Revealing the essential functions of mitochondrial NADPH and NADK2 for cell growt

揭示线粒体 NADPH 和 NADK2 对细胞生长的基本功能

基本信息

批准号：
10797481
负责人：
Gerta N/A Hoxhaj
金额：
$ 20万
依托单位：
UT SOUTHWESTERN MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-15 至 2026-08-31
项目状态：
未结题

项目摘要

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.

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，维持氧化还原稳态。后一个需求对于持续生长，因为细胞增殖过程中细胞代谢活动速率增加导致活性氧 (ROS) 水平，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) 是生物合成还原力的主要提供者大分子和针对氧化应激的保护。 NADPH 的总细胞库受以下因素调节： NAD 激酶 (NADK) 的活性，催化 NAD+ 磷酸化为 NADP+，是限速酶 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 NADK2，产生隔间特定的还原能力。最近我们发现，活动 NADK 受到磷酸肌醇 3 激酶 (PI3K) - Akt 途径的刺激，以促进 NADP(H) 的产生细胞生长，但 NADK2 的重要性以及线粒体 NADPH 在细胞生长中的总体作用尚未明确被成立。在患有各种神经系统疾病和疾病的患者中观察到 NADK2 突变发育障碍。因此，明确 NADK2 和线粒体 NADP(H) 的关键功能至关重要并与人类健康相关。该提议基于我们的发现，即降低线粒体 NADP(H) 水平通过消耗 NADK2，使细胞呈现独特的脯氨酸营养缺陷型。缺失 NADK2 的细胞无法合成脯氨酸并依赖外源脯氨酸生长。脯氨酸对于蛋白质合成至关重要，并且，出乎意料的是，在NADK2缺陷的细胞中，用于核苷酸合成。我们提出三个具体目标 NADK2 和线粒体 NADP(H) 的功能对于细胞生长和增殖至关重要，与人类增殖性疾病和 NADK2 缺乏有关。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并评估其效果 NADK2 患者脯氨酸合成突变。在目标 2 中，我们将确定 NADK2 的机制缺乏和脯氨酸丰度减少会影响从头合成和挽救核苷酸合成的通量途径。在目标 3 中，我们将评估肿瘤生长和线粒体 NADPH 的需求评估靶向 NADK2 与脯氨酸饮食限制相结合的治疗潜力。这该提案将确定线粒体 NADP(H) 和 NADK2 的主要功能，这些功能对细胞至关重要生长和增殖，从而为我们提供对抗增殖性疾病的新治疗策略和人类 NADK2 缺乏症。