Mechanistic role of phosphatidylinositol 5-phosphate 4-kinase beta in GTP-dependent lysosomal acidification for stress-resilient cell growth and metabolism

磷脂酰肌醇5-磷酸4-激酶β在GTP依赖性溶酶体酸化对应激恢复细胞生长和代谢中的机制作用

• 批准号: 10592707
• 负责人: Atsuo Sasaki
• 金额: $ 37.23万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-12-15 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10592707
• 关键词: Acute; Affect; Amino Acid Substitution; Anabolism; Autophagocytosis; Binding; Biogenesis; Catabolism; Cell Proliferation; Cells; Cellular Metabolic Process; Cellular Stress; Collaborations; Coupled; Cytoprotection; Degradation Pathway; Dependence; Disease; Embryo; Energy Metabolism; Enzymes; Exhibits; Family; Fasting; Fibroblasts; Genes; Genetic; Genetic Transcription; Growth; Guanosine Triphosphate; Heart; Hepatic; Hepatocyte; High Fat Diet; Homeostasis; Hypoglycemia; Insulin Resistance; Isoenzymes; Knock-in Mouse; Knock-out; Laboratories; Lipids; Liver; Lysosomes; Malignant Neoplasms; Mediating; Metabolic; Metabolic Diseases; Metabolism; Modeling; Molecular; Mus; Obesity; Organelles; PIK3CG gene; Pathogenesis; Pathology; Phenotype; Phosphatidylinositols; Phosphotransferases; Physiological; Play; Predisposition; Productivity; Property; Proteins; Proteomics; Reactive Oxygen Species; Regulation; Reporting; Research; Role; Second Messenger Systems; Signal Transduction; Stress; System; Testing; Wisconsin; cell growth; human disease; inhibition of autophagy; inhibitor; inorganic phosphate; insulin sensitivity; kinase inhibitor; knock-down; multidisciplinary; novel; nutrient deprivation; pharmacologic; phosphatidylinositol 5-phosphate; public health relevance; recruit; response; reverse genetics; scaffold; screening; sensor; stress resilience; tumor; tumorigenesis; vacuolar H+-ATPase

Increased anabolism is a common feature of tumors and several metabolic diseases. The high anabolic state is typically accompanied by systemic suppression of catabolism (e.g., lysosome biogenesis and autophagy). Paradoxically, the anabolic cells increase dependence on the lysosomal degradation pathways to counteract the obligately increased stresses, such as malfunctioned organelle and reactive oxygen species. However, the molecular mechanism of how cells activate lysosomal functions regardless of their anabolic state remains largely unknown. Phosphatidylinositol 5-phosphate 4-kinase (PI5P4K) is a family of enzymes, consisting of PI5P4Kα, β, γ, and converts the lipid second messenger, phosphatidylinositol 5-phosphate (PI5P), to phosphatidylinositol 4,5-phosphate (PI(4,5)P2). The main function of PI5P4K is considered to control PI5P-dependent signaling, as the bulk of PI(4,5)P2 is generated from another family of enzymes, PI4P5Ks. Genetic deletion studies of the three genes in the PI5P4K family (Pip4k2a, Pip4k2b, and Pip4k2c) in mice indicate that PI5P4Kβ plays distinct and critical roles in mediating cellular responses to stress (e.g., nutrient deprivation, ROS) and ultimately affect whole-body insulin sensitivity, growth, obesity, and cancer. Importantly, PI5P4Ks are atypical kinases that have a unique property to use GTP as a phosphodonor. In particular, PI5P4Kβ preferentially uses GTP rather than ATP, and its kinase activity is regulated by physiological GTP concentrations, acting as a cellular GTP sensor for metabolism and tumorigenesis by mechanisms yet to be defined. Pertaining to this proposal, our group has developed isozyme selective PI5P4K inhibitors using newly developed NMR-based screening, and found that treatment of the PI5P4K inhibitors suppressed lysosome acidification. Newly generated GTP-insensitive Pip4k2bF205L/F205L mice developed severe steatosis compared to WT mice, and exhibited increased hypoglycemia upon fasting, resembling the phenotype of autophagy deficiency. We hypothesize that GTP-dependent PI5P4Kβ activation promotes lysosomal acidification to counterbalance the anabolic stress for stress-resilient cellular growth and hepatic functions. Capitalizing on our long-standing, productive collaborations with a number of cutting-edge laboratories, we will define the mechanistic role of PI5P4Kβ in transcriptionally-independent lysosomal acidification and stress-resilient growth. Using the “structural reverse-genetics” framework that we have developed recently, we will dissect and determine the role of kinase activity and scaffolding functions of PI5P4Kβ (Aim 1). We will test the hypothesis that GTP-dependent PI5P4Kβ activity is required for hepatic lysosomal function and whole-body energy homeostasis (Aim 2). Upon completing the proposed research, we will identify the novel stress counteracting system through which GTP-mediated activation of PI5P4Kβ promotes lysosomal activation to support stress-resilient anabolic cell growth and protect mice from the pathogenesis of metabolic diseases.

合成代谢状态增加是肿瘤和几种代谢疾病的共同特征。 通常是通过全身抑制分解代谢（例如溶酶体生物发生和自噬）来完成的。 矛盾的是，合成代谢细胞增加对溶酶体降解途径的依赖性，以抵消 应当增加应力，例如故障细胞器和活性氧。但是， 细胞如何激活溶酶体功能的分子机制，无论其合成代谢状态如何 未知。磷脂酰肌醇5-磷酸4-激酶（PI5P4K）是一个酶家族，由PI5P4Kα，β，β， γ，并将脂质第二信使磷脂酰肌醇5-磷酸（PI5P）转化为磷脂酰肌醇磷酸（PI5P） 4,5-磷酸（PI（4,5）P2）。 PI5P4K的主要功能被认为控制PI5P依赖性信号传导，作为 PI（4,5）P2的大部分是从另一个酶PI4P5K家族产生的。这三个的遗传缺失研究 PI5P4K家族（PIP4K2A，PIP4K2B和PIP4K2C）中的基因表明PI5P4Kβ在 在介导细胞对压力的反应（例如，营养剥夺，ROS）中的关键作用，最终影响 全身胰岛素敏感性，生长，肥胖和癌症。重要的是，PI5P4K是具有非典型激酶 将GTP用作磷酸的独特特性。特别是，PI5P4Kβ优先使用GTP而不是 ATP及其激酶活性受物理GTP浓度调节，充当细胞GTP传感器 对于尚未定义的机制，代谢和肿瘤发生。与该建议有关，我们的小组有 使用新开发的基于NMR的筛选开发了同工酶选择性PI5P4K抑制剂，发现 PI5P4K抑制剂的治疗抑制了溶酶体酸化。新生成的GTP不敏感 与WT小鼠相比 禁食后，类似于自噬缺陷的表型。我们假设依赖GTP的PI5P4Kβ 激活促进溶酶体酸化，以抵消应力富含胁迫细胞的合成代谢应激 生长和肝功能。利用我们长期以来的产品合作 尖端实验室，我们将定义PI5P4Kβ在转录独立的 溶酶体酸化和应激耐力生长。使用我们的“结构性反向基因学”框架 最近开发了，我们将剖析并确定激酶活性和脚手架功能的作用 PI5P4Kβ（AIM 1）。我们将测试以下假设：肝依赖GTP依赖性PI5P4Kβ活性 溶酶体功能和全身能量稳态（AIM 2）。完成拟议的研究后，我们 将确定GTP介导的PI5P4Kβ激活的新型应力抵消系统促进 溶酶体激活以支持应激抗性合成代谢细胞生长并保护小鼠免受发病机理 代谢疾病。