| Budget Amount *help |
¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2012: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2011: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2010: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Research Abstract |
In previous study, we demonstrated that diacylglycerol kinase DGKzeta which is distributed at nucleus in normal hippocampal neuron in vivo, is translocated to cytoplasm from nucleus by middle cerebral artery infarction in rat. But it is unclear that the mechanisms of ischemia induced translocation of DGKzeta and the physiological functions after localization in cytoplasm of DGKzeta. In this study, we demonstrated that transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin.proteasome system UPSrather than transcriptional inhibition. DGKζ-deficient hippocampus exhibited a significant increase in Ser807811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. From a functional perspective, in vitro gene silencing of D
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GKzeta via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocam-pal neurons of DGKzeta-deficient mice showed vulnerability to kainate-induced seizures. In addition, nucleosome assembly pro-tein NAP1-like 1 NAP1L1and NAP1-like 4 NAP1L4are identified as novel DGKzeta binding partners. The molecular interaction of DGKzeta and NAP1Ls prohibits nuclear import of DGKzeta because binding of NAP1Ls to DGKzeta blocks import carrier proteins, Qip1 and NPI1, to interact with DGKzeta, leading to cytoplasmic tethering of DGKzeta. In addition, overexpression of NAP1Ls ex-erts a protective effect against doxorubicin-induced cytotoxicity. Additionally, we identified a small GTPase effector protein, IQGAP1, as a novel DGKzeta-associated complex protein. A bacterial endotoxin, lipopolysaccharide LPSfacilitated the complex formation in macrophages. RNA interference-mediated knockdown of DGKzeta or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKzeta-mice attenuated LPS-induced phagocytosis of bacteria. DGKzeta is involved in IQGAP1Rac1-mediated phagocytosis upon LPS stimulation in macrophages. Less
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