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

Authors
  • He, Jingxuan1
  • Zou, Ling-Nan2
  • Pareek, Vidhi2
  • Benkovic, Stephen J3
  • 1 Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA.
  • 2 Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania, USA.
  • 3 Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania, USA. Electronic address: [email protected]
Type
Published Article
Journal
Journal of Biological Chemistry
Publisher
American Society for Biochemistry and Molecular Biology
Publication Date
Mar 21, 2022
Volume
298
Issue
5
Pages
101853–101853
Identifiers
DOI: 10.1016/j.jbc.2022.101853
PMID: 35331738
Source
Medline
Keywords
Language
English
License
Unknown

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. Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.

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