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Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling.

ABSTRACT: There is growing evidence that mammalian cells deploy a mitochondria-associated metabolon called the purinosome to perform channeled de novo purine biosynthesis (DNPB). However, the molecular mechanisms of this substrate-channeling pathway are not well defined. Here, we present molecular evidence of protein-protein interactions (PPIs) between the human bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS) and other known DNPB enzymes. We employed two orthogonal approaches: bimolecular fluorescence complementation, to probe PPIs inside live, intact cells, and co-immunoprecipitation using StrepTag-labeled PAICS that was reintegrated into the genome of PAICS-knockout HeLa cells (crPAICS). With the exception of amidophosphoribosyltransferase, the first enzyme of the DNPB pathway, we discovered PAICS interacts with all other known DNPB enzymes and with MTHFD1, an enzyme which supplies the 10-formyltetrahydrofolate cofactor essential for DNPB. We show these interactions are present in cells grown in both purine-depleted and purine-rich conditions, suggesting at least a partial assembly of these enzymes may be present regardless of the activity of the DNPB pathway. We also demonstrate that tagging of PAICS on its C terminus disrupts these interactions and that this disruption is correlated with disturbed DNPB activity. Finally, we show that crPAICS cells with reintegrated N-terminally tagged PAICS regained effective DNPB with metabolic signatures of channeled synthesis, whereas crPAICS cells that reintegrated C-terminally tagged PAICS exhibit reduced DNPB intermediate pools and a perturbed partitioning of inosine monophosphate into AMP and GMP. Our results provide molecular evidence in support of purinosomes and suggest perturbing PPIs between DNPB enzymes negatively impact metabolite flux through this important pathway.

SUBMITTER: He J

PROVIDER: S-EPMC9035706 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Oral squamous cell carcinoma (OSCC) is a prevalent type of oral cancer. While therapeutic innovations have made strides, radioresistance persists as a significant hindrance in OSCC treatment. Despite identifying numerous targets that could potentially suppress the oncogenic attributes of OSCC, the exploration of oncogenic protein kinases for cancer therapy remains limited. Consequently, the functions of many kinase proteins in OSCC continue to be largely undetermined. In this research, we aim to disclose protein kinases that target OSCC and elaborate their roles and molecular mechanisms. Through the examination of the kinome library of radiotherapy-resistant/sensitive OSCC cell lines (HN12 and SAS), we identified a key gene, the tyrosine phosphorylation-regulated kinase 3 (DYRK3), a member of the DYRK family. We developed an in vitro cell model, composed of radiation-resistant OSCC, to scrutinize the clinical implications and contributions of DYRK3 and phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthase (PAICS) signaling in OSCC. This investigation involves bioinformatics and human tissue arrays. We seek to comprehend the role of DYRK3 and PAICS signaling in the development of OSCC and its resistance to radiotherapy. Various in vitro assays are utilized to reveal the essential molecular mechanism behind radiotherapy resistance in connection with the DYRK3 and PAICS interaction. In our study, we quantified the concentrations of DYRK3 and PAICS proteins and tracked the expression levels of key pluripotency markers, particularly PPAT. Furthermore, we extended our investigation to include an analysis of Glut-1, a gene recognized for its linkage to radioresistance in oral squamous cell carcinoma (OSCC). Furthermore, we conducted an in vivo study to affirm the impact of DYRK3 and PAICS on tumor growth and radiotherapy resistance, focusing particularly on the role of DYRK3 in the radiotherapy resistance pathway. This focus leads us to identify new therapeutic agents that can combat radiotherapy resistance by inhibiting DYRK3 (GSK-626616). Our in vitro models showed that inhibiting PAICS disrupts purinosome formation and influences the survival rate of radiation-resistant OSCC cell lines. These outcomes underscore the pivotal role of the DYRK3/PAICS axis in directing OSCC radiotherapy resistance pathways and, as a result, influencing OSCC progression or therapy resistance. Our findings also reveal a significant correlation between DYRK3 expression and the PAICS enzyme in OSCC radiotherapy resistance.

Project description:Although purines and purinergic signaling are crucial for numerous biochemical and cellular processes, their functions during vertebrate embryonic development have not been well characterized. We analyze two recessive zebrafish mutations that affect de novo purine synthesis, gart and paics. gart encodes phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase, a trifunctional enzyme that catalyzes steps 2, 3 and 5 of inosine monophosphate (IMP) synthesis. paics encodes phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase, a bifunctional enzyme that catalyzes steps 6 and 7 of this process. Zygotic gart and paics mutants have pigmentation defects in which xanthophore and iridophore pigmentation is almost completely absent, and melanin-derived pigmentation is significantly decreased, even though pigment cells are present in normal amounts and distributions. Zygotic gart and paics mutants are also microphthalmic, resulting from defects in cell cycle exit of proliferative retinoblasts within the developing eye. Maternal-zygotic and maternal-effect mutants demonstrate a crucial requirement for maternally derived gart and paics; these mutants show more severe developmental defects than their zygotic counterparts. Pigmentation and eye growth phenotypes in zygotic gart and paics mutants can be ascribed to separable biosynthetic pathways: pigmentation defects and microphthalmia result from deficiencies in a GTP synthesis pathway and an ATP synthesis pathway, respectively. In the absence of ATP pathway activity, S phase of proliferative retinoblasts is prolonged and cell cycle exit is compromised, which results in microphthalmia. These results demonstrate crucial maternal and zygotic requirements for de novo purine synthesis during vertebrate embryonic development, and identify independent functions for ATP and GTP pathways in mediating eye growth and pigmentation, respectively.

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Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling.

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