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

肿瘤和几种代谢性疾病的共同特征是血管张力增加。高合成代谢状态是 通常伴有全身性的卡他命抑制（例如，溶酶体生物发生和自噬）。 巧合的是，合成代谢细胞增加了对溶酶体降解途径的依赖性，以抵消细胞的代谢。 强制性增加的压力，如功能失调的细胞器和活性氧。但 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（PI 5 P4 K）是一个酶家族，由PI 5 P4 K α，β， γ，并将脂质第二信使磷脂酰肌醇5-磷酸（PI 5 P）转化为磷脂酰肌醇 4，5-磷酸（PI（4，5）P2）。PI 5 P4 K的主要功能被认为是控制PI 5 P依赖性信号传导， PI（4，5）P2的大部分由另一个酶家族PI 4P 5 Ks产生。三种基因缺失研究 小鼠中PI 5 P4 K家族（Pip 4k 2a、Pip 4k 2b和Pip 4k 2c）中的基因表明PI 5 P4 K β发挥独特的作用， 在介导细胞对应激反应中的关键作用（例如，营养剥夺，ROS），并最终影响 全身胰岛素敏感性、生长、肥胖和癌症。重要的是，PI 5 P4 K是非典型激酶， 使用GTP作为磷酸供体的独特性质。特别是，PI 5 P4 K β优先使用GTP，而不是 ATP，其激酶活性受生理GTP浓度调节，作为细胞GTP传感器 代谢和肿瘤发生的机制尚未确定。关于这项建议，我们的小组已 使用新开发的基于NMR的筛选开发了同工酶选择性PI 5 P4 K抑制剂，并发现 PI 5 P4 K抑制剂的处理抑制了溶酶体酸化。新生成的GTP不敏感 与WT小鼠相比，Pip 4k 2bF 205 L/F205 L小鼠发生了严重的脂肪变性，并表现出低血糖增加 在禁食时，类似于自噬缺陷的表型。我们假设GTP依赖性PI 5 P4 K β 激活促进溶酶体酸化以平衡应激弹性细胞的合成代谢应激 生长和肝功能。利用我们与一些国家和地区的长期、富有成效的合作， 尖端的实验室，我们将定义PI 5 P4 K β在转录独立的机制中的作用， 溶酶体酸化和抗应激生长。利用我们所研究的“结构反向遗传学”框架， 最近发展起来的，我们将解剖和确定激酶活性和支架功能的作用， PI 5 P4 K β（目的1）。我们将检验GTP依赖性PI 5 P4 K β活性是肝细胞凋亡所必需的这一假设。 溶酶体功能和全身能量稳态（目的2）。在完成拟议的研究后，我们 将确定GTP介导的PI 5 P4 K β激活促进的新型应激抵消系统 溶酶体活化以支持应激弹性合成代谢细胞生长并保护小鼠免受 代谢性疾病

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.