPAR/GAPDH protein complex is formed through a direct interaction by performing in vitro binding assays. Together, these data suggest that GAPDH formed a direct protein-protein interaction with the GluR2 subunit through the Y142-K172 region. Since this Y142-K172 region is located on the extracellular part of GluR2 subunit of AMPA receptor, we would like to investigate that through what mechanism (s) would intracellular GAPDH interact with the extracellular region of GluR2, together with the function studies of this protein complex.
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現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
As described above, the project is progressing according to the research plan. In addition, we have found that GAPDH interacts with GluR2 extracellularlly, which triggers more interests in unveiling the mechanism of this protein complex and implies novel findings of the future functional studies.
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今後の研究の推進方策 |
In the coming year, we will continue investigating the function of the GluR2/GAPDH complex, including: 1. The mechanism through which GAPDH interacts with GluR2 extracellularlly. 2. Whether the membrane expression of AMPA receptor is affected by the GluR2/GAPDH interaction. 3. The physiological relevance of this protein complex with focus on AMPA receptor-mediated cell death.
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次年度の研究費の使用計画 |
I was in my childcare leave in April, 2013, therefore there was no spending in that month. In addition, we have found that GluR2 interacts with GAPDH through DIRECT protein-protein interaction, which saves the amount for investigating the possible additional proteins that mediate the interaction. The grant will be spent according to the research plan for 2014 to investigate the physiological functional roles of GluR2/GAPDH protein complex. Most protein-protein interactions occur intracellularly, yet we have discovered that GluR2 interacts with GAPDH extracellularly. Therefore, the amount transferred from 2014 will be spent on investigating the mechanism of this unusual extracellular protein-protein interaction and its possible downstream signaling pathways.
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