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复合物确定，该复合物感应全身养分水平和细胞内营养。我们着手定义控制自噬激活的信号通路，并发现MAP4K3转录因子EB（TFEB）的辐射决定了自噬状态该单元格，并记录了MAP4K3自噬调节位于MTORC1自噬调节的上游。当氨基酸丰富时，MAP4K3激活MTORC1，但是该调节的基础是不确定的。在当前的融资周期中，我们描述了MAP4K3 - MTORC1信号通路，链接MAP4K3 MTORC1激活对AMP激活蛋白激酶的抑制。我们的发现揭示了MAP4K3的抑制 AMPK可能通过SIRTUIN-1的磷酸化而发生，从而导致LKB1失活。了解范围 MAP4K3功能，我们完成了一个Interactome和磷蛋白质组学分析，并在 Gator1/2复合物的调节，该复合物控制MTORC1定位到溶酶体，这是先决条件的步骤用于MTORC1激活。我们还确定MAP4K3可以定位于核，并且可以参与 DNA损伤响应。所有这些发现表明MAP4K3是调节的中心节点细胞稳态，作为新陈代谢，细胞应激途径之间的串扰的联系和细胞存活。我们已经开始检查MAP4K3磷酸调节的物理相关性在TFEB系列中衍生具有磷酸和磷酸氨基酸取代的小鼠线居住（S3）受MAP4K3磷酸化的约束。由于MTORC1失调与癌症有关神经疾病，我们的结果表明，MTOR改变的一种有吸引力的治疗策略信号传导功能是开发抑制MAP4K3的药物。为了实现这一目标，我们在计算机中表演筛选小分子会干扰MAP4K3开放口袋二聚化，并评估13 一系列二级和第三次测定中的化合物，确定了三个诺言。在此续约中项目，我们将定义MAP4K3氨基酸传感依赖途径的组件激活并描绘MAP4K3调节MTORC1激活的分子基础，重点是sirtuin- 1通过MAP4K3和MAP4K3与Gator1/2复合物相互作用的性质。我们通过仔细表征表型和 TFEB S3A和S3E小鼠中的自噬功能，并确定MAP4K3是否参与调节DNA 损坏反应。我们将在MAP4K3抑制剂翻译的药物发现工作上建立磨练通过化合物系列的结构活性关系生成的最有希望的铅化合物，结合有机筛网中的独立和激酶抑制剂效能测试，然后是一条临界路径验证测定法，我们将测试我们的铅MAP4K3抑制剂是否可以挽救小鼠中的疾病表型结节硬化症复合物（TSC）和额颞痴呆 / tauopathy TSC1 +/-小鼠的模型。