Revealing the essential functions of mitochondrial NADPH and NADK2 for cell growth and proliferation

摘要：为了生长和增殖，细胞需要满足三个关键的代谢需求：增加生物合成， 充足的能量供应，并维持氧化还原动态平衡。后一种需求对于 持续生长，因为在细胞增殖过程中细胞代谢活动的增加会导致 活性氧(ROS)水平，这可能对细胞生长产生不利影响。烟酰胺 腺嘌呤二核苷酸磷酸(NADPH)是生物合成的还原动力的主要供应者 大分子和对氧化应激的保护。NADPH的总细胞池受 NAD激酶的活性(NADK)，催化NAD磷酸化为NADP的酶，限速 生产NADPH的底物。哺乳动物细胞表达两种NAD蛋白：胞浆和线粒体 NADK2，它产生特定于车厢的还原功率。最近，我们发现， NADK受磷脂酰肌醇3-激酶(PI3K)-Akt途径的刺激，以促进NADP(H)的产生 细胞生长，但NADK2的重要性和线粒体NADPH在细胞生长中的整体作用尚不清楚 被建立起来。已观察到NADK2突变在各种神经性和非神经性疾病患者中 发育障碍。因此，确定NADK2和线粒体NADP(H)的关键功能是至关重要的 并与人类健康相关。这一建议建立在我们发现线粒体NADP(H)水平下降的基础上 通过NADK2的耗尽，使细胞产生独特的脯氨酸营养缺陷体。删除了NADK2的单元格无法 合成脯氨酸，并依赖外源脯氨酸生长。脯氨酸对蛋白质合成至关重要，而且， 出乎意料的是，对于核苷酸合成，在NADK2缺陷的细胞中。我们提出了三个具体目标来建立 NADK2和线粒体NADP(H)对细胞生长和增殖至关重要的功能和 与人类的增殖性疾病和NADK2缺乏症有关。在目标1中，我们建议定义分子 NADK2和线粒体NADPH支持脯氨酸生物合成的机制及其作用评价 NADK2患者的脯氨酸合成突变。在目标2中，我们将确定NADK2如何 缺乏和减少的脯氨酸丰度影响通过从头合成和挽救核苷酸合成的通量 小路。在目标3中，我们将评估NADK2和线粒体NADPH对肿瘤生长和 评估靶向NADK2与限制饮食的Pro联合治疗的可能性。这 该提案将建立线粒体NADP(H)和NADK2的主要功能，这是细胞所必需的 生长和增殖，从而向我们提供新的治疗策略，以抗击增殖性疾病和 人类NADK2缺乏症。

SUMMARY: To grow and proliferate, cells need to fulfill three key metabolic demands: increased biosynthesis, sufficient energy supply, and maintenance of redox homeostasis. The latter demand is particularly important for sustained growth, because increased rate of cellular metabolic activity during cell proliferation results in elevated levels of reactive oxygen species (ROS), which can have detrimental effects on cell growth. Nicotinamide adenine dinucleotide phosphate (NADPH) is a principal supplier of reducing power for biosynthesis of macromolecules and protection against oxidative stress. The total cellular pool of NADPH is regulated by the activity of NAD kinases (NADK), enzymes that catalyze the phosphorylation of NAD+ to NADP+, the rate-limiting substrate for NADPH production. Mammalian cells express two NAD kinases, cytosolic NADK, and mitochondrial NADK2, which generate compartment-specific reducing power. Recently, we discovered that the activity of NADK is stimulated by the phosphoinositide 3-kinase (PI3K) - Akt pathway to boost the NADP(H) production for cell growth, but the importance of NADK2 and the overall role of mitochondrial NADPH in cell growth has yet to be established. Mutations in NADK2 have been observed in patients with various neurological and developmental disorders. Therefore, defining the key functions of NADK2 and mitochondrial NADP(H) is critical and relevant to human health. This proposal builds on our finding that decreasing mitochondrial NADP(H) levels through depletion of NADK2 renders cells uniquely proline auxotroph. Cells with NADK2 deletion fail to synthesize proline and rely on exogenous proline for their growth. Proline is critical for protein synthesis, and, unexpectedly, for nucleotide synthesis, in NADK2-deficient cells. We propose three Specific Aims to establish the functions of NADK2 and mitochondrial NADP(H) that are essential for cell growth and proliferation and relevant for proliferative diseases and NADK2 deficiency in humans. In Aim 1, we propose to define the molecular mechanisms by which NADK2 and mitochondrial NADPH support proline biosynthesis and evaluate the effects of NADK2 patient mutations on proline synthesis. In Aim 2, we will determine the mechanisms of how NADK2 deficiency and reduced proline abundance affect flux through the de novo and salvage nucleotide synthesis pathways. In Aim 3, we will assess the requirement of NADK2 and mitochondrial NADPH for tumor growth and evaluate the therapeutic potential of targeting NADK2 in combination with dietary restriction of proline. This proposal will establish the primary functions of mitochondrial NADP(H) and NADK2 that are essential for cell growth and proliferation, thereby informing us on new therapeutic strategies to combat proliferative diseases and NADK2 deficiency in humans.