Hypoxia drives the assembly of the multienzyme purinosome complex

Abstract

<jats:p>The purinosome is a dynamic metabolic complex composed of enzymes responsible for <jats:italic>de novo</jats:italic> 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 <jats:italic>de novo</jats:italic> 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 <jats:italic>de novo</jats:italic> 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 <jats:italic>de novo</jats:italic> 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.</jats:p>

The role of liquid–liquid phase separation in regulating enzyme activity

“…Similar trends have been noted in the purine biosynthesis pathway, in which six enzymes required for de novo purine biosynthesis cluster in 'purinosomes' [39][40][41][42][43]. Increased formation of purinosome condensates under hypoxic conditions was demonstrated, similar to G body formation [40].…”

“…Similar trends have been noted in the purine biosynthesis pathway, in which six enzymes required for de novo purine biosynthesis cluster in 'purinosomes' [39][40][41][42][43]. Increased formation of purinosome condensates under hypoxic conditions was demonstrated, similar to G body formation [40]. In fact, treatment with inhibitors of glycolysis and pentose phosphate pathways in cells under hypoxia led to a reduction in purinosome formation pointing to the interconnectivity of these systems.…”

“…In contrast to G bodies [37], it was determined that there was no up-regulation of enzyme activity within the purinosome under hypoxic conditions. This was attributed to a subsequent downregulation of mitochondrial onecarbon metabolism under hypoxia and provides a caveat to previous reports stating that purinosomes increased de novo purine biosynthesis [40][41][42]. The specific reasons as to why purinosome formation is increased under hypoxic conditions therefore remains to be uncovered.…”

The purine nucleoside phosphorylase pnp-1 regulates epithelial cell resistance to infection in C. elegans

“…pnp-1 mutants are defective in the purine salvage pathway, which is less energycostly than the de novo synthesis pathway. Under normal conditions the salvage pathway is preferentially used, but in response to high purine demands such as heat shock, the de novo pathway is activated to increase purine metabolic flux [37][38][39][40][41][42][43]. One possibility is that pnp-1 mutants have constitutive use of the de novo pathway, and so are unable to increase purine metabolic flux upon heat shock, resulting in decreased thermotolerance.…”

Small‐scale hypoxic cultures for monitoring the spatial reorganization of glycolytic enzymes in Saccharomyces cerevisiae

“…Cytoplasmic foci, or G‐bodies, are likely to comprise more than 40 proteins, including metabolic enzymes and transcription factors, under hypoxia (Jin et al, 2017; Miura et al, 2013). Recently, in addition to G‐bodies, another cytoplasmic complex of metabolic enzymes, the purinosome, was found to form in S. cerevisiae under hypoxia (Doigneaux et al, 2020). Here, we propose the highly organized complex of proteins formed under hypoxia as a META body.…”