Phosphoproteomic Analyses of Understudied Protein Kinases that affect Zebrafish Sleep

摘要 最近的两项研究发现，睡眠和清醒对大脑磷酸化蛋白质组有相反的影响。延长 清醒导致大脑中许多蛋白质的过度磷酸化，而睡眠促进 大脑蛋白质组的去磷酸化。盐诱导激酶3（sik3）的功能获得性突变导致 一种类似于长时间清醒的大脑状态，与一个亚群的过度磷酸化有关 野生型动物在长时间的觉醒中过度磷酸化的蛋白质。这些 观察表明，蛋白质磷酸化可能在睡眠期间睡眠压力的积累中起作用。 在睡眠中消散的觉醒，这种磷酸化部分由sik 3介导，尽管它是 很明显，还涉及了其他蛋白激酶。然而，这些蛋白激酶的身份仍然是 未知基于一项大型（> 50万受试者）人类全基因组关联研究（GWAS）， 人类基因组中的许多基因座与人类睡眠变异和睡眠障碍有关， 使用斑马鱼进行靶向遗传筛选以鉴定每个基因座中的相关基因。中的四 在这个筛选中靶向的基因被列为“未充分研究的蛋白质”，作为NIH“阐明可药用 基因组”（IDG）项目。我们发现，在斑马鱼的直系同源物中， 人类基因导致睡眠减少。我们将检验以下假设：（1）其他三个IDG基因靶向 在屏幕上也调节睡眠，（2）这些基因在大脑磷酸化蛋白质组的变化中发挥作用 与睡眠或清醒有关的。我们将通过测定每个突变体的睡眠来检验假设（1 表型使用自发活动和唤醒阈值测定。对于假设（1）正确的基因， 我们将通过描述斑马鱼脑磷酸化蛋白质组对增益和损失的响应来检验假设（2）， 每个基因的功能遗传扰动。这些实验将提供第一个演示功能 对于所测试的IDG基因，表征这些蛋白激酶对脑磷酸化蛋白质组的影响， 并探索一种令人兴奋的新机制，它可能是睡眠稳态的基础。根据这些实验， 人类GWAS数据，该项目还将揭示一些观察到的人类睡眠变化的基础， 一些人类睡眠障碍，并提供了新的药物靶点来治疗这些障碍。

ABSTRACT Two recent studies found that sleep and wake have opposing effects on the brain phosphoproteome. Prolonged wakefulness results in hyperphosphorylation of many proteins in the brain, whereas sleep promotes dephosphorylation of the brain proteome. A gain-of-function mutation in salt-inducible kinase 3 (sik3) results in a brain state similar to that of prolonged wakefulness and is associated with hyperphosphorylation of a subset of the proteins that are hyperphosphorylated in wild-type animals during prolonged wakefulness. These observations suggest that protein phosphorylation may play a role in the accumulation of sleep pressure during wakefulness that dissipates during sleep, and that this phosphorylation is mediated in part by sik3, although it is clear that additional protein kinases are involved. However, the identity of these protein kinases remains unknown. Based on a large (>500,000 subjects) human genome-wide association study (GWAS) that identified many loci in the human genome that are associated with human sleep variation and sleep disorders, we are performing a targeted genetic screen using zebrafish to identify the relevant gene in each locus. Four of the genes targeted in this screen are listed as “Understudied Proteins” as part of the NIH “Illuminating the Druggable Genome” (IDG) project. We have found that loss-of-function mutations in the zebrafish ortholog of one of these human genes results in decreased sleep. We will test the hypotheses that (1) the three other IDG genes targeted in the screen also regulate sleep, and that (2) these genes play roles in changes in the brain phosphoproteome that are associated with sleep or wakefulness. We will test hypothesis (1) by assaying each mutant for sleep phenotypes using locomotor activity and arousal threshold assays. For genes in which hypothesis (1) is correct, we will test hypothesis (2) by characterizing the zebrafish brain phosphoproteome in response to gain- and loss- of-function genetic perturbations of each gene. These experiments will provide the first demonstrated functions for the IDG genes being tested, characterize effects of these protein kinases on the brain phosphoproteome, and explore an exciting new mechanism that may underlie sleep homeostasis. Based on these experiments and human GWAS data, this project will also reveal the basis for some of the observed variation in human sleep and some human sleep disorders and provide new druggable targets to treat these disorders.