Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Alexis Jourdain

LAB HEAD: Alexis Jourdain

PROVIDER: PXD060353 | Pride | 2025-10-29

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	6732_Jourdain_DIA_17268_15SPD.d.zip	Other
	6733_Jourdain_DIA_17269_15SPD.d.zip	Other
	6734_Jourdain_DIA_17272_15SPD.d.zip	Other
	6735_Jourdain_DIA_17260_15SPD.d.zip	Other
	6736_Jourdain_DIA_17261_15SPD.d.zip	Other

Items per page:

1 - 5 of 18

Similar Datasets

NUDT5 restrains PPAT to promote pyrimidine synthesis and effective nucleobase therapy - sgNUDT5 proteomics

Project description: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.

2025-10-29 | PXD060320 | Pride

How does orotic acid affect the growth of R.etli CE3?

Project description:Growth inhibition of Rhizobium etli and other organisms by exogenous OA has not been previously reported. We conductedÂ genome-wide transcriptomic analysisÂ to determine the possible mechanism by which OA exerts its growth-inhibitory effect on R. etli CE3. The observed changes fall into several broad categories. First, the decreased expression of genes involved in protein synthesis (including ribosomal genes), replication and energy production indicates that the cells arrest or at least slow their growth, which agrees with the observed growth arrest induced by OA.Â The upregulation of a key enzyme (PrsAch) forÂ 5'-phosphoribosyl-1'-pyrophosphateÂ (PRPP) synthesis suggests that cells exposed to OA suffer PRPP deprivation. We hypothesized that the PRPP pools were depleted as a result of an increase in the synthesis of OMP stimulated by exogenous OA. PRPP depletion would result in a marked decrease in purine synthesis, which would lead to an imbalance between purine and pyrimidine nucleotides, causing arrest of DNA synthesisÂ and a lack of energetic nucleotides (ADP, ATP). Three independent biological materials with one dyeswap were performed.

2013-10-12 | E-GEOD-46013 | biostudies-arrayexpress

How does orotic acid affect the growth of R.etli CE3?

Project description:Growth inhibition of Rhizobium etli and other organisms by exogenous OA has not been previously reported. We conducted genome-wide transcriptomic analysis to determine the possible mechanism by which OA exerts its growth-inhibitory effect on R. etli CE3. The observed changes fall into several broad categories. First, the decreased expression of genes involved in protein synthesis (including ribosomal genes), replication and energy production indicates that the cells arrest or at least slow their growth, which agrees with the observed growth arrest induced by OA. The upregulation of a key enzyme (PrsAch) for 5'-phosphoribosyl-1'-pyrophosphate (PRPP) synthesis suggests that cells exposed to OA suffer PRPP deprivation. We hypothesized that the PRPP pools were depleted as a result of an increase in the synthesis of OMP stimulated by exogenous OA. PRPP depletion would result in a marked decrease in purine synthesis, which would lead to an imbalance between purine and pyrimidine nucleotides, causing arrest of DNA synthesis and a lack of energetic nucleotides (ADP, ATP).

2013-10-12 | GSE46013 | GEO

The nucleotide messenger (p)ppGpp is an anti-inducer of the purine synthesis transcription regulator PurR in Bacillus

Project description:The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes from soil bacteria to pathogens: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis. We identified PurR as a receptor of (p)ppGpp in Bacillus anthracis and revealed that (p)ppGpp strongly enhances PurR binding to its regulon in the Bacillus subtilis genome. A co-structure reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of PRT enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant fails to downregulate purine synthesis genes upon starvation. Our work establishes the precedent of (p)ppGpp as a classical effector of transcription repressors and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from the human intestinal tract to host-pathogen interfaces.

2021-12-10 | GSE185164 | GEO

An updated inventory of genes essential for oxidative phosphorylation identifies a mitochondrial origin in familial Ménière’s disease - Quantitative patient LCL proteomics

Project description:arise from defects in oxidative phosphorylation (OXPHOS). Their complex mode of inheritance and diverse clinical presentations render the diagnosis of MDs challenging and, to date, most lack a cure. Here, we build on previous efforts to discover genes necessary for OXPHOS and report a highly complementary galactose-sensitized CRISPR-Cas9 “growth” screen, presenting an updated inventory now with 481 OXPHOS genes, including 157 linked to MDs. We further focus on FAM136A, a gene associated with Ménière’s disease and show that it supports inter-membrane space protein homeostasis and OXPHOS in cell lines, mice, and patients. Our study identifies a mitochondrial basis in a familial form of Ménière’s disease (fMD), provides a comprehensive resource of OXPHOS-related genes, and sheds light on the pathways involved in mitochondrial disorders, with the potential to guide future diagnostics and treatments for MDs.

2025-07-30 | PXD060152 | Pride

Genome-wide CRISPR screening reveals nucleotide synthesis negatively regulates autophagy

Project description:Macroautophagy (hereafter, autophagy) is a process that directs the degradation of cytoplasmic material in lysosomes. In addition to its homeostatic roles, autophagy undergoes dynamic positive and negative regulation in response to multiple forms of cellular stress, thus enabling the survival of cells. However, the precise mechanisms of autophagy regulation are not fully understood. To identify potential negative regulators of autophagy, we performed a genome-wide CRISPR screen using the quantitative autophagic flux reporter GFP-LC3-RFP. We identified phosphoribosylformylglycinamidine synthase (PFAS), a component of the de novo purine synthesis pathway, as one such negative regulator of autophagy. Autophagy was activated in cells lacking PFAS or phosphoribosyl pyrophosphate amidotransferase (PPAT), another de novo purine synthesis enzyme, or treated with methotrexate when exogenous levels of purines were insufficient. Purine starvation-induced autophagy activation was concomitant with mTORC1 suppression, and was profoundly suppressed in cells deficient for TSC2, which negatively regulates mTORC1 through inhibition of RHEB, suggesting that purines regulate autophagy through the TSC-RHEB-mTORC1 signaling axis. Moreover, depletion of the pyrimidine synthesis enzymes carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) and dihydroorotate dehydrogenase (DHODH) activated autophagy as well, although mTORC1 activity was not altered by pyrimidine shortage. These results suggest a different mechanism of autophagy induction between purine and pyrimidine starvation. These findings provide novel insights into the regulation of autophagy by nucleotides and possibly the role of autophagy in nucleotide metabolism, leading to further developing anticancer strategies involving nucleotide synthesis and autophagy.

2021-05-11 | GSE164778 | GEO

Subcellular fraction kidney proteomics in MGME1 knockout mice

Project description:Metabolic dysregulation can lead to inflammatory responses. Imbalanced nucleotide synthesis triggers the release of mitochondrial DNA (mtDNA) to the cytosol and an innate immune response by cGAS-STING signaling. However, how nucleotide deficiency drives mtDNA dependent inflammation has not been elucidated. Here, we show that nucleotide imbalance leads to an increased misincorporation of ribonucleotides into mtDNA during age-dependent renal inflammation in a mouse model lacking the mitochondrial exonuclease MGME1 and upon inhibition of pyrimidine synthesis in cells lacking the mitochondrial i-AAA protease YME1L. Similarly, reduced deoxyribonucleotide synthesis increases ribonucleotide content of mtDNA in cell cycle-arrested senescent cells. This leads to mtDNA release into the cytosol, cGAS-STING activation and the mtDNA-dependent senescence-associated secretory phenotype (SASP), which can be suppressed by exogenously added deoxyribonucleotides. Our results explain age- and mtDNA-dependent inflammatory responses by imbalanced nucleotide metabolism and highlight the sensitivity of mtDNA to aberrant ribonucleotide incorporation.

2025-07-25 | PXD053639 | Pride

Effect of mitochondrial electron transport chain blockade on transcriptomic profile in human H460 cells

Project description:Cancer cells reprogram their metabolism to support cell growth and proliferation in harsh environments. To assess how human cells respond to defective mitochondrial respiration, we analyzed metabolomics profiles in cells with deficient electron transport chain (ETC). We found that ETC deficiency induces an accumulation of purine nucleotides. Additionally, we revealed that ETC blockade suppressed de novo purine nucleotide biosynthesis while enhanced purine salvage. This metabolic rewiring of purine nucleotide biosynthesis is not regulated at the transcriptional level. Instead, stable isotope tracing experiments showed that ETC blockade promoted the oxidative branch of pentose phosphate pathway to elevate phosphoribosyl diphosphate to drive purine salvage. In summary, our findings delineate how cells remodel purine metabolism in response to ETC blockade, and uncover a new metabolic vulnerability in tumors with low respiration.

2024-05-09 | GSE265923 | GEO

Tracing retinal progenitors cells and photoreceptors from mouse retinal organoids

Project description:Retinal organoids have become valuable 3D model for translational and developmental research. The knowledge of the limitations of the system ensures its sensible use. All retinal cell types originate from the differentiation of retinal progenitor cells. The properties of retinal progenitors in 3D culture systems are not well studied. In our project, we created a mouse stem cell line with a Rax-mCherry reporter construct that allows retinal progenitors' isolation, tracing, and in vitro imaging during retinogenesis. For proteomic analysis, we used mCherry-positive cells from dissociated embryoid bodies with formed eye field structures on the fourth day after stem cell aggregation. Information about protein content helped to characterize Rax-expressing cells at this stage and their suitability for further applications.

2025-02-14 | PXD039309 | Pride

Cellular pyrimidine imbalance triggers mitochondrial DNA-dependent innate immunity [A33_OE_RNA-seq]

Project description:Cytosolic mitochondrial DNA (mtDNA) elicits a type I interferon response, but signals triggering the release of mtDNA from mitochondria remain enigmatic. Here, we show that mtDNA-dependent immune signalling via the cyclic GMP–AMP syn- thase‒stimulator of interferon genes‒TANK-binding kinase 1 (cGAS–STING–TBK1) pathway is under metabolic control and is induced by cellular pyrimidine deficiency. The mitochondrial protease YME1L preserves pyrimidine pools by supporting de novo nucleotide synthesis and by proteolysis of the pyrimidine nucleotide carrier SLC25A33. Deficiency of YME1L causes inflamma- tion in mouse retinas and in cultured cells. It drives the release of mtDNA and a cGAS–STING–TBK1-dependent inflammatory response, which requires SLC25A33 and is suppressed upon replenishment of cellular pyrimidine pools. Overexpression of SLC25A33 is sufficient to induce immune signalling by mtDNA. Similarly, depletion of cytosolic nucleotides upon inhibition of de novo pyrimidine synthesis triggers mtDNA-dependent immune responses in wild-type cells. Our results thus identify mtDNA release and innate immune signalling as a metabolic response to cellular pyrimidine deficiencies.

2021-02-26 | GSE161732 | GEO