Dynamic Architecture of the Purinosome Involved in Human De Novo Purine Biosynthesis

Abstract

Enzymes in human de novo purine biosynthesis have been demonstrated to form a reversible, transient multienzyme complex, the purinosome, upon purine starvation. However, characterization of purinosomes has been limited to HeLa cells and has heavily relied on qualitative examination of their subcellular localization and reversibility under wide-field fluorescence microscopy. Quantitative approaches, which are particularly compatible with human disease-relevant cell lines, are necessary to explicitly understand the purinosome in live cells. In this work, human breast carcinoma Hs578T cells have been utilized to demonstrate the preferential utilization of the purinosome under purine-depleted conditions. In addition, we have employed a confocal microscopy-based biophysical technique, fluorescence recovery after photobleaching, to characterize kinetic properties of the purinosome in live Hs578T cells. Quantitative characterization of the diffusion coefficients of all de novo purine biosynthetic enzymes reveals the significant reduction of their mobile kinetics upon purinosome formation, the dynamic partitioning of each enzyme into the purinosome, and the existence of three intermediate species in purinosome assembly under purine starvation. We also demonstrate that the diffusion coefficient of the purine salvage enzyme, hypoxanthine phosphoribosyltransferase 1, is not sensitive to purine starvation, indicating exclusion of the salvage pathway from the purinosome. Furthermore, our biophysical characterization of nonmetabolic enzymes clarifies that purinosomes are spatiotemporally different cellular bodies from stress granules and cytoplasmic protein aggregates in both Hs578T and HeLa cells. Collectively, quantitative analyses of the purinosome in Hs578T cells led us to provide novel insights for the dynamic architecture of the purinosome assembly.

Sequestration-Mediated Downregulation of de Novo Purine Biosynthesis by AMPK

“…Due to the slower diffusion kinetics of FGAMS-EGFP inside the self-assembly, we demonstrate FRAP as a biophysical technique to distinguish these two assemblies in live cells. In this regard, we note that, due to different transcriptional and translational profiles in different cell lines, 24 it was not surprising that the diffusion coefficients of FGAMS-EGFP are not only assembly-dependent but also cell-line-dependent (this work versus ref 2). Third, profiling metabolic changes of purine metabolites indicates whether the observed compartments in single cells are purinosomes or FGAMS self-assemblies.…”

A New View into the Regulation of Purine Metabolism: The Purinosome

“…Likewise, HeLa cell lines deficient in specific pathway enzymes resulted either in a complete loss of purinosomes (GART, ADSL, ATIC) or a significant reduction (FGAMS, PAICS) compared to normal HeLa cells [34]. Measurement of the diffusion coefficients of these enzymes in Hs578T breast carcinoma cells using fluorescence recovery after photobleaching substantiated the spatial model of core and peripheral enzymes that assemble stepwise (Figure 2B) [43]. Additionally, adenylsuccinate synthase (ADSS) and inosine monophosphate dehydrogenase (IMPDH) were identified as members of the purinosome [36].…”

“…(B) The first three enzymes in the de novo purine biosynthetic pathway (PPAT, GART, and FGAMS) form the core of the purinosome and assemble first before secondary complexes of [PAICS · ADSL] and [ATIC] interact with the core. (C) Several proteomic studies have uncovered interactions between the enzymes in the de novo purine biosynthetic pathway [33, 39-43]. Captured here is the oligomeric state of the active form of each enzyme in the de novo purine biosynthetic pathway and those protein-protein interactions reported between them where the interaction strength (edge thickness) corresponds to the number of studies referencing the interaction.…”

Spatial Organization of Metabolic Enzyme Complexes in Cells

“…89,90 Biochemical and biophysical studies have also proposed that three enzymes involved in the first half of the pathway (steps 1–5) form a core structure of the purinosome, while the other three enzymes, catalyzing steps 6 through 10, are dynamically associated with the core complex via protein–protein interactions. 91,92 Furthermore, knockout of any purine biosynthetic enzyme resulted in either the reduction or abolishment of purinosome association in human cells. 93 More recently, the functional activity of mechanistic target of rapamycin (mTOR) was linked to the spatial association of purinosomes with the mitochondria as well as purine biosynthesis.…”