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.

合成代谢增加是肿瘤和几种代谢疾病的共同特征。 通常伴随着分解代谢的系统性抑制（例如，溶酶体生物发生和自噬）。 矛盾的是，合成代谢细胞增加了对溶酶体降解途径的依赖，以抵消 必然增加的压力，例如细胞器故障和活性氧。 无论其合成代谢状态如何，细胞如何激活溶酶体功能的分子机制在很大程度上仍然存在 磷脂酰肌醇 5-磷酸 4-激酶 (PI5P4K) 是一个酶家族，由 PI5P4Kα、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 4,5-磷酸 (PI(4,5)P2) PI5P4K 的主要功能被认为是控制 PI5P 依赖性信号传导，如 大部分 PI(4,5)P2 是由另一个酶家族 PI4P5K 产生的，这三个酶的基因缺失研究。 小鼠中 PI5P4K 家族（Pip4k2a、Pip4k2b 和 Pip4k2c）中的基因表明 PI5P4Kβ 发挥独特且 在介导细胞对压力（例如营养剥夺、ROS）反应以及最终的反应中发挥关键作用 重要的是，PI5P4K 是与全身胰岛素敏感性、生长、肥胖和癌症相关的非典型激酶。 使用 GTP 作为磷酸供体的独特特性特别是，PI5P4Kβ 优先使用 GTP 而不是。 ATP 及其激酶活性受生理 GTP 浓度调节，充当细胞 GTP 传感器 关于代谢和肿瘤发生的机制尚未确定，我们的小组已经确定了这一点。 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，并发现 PI5P4K 抑制剂的治疗抑制了新产生的 GTP 不敏感的溶酶体酸化。 与 WT 小鼠相比，Pip4k2bF205L/F205L 小鼠出现严重的脂肪变性，并表现出低血糖增加 禁食后，我们勇敢地重新组装了自噬缺陷的表型，即 GTP 依赖性 PI5P4Kβ。 激活促进溶酶体酸化，以平衡抗应激细胞的合成代谢应激 利用我们与许多公司的长期、富有成效的合作。 尖端实验室，我们将定义 PI5P4Kβ 在转录独立性中的机制作用 使用我们的“结构反向遗传学”框架来研究溶酶体酸化和抗应激生长。 最近发展起来，我们将剖析并确定激酶活性和支架功能的作用 PI5P4Kβ（目标 1）：我们将检验肝脏需要依赖 GTP 的 PI5P4Kβ 活性这一假设。 溶酶体功能和全身能量稳态（目标 2）。 将确定新型压力对抗系统，GTP 介导的 PI5P4Kβ 激活可通过该系统促进 溶酶体激活支持抗应激合成代谢细胞生长并保护小鼠免受以下疾病的发病机制影响 代谢性疾病。