Phosphoproteomic Analyses of Understudied Protein Kinases that affect Zebrafish Sleep

影响斑马鱼睡眠的正在研究的蛋白激酶的磷酸化蛋白质组学分析

• 批准号: 10437190
• 负责人: Tsui-Fen Chou
• 金额: $ 16.84万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-07 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10437190
• 关键词: Affect; Animals; Arousal; Biological Assay; Brain; Data; Disease; Gene Targeting; Genes; Genetic; Genetic Screening; Genetic study; Genome; Homeostasis; Human; Human Genetics; Human Genome; Mediating; Motor Activity; Orthologous Gene; Phenotype; Phosphorylation; Phosphotransferases; Play; Protein Dephosphorylation; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Proteome; Role; Sleep; Sleep Disorders; Testing; United States National Institutes of Health; Vaccinia; Variant; Wakefulness; Zebrafish; base; experimental study; gain of function; gain of function mutation; genome wide association study; loss of function; loss of function mutation; mutant; new therapeutic target; phosphoproteomics; pressure; response; salt-inducible kinase

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.

抽象的 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。 清醒会导致大脑中许多蛋白质过度磷酸化，而睡眠会促进 大脑蛋白质组的去磷酸化导致盐诱导激酶 3 (sik3) 的功能获得性突变。 类似于长时间清醒的大脑状态，并且与子集的过度磷酸化相关 野生型动物在长时间清醒期间过度磷酸化的蛋白质。 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发挥作用。 觉醒在睡眠期间消失，并且这种磷酸化部分是由 sik3 介导的，尽管它是 明确涉及其他蛋白激酶然而，这些蛋白激酶的特性仍然存在。 未知。基于一项大型（> 500,000 名受试者）人类全基因组关联研究 (GWAS)，该研究已确定。 人类基因组中的许多位点与人类睡眠变异和睡眠障碍有关，我们正在 使用斑马鱼进行有针对性的遗传筛选，以确定每个基因座中的四个相关基因。 此屏幕中的目标基因被列为“未充分研究的蛋白质”，作为 NIH“照亮可药物”的一部分 我们发现其中一种斑马鱼的同源基因存在功能丧失突变。 我们将检验以下假设：(1) 其他三个 IDG 基因所针对的。 屏幕中的基因也调节睡眠，并且 (2) 这些基因在大脑磷酸化蛋白质组的变化中发挥作用 我们将通过分析每个突变体的睡眠来检验假设（1）。 对于假设（1）正确的基因，使用运动活性和唤醒阈值测定的表型。 我们将通过表征斑马鱼大脑磷酸蛋白质组对增益和损失的响应来检验假设（2） 每个基因的功能性遗传扰动将提供第一个被证明的功能。 对于正在测试的 IDG 基因，表征这些蛋白激酶对大脑磷酸化蛋白质组的影响， 并根据这些实验探索可能是睡眠稳态基础的令人兴奋的新机制。 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，并提供新的药物靶点来治疗这些疾病。