| 研究実績の概要 |
According to our research plan, we performed quantitative phosphoproteomic studies of whole mouse brains of sleep-deprived mice and Sleepy mutant mice. A combined proteome and phosphoproteome data for 9,410 mouse proteins and 62,384 phosphopeptides were examined. We identifies 80 mostly synaptic Sleep-Need-Index-PhosphoProteins (SNIPPs), whose phosphorylation states closely parallel changes of sleep need due to sleep-deprivation and Sleepy mutation. To examine whether SNIPPs are substrates of SLEEPY kinase, we compared the interactomes of SLEEPY and SIK3 by immunoprecipitation (IP) and mass spectrometric analysis using whole-brain lysates from the Flag-HA-Sik3+ and Flag-HA-Sik3Slp knock-in mice. Accordingly, SLEEPY preferentially associated with synaptic proteins, including 28 of 80 SNIPPs. The 28 SLEEPY-interacting SNIPPs contain 47 putative AMPK sites showing significant changes, of which 40 (85%) become hyper-phosphorylated, in Sleepy brains. Taken together, these observations suggest that SLEEPY may increase phosphorylation of SNIPPs by enhancing kinase-substrate association. Inhibition of SIK3 activity reduces phosphorylation state of SNIPPs and slow wave activity (SWA) during non-rapid-eye-movement sleep (NREMS), in both Sleepy and sleep-deprived wild-type mice. Our results suggest that SNIPPs accumulate/dissipate phosphorylation as the molecular substrate of sleep need.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
1: 当初の計画以上に進展している
理由
We have finished (IP) and mass spectrometric analysis using whole-brain lysates from the Flag-HA-Sik3+ and Flag-HA-Sik3Slp knock-in mice. We also applied the “AMPK Motif Analyzer27” to predict 2,943 phosphopeptides as potential AMPK-like substrates in the Slp/WT phosphoproteome dataset. The 28 SLEEPY-interacting SNIPPs contain 47 putative AMPK sites showing significant changes, of which 40 (85%) become hyper-phosphorylated, in Sleepy brains. Taken together, these observations suggest that SLEEPY may increase phosphorylation of SNIPPs by enhancing kinase-substrate association.
We also attempted to rescue the phenotypes of Sleepy mice by intracerebroventricular (ICV) injection of HG-9-91-01 (HG) to inhibit SLEEPY/SIK3 kinase activity. Administration of HG significantly reduced phosphorylation of AMPK substrates, particularly those from 28 SLEEPY-interacting SNIPPs. Accordingly, inhibition of SLEEPY activity reduced phospho-state of SNIPPs and SWA. Similarly, inhibition of SIK3 activity reduced phosphorylation of AMPK substrates, phospho-state of SNIPPs and SWA of NREMS in sleep-deprived wild-type mice, suggesting a critical role of SIK3-SNIPPs in normal homeostatic sleep regulation.
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