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Identification and characterization of genes and metabolic regulators of nucleotide synthesis

Year: 2025
Type: dissertation
Abstract: Pyrimidine and purine nucleotides are essential molecules for life that participate in cellular metabolism, signal transduction, and storing genetic information. However, they also contribute to diseases, and nucleotide metabolism is a key therapeutic target against cancers, viral infections, and autoinflammatory disorders. Better understanding nucleotide synthesis can explain mechanisms of therapeutic resistance and open new avenues for treatment. We investigate the regulation of pyrimidine de novo synthesis using genome-wide CRISPR-Cas9 screening and uridine auxotrophy to successfully recover both known factors and novel genes. We demonstrate that pyrimidine de novo biosynthesis can continue in the absence of its cofactor coenzyme Q, previously thought to be essential. We then focus on a novel factor, NUDT5, and show that it is required for balanced synthesis of purine and pyrimidine nucleotides, dependent on the common substrate phosphoribosyl pyrophosphate (PRPP). In absence of NUDT5, purine synthesis depletes the cellular PRPP pool to drive nucleotide imbalance and resistance to nucleobase analog chemotherapies. We elucidate this mechanism by showing NUDT5 exerts an inhibitory effect on purine enzyme PPAT through direct binding, sensitive to PPAT allosteric activators. Finally, we present preliminary findings on a second regulatory factor, the protein phosphatase PP1b. Linking this protein to pyrimidine de novo biosynthesis opens therapeutic options for the rare gentic disorder Noonan Syndrome-Like Disorder with Loose Anagen Hair 2 (NSLH2), caused by mutations in the gene encoding PP1b. Together, we reveal fundamental mechanisms of nucleotide regulation with important implications for therapy.
Source: IRIS
Language: English
Open Access status: green
OpenAlex ID: W7133034162
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