Songon An

Reversible Compartmentalization of de Novo Purine Biosynthetic Complexes in Living Cells

Purines are synthesized de novo in 10 chemical steps that are catalyzed by six enzymes in eukaryotes. Studies in vitro have provided little evidence of anticipated protein-protein interactions that would enable substrate channeling and regulation of the metabolic flux. We applied fluorescence microscopy to HeLa cells and discovered that all six enzymes colocalize to form clusters in the cellular cytoplasm. The association and dissociation of these enzyme clusters can be regulated dynamically, by either changing the purine levels of or adding exogenous agents to the culture media. Collectively, the data provide strong evidence for the formation of a multi-enzyme complex, the “purinosome,” to carry out de novo purine biosynthesis in cells.

Trans-editing of Cys-tRNAPro by Haemophilus influenzae YbaK Protein

Prolyl-tRNA synthetases (ProRSs) from all three domains of life have been shown to misactivate cysteine and to mischarge cysteine onto tRNAPro. Although most bacterial ProRSs possess an amino acid editing domain that deacylates mischarged Ala-tRNAPro, editing of Cys-tRNAPro has not been demonstrated and a double-sieve mechanism of editing does not appear to be sufficient to eliminate all misacylated tRNAPro species from the cell. It was recently shown that a ProRS paralog, the YbaK protein from Haemophilus influenzae, which is homologous to the ProRS editing domain, is capable of weakly deacylating Ala-tRNAPro. This function appears to be redundant with that of its corresponding ProRS, which contains a canonical bacterial editing domain. In the present study, we test the specificity of editing by H. influenzae YbaK and show that it efficiently edits Cys-tRNAPro and that a conserved Lys residue is essential for this activity. These findings represent the first example of an editing domain paralog possessing altered specificity and suggest that similar autonomous editing domains could act upon different mischarged tRNAs thus providing cells with enhanced proofreading potential. This work also suggests a novel mechanism of editing wherein a third sieve is used to clear Cys-tRNAPro in at least some organisms.

GPCRs regulate the assembly of a multienzyme complex for purine biosynthesis

G protein-coupled receptors (GPCRs) transmit exogenous signals to the nucleus, promoting a myriad of biological responses via multiple signaling pathways in both normal and cancer cells. However, little is known about the response in cytosolic metabolic pathways to GPCR-mediated signaling. Here, we applied fluorescent live-cell imaging and label-free dynamic mass redistribution assays to study whether purine metabolism is associated with GPCR signaling. By screening a library of GPCR ligands in conjunction with live-cell imaging of a metabolic multienzyme complex for de novo purine biosynthesis, the purinosome, we demonstrated that the activation of endogenous Gαi-coupled receptors correlates with purinosome assembly/disassembly in native HeLa cells. Given the implications of GPCRs in mitogenic signaling as well as the purinosome in controlling metabolic flux via de novo purine biosynthesis, we hypothesize that regulation of purinosome assembly/disassembly may represent one of downstream events of mitogenic GPCR signaling in human cancer cells.

Microtubule-assisted mechanism for functional metabolic macromolecular complex formation

Evidence has been presented for a metabolic multienzyme complex, the purinosome, that participates in de novo purine biosynthesis to form clusters in the cytoplasm of living cells under purine-depleted conditions. Here we identified, using fluorescent live cell imaging, that a microtubule network appears to physically control the spatial distribution of purinosomes in the cytoplasm. Application of a cell-based assay measuring the rate of de novo purine biosynthesis confirmed that the metabolic activity of purinosomes was significantly suppressed in the absence of microtubules. Collectively, we propose a microtubule-assisted mechanism for functional purinosome formation in HeLa cells.

Dynamic Regulation of a Metabolic Multi-enzyme Complex by Protein Kinase CK2

The reversible association and dissociation of a metabolic multi-enzyme complex participating in de novo purine biosynthesis, the purinosome, was demonstrated in live cells to respond to the levels of purine nucleotides in the culture media. We also took advantage of in vitro proteomic scale studies of cellular substrates of human protein kinases (e.g. casein kinase II (CK2) and Akt), that implicated several de novo purine biosynthetic enzymes as kinase substrates. Here, we successfully identified that purinosome formation in vivo was significantly promoted in HeLa cells by the addition of small-molecule CK2-specific inhibitors (i.e. 4,5,6,7-tetrabromo-1H-benzimidazole, 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole, tetrabromocinammic acid, 4,4′,5,5′,6,6′-hexahydroxydiphenic acid 2,2′,6,6′-dilactone (ellagic acid) as well as by silencing the endogenous human CK2α catalytic subunit with small interfering RNA. However, 4,5,6,7-tetrabromobenzotriazole, another CK2-specific inhibitor, triggered the dissociation of purinosome clusters in HeLa cells. Although the mechanism by which 4,5,6,7-tetrabromobenzotriazole affects purinosome clustering is not clear, we were capable of chemically reversing purinosome formation in cells by the sequential addition of two CK2 inhibitors. Collectively, we provide compelling cellular evidence that CK2-mediated pathways reversibly regulate purinosome assembly, and thus the purinosome may be one of the ultimate targets of kinase inhibitors.

Hsp70/Hsp90 chaperone machinery is involved in the assembly of the purinosome

The de novo biosynthesis of purines is carried out by a highly conserved metabolic pathway that includes several validated targets for anticancer, immunosuppressant, and anti-inflammatory chemotherapeutics. The six enzymes in humans that catalyze the 10 chemical steps from phosphoribosylpyrophosphate to inosine monophosphate were recently shown to associate into a dynamic multiprotein complex called the purinosome. Here, we demonstrate that heat shock protein 90 (Hsp90), heat shock protein 70 (Hsp70), and several cochaperones functionally colocalize with this protein complex. Knockdown of expression levels of the identified cochaperones leads to disruption of purinosomes. In addition, small molecule inhibitors of Hsp90 and Hsp70 reversibly disrupt purinosomes and are shown to have a synergistic effect with methotrexate, an anticancer agent that targets purine biosynthesis. These data implicate the Hsp90/Hsp70 chaperone machinery in the assembly of the purinosome and provide a strategy for the development of improved anticancer therapies that disrupt purine biosynthesis.

Mapping Protein-Protein Proximity in the Purinosome

Background: Six enzymes in the human de novo purine synthesis pathway form a cellular enzyme complex, the purinosome, to increase the purine synthesis rate under a purine-depleted condition. Results: Cellular protein-protein proximity was detected among six enzymes in the human de novo purine synthesis pathway. Conclusion: All six enzymes in the human de novo purine synthesis pathway are in proximity during purinosome formation, which could be achieved by either direct or indirect protein-protein interactions. Significance: Probing cellular protein-protein proximity provides an approach to examine the factors modulating purinosome formation and disassembly. The enzymes in the human de novo purine synthesis pathway were found to form a cellular complex, the purinosome, upon culturing cells in purine-depleted medium (An, S., Kumar R., Sheets, E. D., and Benkovic, S. J. (2008) Science 320, 103–106). Purinosome formation and dissociation were found to be modulated by several factors, including the microtubule network and cell signaling involving protein phosphorylation. To determine whether the pathway enzymes are in physical contact, we probed for the protein-protein interactions (PPIs) within the purinosome with a novel application of the Tango PPI reporter system (Barnea, G., Strapps, W., Herrada, G., Berman, Y., Ong, J., Kloss, B., Axel, R., and Lee, K. J. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 64–69). We found PPIs among all six enzymes within the pathway and evidence for a core involving the first three enzymes. We also captured purinosomes under both purine-rich and purine-depleted conditions. The results provide additional insights into the transient nature and topography of the purinosome.

Substrate-mediated fidelity mechanism ensures accurate decoding of proline codons

Aminoacyl-tRNA synthetases attach specific amino acids to cognate tRNAs. Prolyl-tRNA synthetases are known to mischarge tRNAPro with the smaller amino acid alanine and with cysteine, which is the same size as proline. Quality control in proline codon translation is partly ensured by an editing domain (INS) present in most bacterial prolyl-tRNA synthetases that hydrolyzes smaller Ala-tRNAPro and excludes Pro-tRNAPro. In contrast, Cys-tRNAPro is cleared by a freestanding INS domain homolog, YbaK. Here, we have investigated the molecular mechanism of catalysis and substrate recognition by Hemophilus influenzae YbaK using site-directed mutagenesis, enzymatic assays of isosteric substrates and functional group analogs, and computational modeling. These studies together with mass spectrometric characterization of the YbaK-catalyzed reaction products support a novel substrate-assisted mechanism of Cys-tRNAPro deacylation that prevents nonspecific Pro-tRNAPro hydrolysis. Collectively, we propose that the INS and YbaK domains co-evolved distinct mechanisms involving steric exclusion and thiol-specific chemistry, respectively, to ensure accurate decoding of proline codons.

Dynamic architecture of the purinosome involved in human de novo purine biosynthesis.

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.

Spatial Organization of Metabolic Enzyme Complexes in Cells

The organization of metabolic multienzyme complexes has been hypothesized to benefit metabolic processes and provide a coordinated way for the cell to regulate metabolism. Historically, their existence has been supported by various in vitro techniques. However, it is only recently that the existence of metabolic complexes inside living cells has come to light to corroborate this long-standing hypothesis. Indeed, subcellular compartmentalization of metabolic enzymes appears to be widespread and highly regulated. On the other hand, it is still challenging to demonstrate the functional significance of these enzyme complexes in the context of the cellular milieu. In this review, we discuss the current understanding of metabolic enzyme complexes by primarily focusing on central carbon metabolism and closely associated metabolic pathways in a variety of organisms, as well as their regulation and functional contributions to cells.