PHGDH的氧化还原修饰通过调控丝氨酸生物合成促进肿瘤生长的机制研究

The serine biosynthesis pathway is critical for tumor cell survival via production of metabolic intermediates for macromolecular biosynthesis and maintenance of intracellular redox homeostasis. PHGDH (phosphoglycerate dehydrogenase), which catalyzes the first and rate-limiting step in serine biosynthesis pathway, has been demonstrated to promote tumor cell survival and growth. However, the regulatory mechanisms of PHGDH in tumorigenesis remain largely unknown. Our “OxiodoTMT” redox proteomics analysis has identified PHGDH could act as a redox sensor undergoing redox modifications in response to ROS (reactive oxygen species). The increase in intracellular ROS levels resulted in PHGDH tetramerization via the formation of intermolecular disulfide bond, thereby elevating the enzymatic activity of PHGDH. In addition, PHGDH knockdown significantly inhibits the growth and proliferation of tumor cells. In this project, we will identify the oxidized cysteine residues using site-directed mutagenesis strategy. Moreover, we will investigate the molecular mechanisms underlying PHGDH oxidation-mediated promotion of tumor growth via provoking serine biosynthesis. Finally, the roles of PHGDH and its redox modifications in sensitize tumor cells to chemotherapeutic agents will be evaluated in vitro and in vivo. Our study will provide new insights into the redox regulation mechanisms in tumorigenesis, and pave the road for the development of novel cancer therapeutic strategies based on redox manipulation.

丝氨酸生物合成可促进生物大分子合成并维持氧化还原稳态，在肿瘤生长和存活过程中发挥重要作用。PHGDH是丝氨酸合成的关键限速酶，但其参与肿瘤发生的分子机制尚不清楚。我们前期通过氧化还原蛋白组学技术发现PHGDH可受氧化还原修饰调控。活性氧ROS可导致PHGDH通过分子间二硫键形成同源四聚体，从而增强了PHGDH丝氨酸生物合成的酶活性。进一步研究发现，在氧化应激条件下，敲降PHGDH可显著抑制肿瘤细胞生长和增殖。在此基础上，本项目拟通过半胱氨酸定点突变技术明确PHGDH的半胱氨酸修饰位点和修饰模式；结合细胞与动物模型，阐明PHGDH的氧化还原修饰通过调控丝氨酸生物合成促进肿瘤生长的分子机制；明确PHGDH或其氧化还原修饰在化疗药物杀伤肿瘤细胞中的潜在应用价值。通过本项目的实施，有望从氧化还原信号调控的角度阐明PHGDH在肿瘤发生过程中的生物学功能，为基于抗氧化调变的肿瘤治疗策略提供新思路。

丝氨酸从头合成可促进生物大分子合成并维持氧化还原稳态，在肿瘤生长和存活过程中发挥重要作用。PHGDH是丝氨酸合成通路第一步反应的关键限速酶，已有研究表明PHGDH可促进肿瘤发生，但其中的具体机制尚不清楚。我们发现肝癌细胞内活性氧ROS积累可促进PHGDH形成同源四聚体，从而增强PHGDH酶活。机制研究表明，ROS可导致Cys369和Cys421氧化形成分子间二硫键，从而促进PHGDH同源四聚体的形成。突变Cys369和Cys421两个半胱氨酸位点对糖酵解无明显影响，但可显著降低细胞内NADPH/NADP+比值和GSH水平，从而导致肝癌细胞铁死亡的发生。综上所述，我们的研究表明PHGDH的氧化还原修饰可通过维持氧化还原稳态促进肝癌细胞存活和生长。