NUDT5 restrains PPAT to promote pyrimidine synthesis and effective nucleobase therapy - NUDT5 co-immunoprecipitation

Nucleotides are essential building blocks for nucleic acid synthesis, signaling, and metabolism. Rapidly proliferating cells require large amounts of nucleotides, making nucleotide metabolism a widely exploited target for cancer therapy. However, resistance frequently emerges, highlighting the need for a deeper understanding of nucleotide regulation. Here, we use uridine-sensitized CRISPR-Cas9 screening to reveal novel regulators of pyrimidine synthesis. We identify NUDT5 which we show binds to and negatively regulates PPAT, the rate-limiting enzyme in purine biosynthesis. We demonstrate the NUDT5-PPAT interaction prevents excessive purine production, maintains phosphoribosyl pyrophosphate (PRPP), a critical substrate for pyrimidine synthesis, and thus enhances conversion of nucleobase analogs into active anti-cancer molecules. We further report that NUDT5 regulates PPAT independently of its enzymatic activity and can be displaced by PRPP, revealing intricate allosteric regulation. Our findings reveal a previously unrecognized mechanism for maintaining nucleotide balance and positions NUDT5 as a potential therapeutic target for overcoming resistance to chemotherapy in proliferative diseases.

核苷酸是核酸合成、信号转导与代谢过程的必需构建单元。快速增殖的细胞需要大量核苷酸，因此核苷酸代谢成为癌症治疗中被广泛开发的靶点。然而耐药性频繁出现，凸显了深入解析核苷酸调控机制的必要性。本研究借助尿苷敏感型CRISPR-Cas9筛选技术，揭示了嘧啶合成的新型调控因子。我们鉴定出NUDT5，并证实其可结合并负调控嘌呤生物合成的限速酶PPAT。实验证明，NUDT5与PPAT的相互作用能够阻止嘌呤过度生成，维持磷酸核糖焦磷酸（phosphoribosyl pyrophosphate, PRPP）——嘧啶合成的关键底物——的稳态，从而提升核苷类似物转化为活性抗癌分子的效率。我们进一步发现，NUDT5对PPAT的调控不依赖其自身酶活性，且可被PRPP竞争性置换，揭示了复杂的别构调控机制。本研究揭示了一条此前未被发现的核苷酸稳态维持途径，并将NUDT5确立为克服增殖性疾病化疗耐药的潜在治疗靶点。