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

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， 充足的能量供应和维持氧化还原稳态。后一项要求特别重要， 持续生长，因为在细胞增殖期间细胞代谢活性的速率增加导致 活性氧（ROS）水平，这可能对细胞生长产生有害影响。烟酰胺 腺嘌呤二核苷酸磷酸（NADPH）是生物合成的还原力的主要供应者。 大分子和保护免受氧化应激。NADPH的总细胞库由 NAD激酶（NADK）的活性，催化NAD+磷酸化为NADP+的酶， 用于NADPH生产的底物。哺乳动物细胞表达两种NAD激酶，胞质NADK和线粒体NADK。 NADK 2，其产生隔室特异性还原能力。最近，我们发现， 磷酸肌醇3-激酶（PI 3 K）- Akt途径刺激NADK以促进NADP（H）的产生， 细胞生长，但在细胞生长中的重要性和线粒体NADPH的整体作用还没有， 建立。已经在各种神经系统疾病患者中观察到了NADK 2的突变， 发育障碍因此，确定NADK 2和线粒体NADP（H）的关键功能至关重要 与人类健康有关。该提案建立在我们的发现基础上，即线粒体NADP（H）水平降低 通过耗竭NADK 2使细胞成为唯一的脯氨酸营养缺陷型。具有NADK 2缺失的细胞不能 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， 出乎意料地，对于核苷酸合成，在NADK 2缺陷细胞中。我们提出三个具体目标，以建立 细胞生长和增殖所必需的NADK 2和线粒体NADP（H）的功能， 与人类增殖性疾病和NADK 2缺乏相关。在目标1中，我们建议定义分子 NADK 2和线粒体NADPH支持脯氨酸生物合成的机制，并评估其作用 NADK 2患者突变对脯氨酸合成的影响。在目标2中，我们将确定NADK 2 缺乏和减少脯氨酸丰度影响通量通过从头和补救核苷酸合成 途径。在目标3中，我们将评估肿瘤生长对NADPH 2和线粒体NADPH的需求， 评估靶向NADK 2与脯氨酸饮食限制组合的治疗潜力。这 该提案将确定线粒体NADP（H）和NADK 2的主要功能，这对细胞生长至关重要。 生长和增殖，从而为我们提供新的治疗策略，以打击增殖性疾病， 人的NADK 2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.