MAP4K3 regulation of cellular stress response pathways in health and disease

MAP4K3 对健康和疾病中细胞应激反应途径的调节

基本信息

批准号：
10530025
负责人：
ALBERT R LA SPADA
金额：
$ 202.26万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10530025
关键词：
5&apos -AMP-activated protein kinase Age Amino Acid Substitution Amino Acids Autophagocytosis Biological Biological Assay Brain Cell Cycle Checkpoint Cell Line Cell Nucleus Cell Survival Cells Cellular Stress Cellular Stress Response Complex Coupled Critical Pathways DNA DNA Damage Dimerization Disease Disinhibition Dose Embryo FRAP1 gene Frontotemporal Dementia Funding Generations Goals Health Homeostasis In Vitro Knock-in Mouse Lead Link Lysosomes Malignant Neoplasms Metabolic Metabolism Molecular Molecular Conformation Mus Nature Nerve Degeneration Nutrient Organ Pathway interactions Penetration Pharmaceutical Preparations Phenotype Phosphorylation Physiological Process Protein Kinase Proteomics Regimen Regulation Risk Factors Role SIRT1 gene STK11 gene Series Serine Signal Pathway Signal Transduction Site Structure-Activity Relationship Surface TSC1 gene TSC2 gene Tauopathies Testing Therapeutic Threonine Tissues Tuberous Sclerosis Validation Work base cancer therapy cell growth regulation disease phenotype drug discovery extracellular in silico inhibitor inhibitor therapy kinase inhibitor mouse model nervous system disorder pharmacokinetics and pharmacodynamics phosphoproteomics potency testing preclinical trial protein complex recruit response screening small molecule transcription factor

项目摘要

Regulation of cellular metabolic status is determined by the mTORC1 complex, which senses systemic nutrient levels and intracellular nutrients. We set out to define the signaling pathways controlling autophagy activation, and discovered that MAP4K3 phosphorylation of transcription factor EB (TFEB) dictates autophagy status in the cell, and documented that MAP4K3 autophagy regulation lies upstream of mTORC1 autophagy regulation. MAP4K3 activates mTORC1 when amino acids are plentiful, but the basis for this regulation is ill-defined. During the current funding cycle, we delineated the MAP4K3 – mTORC1 signaling pathway, linking MAP4K3 activation of mTORC1 to inhibition of AMP-activated protein kinase. Our findings reveal MAP4K3 inhibition of AMPK may occur via phosphorylation of Sirtuin-1 resulting in LKB1 inactivation. To understand the scope of MAP4K3 function, we completed an interactome and phosphoproteomics analysis, and implicated MAP4K3 in regulation of the GATOR1/2 complex, which controls mTORC1 localization to the lysosome, a prerequisite step for mTORC1 activation. We also determined that MAP4K3 can localize to the nucleus, and may participate in the DNA damage response. All these findings indicate that MAP4K3 is a central node for the regulation of cellular homeostasis, serving as a nexus for cross-talk between pathways of metabolism, cell stress, and cell survival. We have begun to examine the physiological relevance of MAP4K3 phosphoregulation by deriving lines of mice with phosphoresistant and phosphomimetic amino acid substitutions at the TFEB serine residue (S3) subject to MAP4K3 phosphorylation. As mTORC1 dysregulation is implicated in cancer and neurological disease, our results suggest that one appealing therapeutic strategy for diseases of altered mTOR signaling function would be to develop drugs to inhibit MAP4K3. To achieve this goal, we performed in silico screening for small molecules that interfere with MAP4K3 open pocket dimerization, and evaluated 13 compounds in a series of secondary and tertiary assays, identifying three promising hits. In this renewal project, we will define components of the MAP4K3 amino acid sensing dependent pathway of mTORC1 activation and delineate the molecular basis for MAP4K3 regulation of mTORC1 activation, focusing on Sirtuin- 1 phosphoregulation by MAP4K3 and the nature of MAP4K3 interaction with the GATOR1/2 complex. We propose to assess MAP4K3 regulation of autophagy and cell stress by carefully characterizing phenotypes and autophagy function in TFEB S3A and S3E mice, and determining if MAP4K3 is involved in modulating DNA damage responses. We will build on our MAP4K3 inhibitor translational drug discovery work by honing in on the most promising lead compounds through structure-activity relationship generation of a compound series, coupled with an independent in silico screen and kinase inhibitor potency testing followed by a critical path of validation assays, and we will test if our lead MAP4K3 inhibitor(s) can rescue disease phenotypes in a mouse model of tuberous sclerosis complex (TSC) and in frontotemporal dementia / tauopathy Tsc1 +/- mice .

细胞代谢状态的调节由 mTORC1 复合物决定，该复合物可感知全身营养我们着手定义控制自噬激活的信号通路，并发现转录因子 EB (TFEB) 的 MAP4K3 磷酸化决定了自噬状态细胞，并记录了 MAP4K3 自噬调节位于 mTORC1 自噬调节的上游。当氨基酸充足时，MAP4K3 会激活 mTORC1，但这种调节的基础尚不明确。在当前的融资周期中，我们描绘了 MAP4K3 – mTORC1 信号通路，将 MAP4K3 连接起来 mTORC1 的激活抑制 AMP 激活的蛋白激酶我们的研究结果揭示了 MAP4K3 的抑制作用。 AMPK 可能通过 Sirtuin-1 磷酸化而发生，导致 LKB1 失活了解其范围。 MAP4K3 功能，我们完成了相互作用组和磷酸蛋白质组分析，并暗示 MAP4K3 GATOR1/2 复合体的调节，控制 mTORC1 定位到溶酶体，这是一个先决步骤我们还确定 MAP4K3 可以定位于细胞核，并可能参与 mTORC1 激活。所有这些发现表明 MAP4K3 是调节 DNA 损伤反应的中心节点。细胞稳态，作为代谢途径、细胞应激、我们已经开始通过以下方法研究 MAP4K3 磷酸调节的生理相关性。衍生出 TFEB 丝氨酸具有抗磷性和拟磷性氨基酸取代的小鼠品系残基 (S3) 会被 MAP4K3 磷酸化，因为 mTORC1 失调与癌症有关。神经系统疾病，我们的结果表明 mTOR 疾病的一种有吸引力的治疗策略信号传导功能将是开发抑制 MAP4K3 的药物。为了实现这一目标，我们进行了计算机模拟。筛选干扰 MAP4K3 开放口袋二聚化的小分子，并评估 13 化合物进行了一系列二级和三级测定，在这次更新中确定了三个有希望的化合物。项目中，我们将定义 mTORC1 MAP4K3 氨基酸传感依赖性通路的组成部分激活并描述 MAP4K3 调节 mTORC1 激活的分子基础，重点关注 Sirtuin- 1 MAP4K3 的磷酸调节以及 MAP4K3 与 GATOR1/2 复合物相互作用的性质。建议通过仔细表征表型和细胞应激来评估 MAP4K3 对自噬和细胞应激的调节 TFEB S3A 和 S3E 小鼠的自噬功能，并确定 MAP4K3 是否参与 DNA 调节我们将通过磨练我们的 MAP4K3 抑制剂转化药物发现工作。通过化合物系列的构效关系生成最有前途的先导化合物，加上独立的计算机筛选和激酶抑制剂效力测试，然后是关键路径验证测定，我们将测试我们的主要 MAP4K3 抑制剂是否可以挽救小鼠的疾病表型结节性硬化症 (TSC) 模型和额颞叶痴呆/tau 蛋白病 Tsc1 +/- 小鼠。