The de novo purine biosynthetic pathway generates inosine monophosphate, the precursor to purines and their derivatives that act in numerous cellular anabolic and catabolic networks. The pathway consists of enzymes that catalyze ten chemical steps and, except for being mono- or multifunctional, are highly conserved from pro- to eukaryotes. Although the enzymes have long been hypothesized to exist in a multienzyme complex, only recently have we been able to demonstrate their clustering in vivo to form the purinosome. We propose to investigate the protein components of this cluster in terms of their stoichiometry and the possible inclusion of non-pathway proteins;of their protein-protein proximity within the purinosome;of putative post-translational modifications that drive purinosome assembly, and of their effect when clustered on the formation of pathway generated metabolite levels. Detection, identification, and quantification of the purinosome associated enzymes and non-pathway proteins will make extensive use of cellular imaging of chimeric fluorescent protein constructs, of co- immunoprecipitation, and of blue native polyacrylamide gel electrophoresis, the latter two linked to MALDI/MS/MS. Tandem MS/MS will be the method of choice for investigations of putative post- translational modifications. The locus and stoichiometry of purinosome protein members within the complex will be sought by in vivo fluorescence resonance energy transfer (FRET), stochastic optical reconstruction microscopy (STORM), and intracellular reporter assays. The functional advantage of the purinosome to affect metabolite levels within cells will be probed by MALDI/MS/MS as well as secondary ion mass spectrometry (SIMS) and correlated with purinosome cluster density. Many of the individual enzymes, particularly those that utilize folate cofactors, have been biologically validated as chemotherapeutic targets. The proposed studies in the long term have the potential to expand greatly our understanding of how biosynthetic or catabolic pathways may organize intracellularly and the biochemical advantages to the cell of such multienzyme clusters as our approach is extended to other pathways.
The purinosome, consisting of the enzymes responsible for de novo purine biosynthesis, is a novel subcellular organization and serves as a prototype for how a cellular, enzymatic, metabolic pathway may be transiently organized. An understanding of its assembly and function in response to the cellular environment will provide profound insights into how cell viability is maintained in normal and diseased states.
| Chan, Chung Yu; Pedley, Anthony M; Kim, Doory et al. (2018) Microtubule-directed transport of purine metabolons drives their cytosolic transit to mitochondria. Proc Natl Acad Sci U S A 115:13009-13014 |
| Pedley, Anthony M; Karras, Georgios I; Zhang, Xin et al. (2018) Role of HSP90 in the Regulation of de Novo Purine Biosynthesis. Biochemistry 57:3217-3221 |
| Mangold, Colleen A; Yao, Pamela J; Du, Mei et al. (2018) Expression of the purine biosynthetic enzyme phosphoribosyl formylglycinamidine synthase in neurons. J Neurochem 144:723-735 |
| Pedley, Anthony M; Benkovic, Stephen J (2017) A New View into the Regulation of Purine Metabolism: The Purinosome. Trends Biochem Sci 42:141-154 |
| French, Jarrod B; Jones, Sara A; Deng, Huayun et al. (2016) Spatial colocalization and functional link of purinosomes with mitochondria. Science 351:733-7 |
| Fu, Rong; Sutcliffe, Diane; Zhao, Hong et al. (2015) Clinical severity in Lesch-Nyhan disease: the role of residual enzyme and compensatory pathways. Mol Genet Metab 114:55-61 |
| Chan, Chung Yu; Zhao, Hong; Pugh, Raymond J et al. (2015) Purinosome formation as a function of the cell cycle. Proc Natl Acad Sci U S A 112:1368-73 |
| Zhao, Hong; Chiaro, Christopher R; Zhang, Limin et al. (2015) Quantitative analysis of purine nucleotides indicates that purinosomes increase de novo purine biosynthesis. J Biol Chem 290:6705-13 |
| French, Jarrod B; Zhao, Hong; An, Songon et al. (2013) Hsp70/Hsp90 chaperone machinery is involved in the assembly of the purinosome. Proc Natl Acad Sci U S A 110:2528-33 |
| Zhao, Hong; French, Jarrod B; Fang, Ye et al. (2013) The purinosome, a multi-protein complex involved in the de novo biosynthesis of purines in humans. Chem Commun (Camb) 49:4444-52 |
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