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

合成代谢增加是肿瘤和几种代谢性疾病的共同特征。高合成代谢状态是 通常伴随着分解代谢的全身性抑制(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 不可避免地增加压力，如细胞器故障和活性氧物种。然而， 细胞如何激活溶酶体功能而与其合成代谢状态无关的分子机制在很大程度上仍然存在 未知。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依赖于PI5P的信号，AS PI(4，5)P2的大部分是由另一个酶家族PI4P5Ks产生的。三者的基因缺失研究 小鼠PI5P4K家族的基因(PIP4K2a、Pip4k2b和Pip4k2c)表明，PI5P4Kβ发挥着独特的 在调节细胞对压力的反应(例如，营养缺乏，ROS)中的关键作用并最终影响 全身胰岛素敏感性、生长、肥胖和癌症。重要的是，PI5P4K是一种非典型的具有 将GTP用作磷光体的独特性质。特别是，PI5P4Kβ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感受器 对新陈代谢和肿瘤发生的作用机制尚未确定。关于这项建议，我们小组已经 开发了同工酶选择性PI5P4K抑制剂，使用新开发的基于核磁共振的筛选，并发现 PI5P4K抑制剂处理可抑制溶酶体酸化。新生成的GTP不敏感 与WT小鼠相比，PIP4K2bF205L/F205L小鼠发生了严重的脂肪变性，并表现出更高的低血糖 禁食时，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激弹性细胞的合成代谢应激 生长和肝功能。充分利用我们与多家公司长期高效的合作关系 尖端实验室，我们将定义PI5P4Kβ在转录独立的机制中的作用 溶酶体酸化和抗逆境生长。使用“结构反向遗传学”框架，我们 最近发展起来的，我们将解剖和确定激酶活性的作用和支架功能 PI5P4Kβ(目标1)。我们将验证肝脏需要依赖GTP的PI5P4Kβ活性的假设 溶酶体功能和全身能量平衡(目标2)。在完成建议的研究后，我们 将确定GTP介导的PI5P4Kβ激活促进PI5P4K蛋白表达的新的应激抵消系统 溶酶体激活以支持应激恢复合成代谢细胞的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.