Hypoxia drives the assembly of the multienzyme purinosome complex
2020; Elsevier BV; Volume: 295; Issue: 28 Linguagem: Inglês
10.1074/jbc.ra119.012175
ISSN1083-351X
AutoresCyrielle Doigneaux, Anthony M. Pedley, Ishna N. Mistry, Monika Papayova, Stephen J. Benkovic, Ali Tavassoli,
Tópico(s)RNA modifications and cancer
ResumoThe purinosome is a dynamic metabolic complex composed of enzymes responsible for de novo purine biosynthesis, whose formation has been associated with elevated purine demand. However, the physiological conditions that govern purinosome formation in cells remain unknown. Here, we report that purinosome formation is up-regulated in cells in response to a low-oxygen microenvironment (hypoxia). We demonstrate that increased purinosome assembly in hypoxic human cells requires the activation of hypoxia inducible factor 1 (HIF-1) and not HIF-2. Hypoxia-driven purinosome assembly was inhibited in cells lacking 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a single enzyme in de novo purine biosynthesis, and in cells treated with a small molecule inhibitor of ATIC homodimerization. However, despite the increase in purinosome assembly in hypoxia, we observed no associated increase in de novo purine biosynthesis in cells. Our results indicate that this was likely due to a reduction in mitochondrial one-carbon metabolism, resulting in reduced mitochondrion-derived one-carbon units needed for de novo purine biosynthesis. The findings of our study further clarify and deepen our understanding of purinosome formation by revealing that this process does not solely depend on cellular purine demand. The purinosome is a dynamic metabolic complex composed of enzymes responsible for de novo purine biosynthesis, whose formation has been associated with elevated purine demand. However, the physiological conditions that govern purinosome formation in cells remain unknown. Here, we report that purinosome formation is up-regulated in cells in response to a low-oxygen microenvironment (hypoxia). We demonstrate that increased purinosome assembly in hypoxic human cells requires the activation of hypoxia inducible factor 1 (HIF-1) and not HIF-2. Hypoxia-driven purinosome assembly was inhibited in cells lacking 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a single enzyme in de novo purine biosynthesis, and in cells treated with a small molecule inhibitor of ATIC homodimerization. However, despite the increase in purinosome assembly in hypoxia, we observed no associated increase in de novo purine biosynthesis in cells. Our results indicate that this was likely due to a reduction in mitochondrial one-carbon metabolism, resulting in reduced mitochondrion-derived one-carbon units needed for de novo purine biosynthesis. The findings of our study further clarify and deepen our understanding of purinosome formation by revealing that this process does not solely depend on cellular purine demand. Purines are more than just the building blocks for DNA and RNA; they are key metabolites that are critical for cellular function. Purines constitute the cellular energy unit ATP, the key signaling molecule GTP, and the substrates and cofactors for a variety of cellular pathways. There are two paths for purine production in cells: recycling of existing bases via a salvage pathway and de novo synthesis of the purine precursor inosine monophosphate (IMP) from phosphoribosyl pyrophosphate (PRPP) by six enzymes in 10 steps. Purine salvage is the predominant path for purine production in nonmalignant human cells as it is more resource efficient (1Murray A.W. The biological significance of purine salvage.Annu. Rev. Biochem. 1971; 40 (4330582): 811-82610.1146/annurev.bi.40.070171.004115Crossref PubMed Scopus (279) Google Scholar). It has been hypothesized that during periods of rapid cell growth, the de novo purine biosynthesis pathway is up-regulated, but little is known about other factors that affect the balance between these two pathways (1Murray A.W. The biological significance of purine salvage.Annu. Rev. Biochem. 1971; 40 (4330582): 811-82610.1146/annurev.bi.40.070171.004115Crossref PubMed Scopus (279) Google Scholar). As with other multienzyme pathways, it is difficult to explain the intracellular kinetics of de novo purine biosynthesis and the chemical stability of several intermediates if the six enzymes of this pathway were randomly dispersed within the cytosol. The association of these enzymes in a functional multienzyme complex or "metabolon" has therefore been a longstanding hypothesis. Using a combination of microscopy-based techniques, the six de novo purine biosynthetic enzymes were shown to assemble into a dynamic complex in cells named the purinosome, in response to purine depletion from the cell culture medium (2An S. Kumar R. Sheets E.D. Benkovic S.J. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells.Science. 2008; 320 (18388293): 103-10610.1126/science.1152241Crossref PubMed Scopus (368) Google Scholar, 3Kyoung M. Russell S.J. 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As purinosome formation has been correlated to increased de novo purine biosynthesis, and considering the significant metabolic reprogramming that occurs in hypoxic cells, we hypothesized that a hypoxic environment would lead to increased purinosome formation in cells. We first investigated the possibility that hypoxia enhances the assembly of the multienzyme purinosome complex. HeLa cells were transfected with a construct encoding formylglycinamidine ribonucleotide synthase (FGAMS), which catalyzes step 4 of de novo purine biosynthesis, as a fusion with the fluorescent protein mCherry (FGAMS-mCherry). These cells were cultured for 24 h in hypoxia (1% environmental oxygen) in the presence of purines (so far, purinosome formation had only been observed in cells cultured in purine-depleted media), and the degree of purinosome formation, as noted by enzyme clustering into distinct punctate structures, was assessed by fluorescence microscopy (2An S. Kumar R. Sheets E.D. Benkovic S.J. Reversible compartmentalization of de novo purine biosynthetic complexes in living cells.Science. 2008; 320 (18388293): 103-10610.1126/science.1152241Crossref PubMed Scopus (368) Google Scholar, 22Chan C.Y. Zhao H. Pugh R.J. Pedley A.M. French J. Jones S.A. Zhuang X. Jinnah H. Huang T.J. Benkovic S.J. Purinosome formation as a function of the cell cycle.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (25605889): 1368-137310.1073/pnas.1423009112Crossref PubMed Google Scholar, 23Pedley A.M. Benkovic S.J. Detecting purinosome metabolon formation with fluorescence microscopy.Methods Mol. Biol. 2018; 1764 (29605921): 279-28910.1007/978-1-4939-7759-8_17Crossref PubMed Scopus (6) Google Scholar). We observed 40% of cells showing the clustering of FGAMS-mCherry in response to hypoxia compared with the 19% of cells in normoxia (Fig. 1, a and c). This was comparable with the ∼2-fold increase in purinosome-containing cells observed when cells were cultured in purine-depleted medium in normoxia (Fig. 1c). This experiment was repeated with ADSL, which catalyzes step 8 of de novo purine biosynthesis, tagged with GFP (ADSL-EGFP). Similar to that of FGAMS-mCherry, we observed a 2-fold increase in cells showing clustering of ADSL-EGFP in hypoxia (Fig. 1, b and d). Colocalization analysis between FGAMS-mCherry and ADSL-EGFP by confocal microscopy showed a high degree of colocalization in hypoxia (Pearson's coefficient of 0.83) and was validated by three-dimensional volume reconstitution showing clear isolated peaks within the cytoplasm, supporting the notion that purinosomes form in response to hypoxia (Fig. S1, a and b). The number of fluorescent clusters per cell was next measured using HeLa cells transfected with either FGAMS-mCherry or ADSL-EGFP (24Chan C.Y. Zhao H. Pugh R.J. Pedley A.M. French J. Jones S.A. Zhuang X.W. Jinnah H. Huang T.J. Benkovic S.J. Purinosome formation as a function of the cell cycle.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (25605889): 1368-137310.1073/pnas.1423009112Crossref PubMed Scopus (60) Google Scholar). We found that the number of clusters per cell varied between 6 and 220 with the median being 42 clusters per cell (Fig. 1e). In addition, we analyzed the diameter of the clusters and found that these ranged from 0.6 to 1.9 μm, with an average diameter of 0.96 ± 0.25 μm (Fig. 1e). To ensure that our observation of hypoxia-mediated purinosome formation was not cell line-specific, we transfected the plasmid encoding FGAMS-mCherry in MDA-MB-231 cells and observed an increase in purinosome-positive cells in hypoxia similar to that observed in HeLa (Fig. S2, a and c). Hypoxia is known to induce oxidative stress (25Majmundar A.J. Wong W.J. Simon M.C. Hypoxia-inducible factors and the response to hypoxic stress.Mol. Cell. 2010; 40 (20965423): 294-30910.1016/j.molcel.2010.09.022Abstract Full Text Full Text PDF PubMed Scopus (1633) Google Scholar), so we investigated whether these intracellular puncta were distinct from stress granules. HeLa cells were co-transfected with plasmids encoding FGAMS-mCherry and GFP-tagged RasGAP-associated endoribonuclease (GFP-G3BP), an intracellular marker for stress granules (3Kyoung M. Russell S.J. Kohnhorst C.L. Esemoto N.N. An S. Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.Biochemistry. 2015; 54 (25540829): 870-88010.1021/bi501480dCrossref PubMed Scopus (32) Google Scholar, 26French J.B. Zhao H. An S.O. Niessen S. Deng Y.J. Cravatt B.F. Benkovic S.J. Hsp70/Hsp90 chaperone machinery is involved in the assembly of the purinosome.Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23359685): 2528-253310.1073/pnas.1300173110Crossref PubMed Scopus (49) Google Scholar), and colocalization was assessed by fluorescence microscopy. No overlap in the signals from these two markers was observed (Pearson's coefficient of −0.003), indicating that the observed clusters of FGAMS-mCherry in hypoxia are not associated with stress granules (Fig. S1c). In addition, we assessed whether hypoxia increases the number of stress granule-positive cells (Fig. S1d). We observed comparable numbers of cells containing stress granules in normoxia and hypoxia, indicating that the formation of the purinosome complexes in hypoxia is not a result of enhanced cellular stress. Previous reports note that the formation of the purinosome complex correlates to the cell cycle, reaching a maximum in G1 phase (22Chan C.Y. Zhao H. Pugh R.J. Pedley A.M. French J. Jones S.A. Zhuang X. Jinnah H. Huang T.J. Benkovic S.J. Purinosome formation as a function of the cell cycle.Proc. Natl. Acad. Sci. U.S.A. 2015; 112 (25605889): 1368-137310.1073/pnas.1423009112Crossref PubMed Google Scholar). We therefore quantified purinosome formation in the G1 phase of synchronized HeLa cells in hypoxia, however, we observed a similar ratio of purinosome containing cells in hypoxia (28%) as when using nonsynchronized cells (Fig. S1e). To verify that hypoxia-induced purinosome formation occurs on the endogenous level, we developed a proximity ligation assay (PLA) to visualize and quantify the association of pathway enzymes, FGAMS and ADSL, within purinosomes (27Söderberg O. Gullberg M. Jarvius M. Ridderstråle K. Leuchowius K.J. Jarvius J. Wester K. Hydbring P. Bahram F. Larsson L.G. Landegren U. Direct observation of individual endogenous protein complexes in situ by proximity ligation.Nat Methods. 2006; 3 (17072308): 995-100010.1038/nmeth947Crossref PubMed Scopus (1754) Google Scholar). As this method probes endogenous proteins, any observed clustering of these enzymes cannot be a consequence of their transient overexpression nor due to interactions between the fluorescent protein tag(s). We observed 88% of hypoxic cells having a colocalization between FGAMS and ADSL, whereas few (10%) normoxic cells showed colocalization (Fig. 1, f and g). As a positive control, we confirmed an association of endogenous FGAMS and ADSL in 87% of normoxic cells cultured in purine-depleted medium (Fig. 1, f and g). To further support this, we assessed the colocalization of FGAMS and GART (a trifunctional protein that catalyses steps 2, 3, and 5 of the de novo purine biosynthetic pathway), and observed an increase in the number of PLA signal-positive cells in hypoxia (81%) compared with normoxia (4%) (Fig. S1, f and g), which is in line with the results observed for ADSL and FGAMS. These data suggest that a substantially higher subset of cells form purinosomes in hypoxia (and in purine-depleted normoxia) than observed when using fluorescently-tagged proteins (88 versus 40%). This may be a consequence of the need for multiple copies of a given fluorescently tagged proteins to be present in a purinosome cluster before it can be observed by fluorescent microscopy. Alternatively, the presence of the fluorescent protein may impede complete purinosome formation or be less favorable than the complexation of endogenous proteins. Together the above data demonstrates that purinosome assembly is up-regulated by hypoxia in HeLa cells. HIF-1 plays a central role in the cellular hypoxia response, and we therefore sought to assess whether activation of HIF-1 correlates with purinosome formation in hypoxia. HeLa cells transiently expressing FGAMS-EGFP were treated with HIF-1α siRNA prior to incubation in hypoxia for 24 h. Consistent with before, a 2-fold increase in purinosome formation was observed in hypoxic cells (Fig. 1c and Fig. 2a); however, in cells lacking HIF-1α, purinosome formation was not detected (Fig. 2a). The role of HIF-1 in this process was further probed using desferrioxamine (DFX), which chemically stabilizes HIF-1α in normoxia (28Woo K.J. Lee T.J. Park J.W. Kwon T.K. Desferrioxamine, an iron chelator, enhances HIF-1alpha accumulation via cyclooxygenase-2 signaling pathway.Biochem. Biophys. Res. Commun. 2006; 343 (16527254): 8-1410.1016/j.bbrc.2006.02.116Crossref PubMed Scopus (86) Google Scholar). If purinosome formation in hypoxia is mediated through HIF-1α stabilization, then DFX treatment will be sufficient to enhance purinosome formation in normoxia. We observed a ∼3-fold increase in purinosome formation in DFX-treated cells cultured in normoxia (Fig. 2a). The isoform specificity of HIF-1 in promoting purinosome formation was probed using 786-O cells, which under hypoxic conditions use the HIF-2α isoform, instead of HIF-1α, and the HIF-2 transcription factor (29Maxwell P.H. Wiesener M.S. Chang G.W. Clifford S.C. Vaux E.C. Cockman M.E. Wykoff C.C. Pugh C.W. Maher E.R. Ratcliffe P.J. The tumour suppressor protein VHL targets hypoxia-inducible factors for oxygen-dependent proteolysis.Nature. 1999; 399 (10353251): 271-27510.1038/20459Crossref PubMed Scopus (4117) Google Scholar). We did not observe any hypoxia-driven increase in purinosome formation in these cells (Fig. S2, b and c). To ensure that our data were not a result of an inherent inability of 786-O cells to form purinosomes, we incubated these cells under normoxic purine-depleted conditions and observed a ∼6-fold increase in purinosome formation (Fig. S2b). This confirms the necessity of the HIF-1α isoform in driving purinosome formation in hypoxic cells, and this phenomenon is not replaceable with the HIF-2α isoform. To further probe the link between HIF-1α stabilization in hypoxia and purinosome formation, we carried out a time course experiment. Purinosome assembly and HIF-1α levels were assessed in HeLa cells incubated under hypoxic conditions at intervals over a 10 h period. We observed a ∼50% reduction in the number of purinosome-containing cells within the first 2 h of hypoxia exposure followed by a steady increase where it eventually levelled off after 8-10 h (Fig. 2b, Video S1). During the same time course, HIF-1α protein levels showed the highest expression at 3 h and gradually decreased over time (Fig. 2c), consistent with prior reports (30Asby D.J. Cuda F. Hoakwie F. Miranda E. Tavassoli A. HIF-1 promotes the expression of its α-subunit via an epigenetically regulated transactivation loop.Mol. 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Reversible compartmentalization of de novo purine biosynthetic complexes in living cells.Science. 2008; 320 (18388293): 103-10610.1126/science.1152241Crossref PubMed Scopus (368) Google Scholar). We therefore investigated whether purinosomes formed in hypoxic cells were similarly dynamic and could be disrupted by reoxygenation. After 10 h in hypoxia, cells were reincubated in normoxia for 2 h, and the number of purinosome containing cells counted. Upon reoxygenation, the ratio of purinosome-containing cells had reverted back to normoxic levels (Fig. 2b), demonstrating the reversible and transient nature of this multienzyme complex. Cellular adaptation to hypoxia is driven by HIF-1–mediated transcription of multiple genes, several of which encode metabolic enzymes (14Greijer A.E. van der Groep P. Kemming D. Shvarts A. Semenza G.L. Meijer G.A. van de Wiel M.A. Belien J.A.M. van Diest P.J. van der Wall E. Up-regulation of gene expression by hypoxia is mediated predominantly by hypoxia-inducible factor 1 (HIF-1).J. Pathol. 2005; 206 (15906272): 291-30410.1002/path.1778Crossref PubMed Scopus (360) Google Scholar, 32Lane A.N. Fan T.W. Regulation of mammalian nucleotide metabolism and biosynthesis.Nucleic Acids Res. 2015; 43 (25628363): 2466-248510.1093/nar/gkv047Crossref PubMed Scopus (444) Google Scholar). We therefore assessed whether the observed increase in purinosome formation in hypoxia is a result of an up-regulation in gene or protein expression of its enzymes. The expression levels of the six genes encoding the enzymes in de novo purine biosynthesis remained unchanged after exposure to hypoxia for 24 h (Fig. 2d). Protein expression by immunoblotting also showed no changes in the levels of the purinosome enzymes except for a slight increase in intracellular ATIC protein levels (Fig. 2e). These data eliminated the possibility that the increase in purinosome formation under hypoxic conditions is caused by an up-regulation in transcription or translation of the enzymes within the de novo purine biosynthesis pathway. It has been previously demonstrated that the formation of the purinosome complex is disrupted in cells lacking one of the enzymes in de novo purine biosynthesis or by introducing loss-of-function mutations in ADSL and/or ATIC (7Baresova V. Skopova V. Sikora J. Patterson D. Sovova J. Zikanova M. Kmoch S. Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency.Hum. Mol. Genet. 2012; 21 (22180458): 1534-154310.1093/hmg/ddr591Crossref PubMed Scopus (52) Google Scholar, 33Baresova V. Krijt M. Skopova V. Souckova O. Kmoch S. Zikanova M. 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Targeting tumour proliferation with a small-molecule inhibitor of AICAR transformylase homodimerization.ChemBioChem. 2012; 13 (22764122): 1628-163410.1002/cbic.201200279Crossref PubMed Scopus (48) Google Scholar). We used Cpd14 to probe whether disruption of ATIC homodimerization leads to inhibition of purinosome formation in hypoxia. HeLa cells were transfected with a plasmid encoding FGAMS-EGFP and treated with Cpd14 for 24 h in purine-rich media in hypoxia or normoxia supplemented with DFX. In both cases, treatment with Cpd14 prevented hypoxia- and DFX-induced purinosome formation (Fig. 3a). Similarly, purinosome formation was assessed in an ATIC knockout HeLa cell line (33Baresova V. Krijt M. Skopova V. Souckova O. Kmoch S. Zikanova M. CRISPR-Cas9 induced mutations along de novo purine synthesis in HeLa cells result in accumulation of individual enz
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