Structural basis of the T4 bacteriophage primosome assembly and primer synthesis.

The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.

Structural basis of the T4 bacteriophage primosome assembly and primer synthesis.

The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome complex to couple DNA unwinding with RNA primer synthesis for DNA replication. How a primosome is assembled and how the length of the RNA primer is defined in the T4 bacteriophage, or in any model system, are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates at resolutions up to 2.7 Å. We show that the gp41 helicase is an open spiral in the absence of ssDNA, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the open spiral to a closed ring that activates the helicase. We found that the activation of the gp41 helicase exposes a cryptic hydrophobic primase-binding surface allowing for the recruitment of the gp61 primase. The primase binds the gp41 helicase in a bipartite mode in which the N-terminal Zn-binding domain (ZBD) and the C-terminal RNA polymerase domain (RPD) each contain a helicase-interacting motif (HIM1 and HIM2, respectively) that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Based on two observed primosome conformations - one in a DNA-scanning mode and the other in a post RNA primer-synthesis mode - we suggest that the linker loop between the gp61 ZBD and RPD contributes to the T4 pentaribonucleotide primer. Our study reveals T4 primosome assembly process and sheds light on RNA primer synthesis mechanism.

Purine synthesis suppression reduces the development and progression of pulmonary hypertension in rodent models.

AIMS: Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of pulmonary hypertension (PH). Proliferative cells utilize purine bases from the de novo purine synthesis (DNPS) pathways for nucleotide synthesis; however, it is unclear whether DNPS plays a critical role in VSMC proliferation during development of PH. The last two steps of DNPS are catalysed by the enzyme 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC). This study investigated whether ATIC-driven DNPS affects the proliferation of pulmonary artery smooth muscle cells (PASMCs) and the development of PH. METHODS AND RESULTS: Metabolites of DNPS in proliferative PASMCs were measured by liquid chromatography-tandem mass spectrometry. ATIC expression was assessed in platelet-derived growth factor-treated PASMCs and in the lungs of PH rodents and patients with pulmonary arterial hypertension. Mice with global and VSMC-specific knockout of Atic were utilized to investigate the role of ATIC in both hypoxia- and lung interleukin-6/hypoxia-induced murine PH. ATIC-mediated DNPS at the mRNA, protein, and enzymatic activity levels were increased in platelet-derived growth factor-treated PASMCs or PASMCs from PH rodents and patients with pulmonary arterial hypertension. In cultured PASMCs, ATIC knockdown decreased DNPS and nucleic acid DNA/RNA synthesis, and reduced cell proliferation. Global or VSMC-specific knockout of Atic attenuated vascular remodelling and inhibited the development and progression of both hypoxia- and lung IL-6/hypoxia-induced PH in mice. CONCLUSION: Targeting ATIC-mediated DNPS compromises the availability of purine nucleotides for incorporation into DNA/RNA, reducing PASMC proliferation and pulmonary vascular remodelling and ameliorating the development and progression of PH.

ATIC-Associated De Novo Purine Synthesis Is Critically Involved in Proliferative Arterial Disease.

BACKGROUND: Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arterial diseases, especially in arterial restenosis after angioplasty or stent placement. VSMCs reprogram their metabolism to meet the increased requirements of lipids, proteins, and nucleotides for their proliferation. De novo purine synthesis is one of critical pathways for nucleotide synthesis. However, its role in proliferation of VSMCs in these arterial diseases has not been defined. METHODS: De novo purine synthesis in proliferative VSMCs was evaluated by liquid chromatography-tandem mass spectrometry. The expression of ATIC (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase), the critical bifunctional enzyme in the last 2 steps of the de novo purine synthesis pathway, was assessed in VSMCs of proliferative arterial neointima. Global and VSMC-specific knockout of Atic mice were generated and used for examining the role of ATIC-associated purine metabolism in the formation of arterial neointima and atherosclerotic lesions. RESULTS: In this study, we found that de novo purine synthesis was increased in proliferative VSMCs. Upregulated purine synthesis genes, including ATIC, were observed in the neointima of the injured vessels and atherosclerotic lesions both in mice and humans. Global or specific knockout of Atic in VSMCs inhibited cell proliferation, attenuating the arterial neointima in models of mouse atherosclerosis and arterial restenosis. CONCLUSIONS: These results reveal that de novo purine synthesis plays an important role in VSMC proliferation in arterial disease. These findings suggest that targeting ATIC is a promising therapeutic approach to combat arterial diseases.

Multienzyme interactions of the de novo purine biosynthetic protein PAICS facilitate purinosome formation and metabolic channeling.

There is growing evidence that mammalian cells deploy a mitochondria-associated metabolon called the purinosome to perform channeled de novo purine biosynthesis (DNPB). However, the molecular mechanisms of this substrate-channeling pathway are not well defined. Here, we present molecular evidence of protein-protein interactions (PPIs) between the human bifunctional phosphoribosylaminoimidazole carboxylase/succinocarboxamide synthetase (PAICS) and other known DNPB enzymes. We employed two orthogonal approaches: bimolecular fluorescence complementation, to probe PPIs inside live, intact cells, and co-immunoprecipitation using StrepTag-labeled PAICS that was reintegrated into the genome of PAICS-knockout HeLa cells (crPAICS). With the exception of amidophosphoribosyltransferase, the first enzyme of the DNPB pathway, we discovered PAICS interacts with all other known DNPB enzymes and with MTHFD1, an enzyme which supplies the 10-formyltetrahydrofolate cofactor essential for DNPB. We show these interactions are present in cells grown in both purine-depleted and purine-rich conditions, suggesting at least a partial assembly of these enzymes may be present regardless of the activity of the DNPB pathway. We also demonstrate that tagging of PAICS on its C terminus disrupts these interactions and that this disruption is correlated with disturbed DNPB activity. Finally, we show that crPAICS cells with reintegrated N-terminally tagged PAICS regained effective DNPB with metabolic signatures of channeled synthesis, whereas crPAICS cells that reintegrated C-terminally tagged PAICS exhibit reduced DNPB intermediate pools and a perturbed partitioning of inosine monophosphate into AMP and GMP. Our results provide molecular evidence in support of purinosomes and suggest perturbing PPIs between DNPB enzymes negatively impact metabolite flux through this important pathway.

Purine biosynthetic enzymes assemble into liquid-like condensates dependent on the activity of chaperone protein HSP90.

Enzymes within the de novo purine biosynthetic pathway spatially organize into dynamic intracellular assemblies called purinosomes. The formation of purinosomes has been correlated with growth conditions resulting in high purine demand, and therefore, the cellular advantage of complexation has been hypothesized to enhance metabolite flux through the pathway. However, the properties of this cellular structure are unclear. Here, we define the purinosome in a transient expression system as a biomolecular condensate using fluorescence microscopy. We show that purinosomes, as denoted by formylglycinamidine ribonucleotide synthase granules in purine-depleted HeLa cells, are spherical and appear to coalesce when two come into contact, all liquid-like characteristics that are consistent with previously reported condensates. We further explored the biophysical and biochemical means that drive the liquid-liquid phase separation of these structures. We found that the process of enzyme condensation into purinosomes is likely driven by the oligomeric state of the pathway enzymes and not a result of intrinsic disorder, the presence of low-complexity domains, the assistance of RNA scaffolds, or changes in intracellular pH. Finally, we demonstrate that the heat shock protein 90 KDa helps to regulate the physical properties of the condensate and maintain their liquid-like state inside HeLa cells. We show that disruption of heat shock protein 90 KDa activity induced the transformation of formylglycinamidine ribonucleotide synthase clusters into more irregularly shaped condensates, suggesting that its chaperone activity is essential for purinosomes to retain their liquid-like properties. This refined view of the purinosome offers new insight into how metabolic enzymes spatially organize into dynamic condensates within human cells.

The Purinosome: A Case Study for a Mammalian Metabolon.

Over the past fifteen years, we have unveiled a new mechanism by which cells achieve greater efficiency in de novo purine biosynthesis. This mechanism relies on the compartmentalization of de novo purine biosynthetic enzymes into a dynamic complex called the purinosome. In this review, we highlight our current understanding of the purinosome with emphasis on its biophysical properties and function and on the cellular mechanisms that regulate its assembly. We propose a model for functional purinosomes in which they consist of at least ten enzymes that localize near mitochondria and carry out de novo purine biosynthesis by metabolic channeling. We conclude by discussing challenges and opportunities associated with studying the purinosome and analogous metabolons.

Metabolic channeling: predictions, deductions, and evidence.

With the elucidation of myriad anabolic and catabolic enzyme-catalyzed cellular pathways crisscrossing each other, an obvious question arose: how could these networks operate with maximal catalytic efficiency and minimal interference? A logical answer was the postulate of metabolic channeling, which in its simplest embodiment assumes that the product generated by one enzyme passes directly to a second without diffusion into the surrounding medium. This tight coupling of activities might increase a pathway's metabolic flux and/or serve to sequester unstable/toxic/reactive intermediates as well as prevent their access to other networks. Here, we present evidence for this concept, commencing with enzymes that feature a physical molecular tunnel, to multi-enzyme complexes that retain pathway substrates through electrostatics or enclosures, and finally to metabolons that feature collections of enzymes assembled into clusters with variable stoichiometric composition. Lastly, we discuss the advantages of reversibly assembled metabolons in the context of the purinosome, the purine biosynthesis metabolon.

From Bioorganic Models to Cells.

I endeavor to share how various choices-some deliberate, some unconscious-and the unmistakable influence of many others shaped my scientific pursuits. I am fascinated by how two long-term, major streams of my research, DNA replication and purine biosynthesis, have merged with unexpected interconnections. If I have imparted to many of the talented individuals who have passed through my lab a degree of my passion for uncloaking the mysteries hidden in scientific research and an understanding of the honesty and rigor it demands and its impact on the world community, then my mentorship has been successful.

Human de novo purine biosynthesis.

The focus of this review is the human de novo purine biosynthetic pathway. The pathway enzymes are enumerated, as well as the reactions they catalyze and their physical properties. Early literature evidence suggested that they might assemble into a multi-enzyme complex called a metabolon. The finding that fluorescently-tagged chimeras of the pathway enzymes form discrete puncta, now called purinosomes, is further elaborated in this review to include: a discussion of their assembly; the role of ancillary proteins; their locus at the microtubule/mitochondria interface; the elucidation that at endogenous levels, purinosomes function to channel intermediates from phosphoribosyl pyrophosphate to AMP and GMP; and the evidence for the purinosomes to exist as a protein condensate. The review concludes with a consideration of probable signaling pathways that might promote the assembly and disassembly of the purinosome, in particular the identification of candidate kinases given the extensive phosphorylation of the enzymes. These collective findings substantiate our current view of the de novo purine biosynthetic metabolon whose properties will be representative of how other metabolic pathways might be organized for their function.

PCNA Monoubiquitination Is Regulated by Diffusion of Rad6/Rad18 Complexes along RPA Filaments.

Translesion DNA synthesis (TLS) enables DNA replication through damaging modifications to template DNA and requires monoubiquitination of the proliferating cell nuclear antigen (PCNA) sliding clamp by the Rad6/Rad18 complex. This posttranslational modification is critical to cell survival following exposure to DNA-damaging agents and is tightly regulated to restrict TLS to damaged DNA. Replication protein A (RPA), the major single-strand DNA (ssDNA) binding protein complex, forms filaments on ssDNA exposed at TLS sites and plays critical yet undefined roles in regulating PCNA monoubiquitination. Here, we utilize kinetic assays and single-molecule FRET microscopy to monitor PCNA monoubiquitination and Rad6/Rad18 complex dynamics on RPA filaments, respectively. Results reveal that a Rad6/Rad18 complex is recruited to an RPA filament via Rad18·RPA interactions and randomly translocates along the filament. These translocations promote productive interactions between the Rad6/Rad18 complex and the resident PCNA, significantly enhancing monoubiquitination. These results illuminate critical roles of RPA in the specificity and efficiency of PCNA monoubiquitination and represent, to the best of our knowledge, the first example of ATP-independent translocation of a protein complex along a protein filament.

Hypoxia drives the assembly of the multienzyme purinosome complex.

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.

Metabolomics and mass spectrometry imaging reveal channeled de novo purine synthesis in cells.

Metabolons, multiprotein complexes consisting of sequential enzymes of a metabolic pathway, are proposed to be biosynthetic "hotspots" within the cell. However, experimental demonstration of their presence and functions has remained challenging. We used metabolomics and in situ three-dimensional submicrometer chemical imaging of single cells by gas cluster ion beam secondary ion mass spectrometry (GCIB-SIMS) to directly visualize de novo purine biosynthesis by a multienzyme complex, the purinosome. We found that purinosomes comprise nine enzymes that act synergistically, channeling the pathway intermediates to synthesize purine nucleotides, increasing the pathway flux, and influencing the adenosine monophosphate/guanosine monophosphate ratio. Our work also highlights the application of high-resolution GCIB-SIMS for multiplexed biomolecular analysis at the level of single cells.

Recognition of a Key Anchor Residue by a Conserved Hydrophobic Pocket Ensures Subunit Interface Integrity in DNA Clamps.

Sliding clamp proteins encircle duplex DNA and are involved in processive DNA replication and the DNA damage response. Clamp proteins are ring-shaped oligomers (dimers or trimers) and are loaded onto DNA by an ATP-dependent clamp loader complex that ruptures the interface between two adjacent subunits. Here we measured the solution dynamics of the human clamp protein, proliferating cell nuclear antigen, by monitoring the change in the fluorescence of a site-specifically labeled. To unravel the origins of clamp subunit interface stability, we carried out comprehensive comparative analysis of the interfaces of seven sliding clamps. We used computational modeling (molecular dynamic simulations and MM/GBSA binding energy decomposition analyses) to identify conserved networks of hydrophobic residues critical for clamp stability and ring-opening dynamics. The hydrophobic network is shared among clamp proteins and exhibits a "key in a keyhole" pattern where a bulky aromatic residue from one clamp subunit is anchored into a hydrophobic pocket of the opposing subunit. Bioinformatics and dynamic network analyses showed that this oligomeric latch is conserved across DNA sliding clamps from all domains of life and dictates the dynamics of clamp opening and closing.

Mapping Post-Translational Modifications of de Novo Purine Biosynthetic Enzymes: Implications for Pathway Regulation.

Purines represent a class of essential metabolites produced by the cell to maintain cellular homeostasis and facilitate cell proliferation. In times of high purine demand, the de novo purine biosynthetic pathway is activated; however, the mechanisms that facilitate this process are largely unknown. One plausible mechanism is through intracellular signaling, which results in enzymes within the pathway becoming post-translationally modified to enhance their individual enzyme activities and the overall pathway metabolic flux. Here, we employ a proteomic strategy to investigate the extent to which de novo purine biosynthetic pathway enzymes are post-translationally modified in 293T cells. We identified 7 post-translational modifications on 135 residues across the 6 human pathway enzymes. We further asked whether there were differences in the post-translational modification state of each pathway enzyme isolated from cells cultured in the presence or absence of purines. Of the 174 assigned modifications, 67% of them were only detected in one experimental growth condition in which a significant number of serine and threonine phosphorylations were noted. A survey of the most-probable kinases responsible for these phosphorylation events uncovered a likely AKT phosphorylation site at residue Thr397 of PPAT, which was only detected in cells under purine-supplemented growth conditions. These data suggest that this modification might alter enzyme activity or modulate its interaction(s) with downstream pathway enzymes. Together, these findings propose a role for post-translational modifications in pathway regulation and activation to meet intracellular purine demand.

Replication protein A dynamically regulates monoubiquitination of proliferating cell nuclear antigen.

DNA damage tolerance permits bypass of DNA lesions encountered during S-phase and may be carried out by translesion DNA synthesis (TLS). Human TLS requires selective monoubiquitination of proliferating cell nuclear antigen (PCNA) sliding clamps encircling damaged DNA. This posttranslational modification (PTM) is catalyzed by Rad6/Rad18. Recent studies revealed that replication protein A (RPA), the major ssDNA-binding protein, is involved in the regulation of PCNA monoubiquitination and interacts directly with Rad18 on chromatin and in the nucleoplasm. However, it is unclear how RPA regulates this critical PTM and what functional role(s) these interactions serve. Here, we developed an in vitro assay to quantitatively monitor PCNA monoubiquitination under in vivo scenarios. Results from extensive experiments revealed that RPA regulates Rad6/Rad18 activity in an ssDNA-dependent manner. We found that "DNA-free" RPA inhibits monoubiquitination of free PCNA by directly interacting with Rad18. This interaction is promoted under native conditions when there is an overabundance of free RPA in the nucleoplasm where Rad6/Rad18 and a significant fraction of PCNA reside. During DNA replication stress, RPA binds the ssDNA exposed downstream of stalled primer/template (P/T) junctions, releasing Rad6/Rad18. RPA restricted the resident PCNAs to the upstream duplex regions by physically blocking diffusion of PCNA along ssDNA, and this activity was required for efficient monoubiquitination of PCNA on DNA. Furthermore, upon binding ssDNA, RPA underwent a conformational change that increased its affinity for Rad18. Rad6/Rad18 complexed with ssDNA-bound RPA was active, and this interaction may selectively promote monoubiquitination of PCNA on long RPA-coated ssDNA.

Microtubule-directed transport of purine metabolons drives their cytosolic transit to mitochondria.

To meet their purine demand, cells activate the de novo purine biosynthetic pathway and transiently cluster the pathway enzymes into metabolons called purinosomes. Recently, we have shown that purinosomes were spatially colocalized with mitochondria and microtubules, yet it remained unclear as to what drives these associations and whether a relationship between them exist. Here, we employed superresolution imaging methods to describe purinosome transit in the context of subcellular localization. Time-resolved imaging of purinosomes showed that these assemblies exhibit directed motion as they move along a microtubule toward mitochondria, where upon colocalization, a change in purinosome motion was observed. A majority of purinosomes colocalized with mitochondria were also deemed colocalized with microtubules. Nocodazole-dependent microtubule depolymerization resulted in a loss in the purinosome-mitochondria colocalization, suggesting that the association of purinosomes with mitochondria is facilitated by microtubule-directed transport, and thereby supporting our notion of an interdependency between these subcellular components in maximizing purine production through the de novo purine biosynthetic pathway.

Detecting Purinosome Metabolon Formation with Fluorescence Microscopy.

A long-standing hypothesis in the de novo purine biosynthetic pathway is that there must be highly coordinated processes to allow for enhanced metabolic flux when a cell demands purines. One mechanism by which the pathway meets its cellular demand is through the spatial organization of pathway enzymes into multienzyme complexes called purinosomes. Cellular conditions known to impact the activity of enzymes in the pathway or overall pathway flux have been reflected in a change in the number of purinosome-positive cells or the density of purinosomes in a given cell. The following general protocols outline the steps needed for purinosome detection through transient expression of fluorescent protein chimeras or through immunofluorescence in purine-depleted HeLa cells using confocal laser scanning microscopy. These protocols define a purinosome as a colocalization of FGAMS with one additional pathway enzyme, such as PPAT or GART, and provide insights into the proper identification of a purinosome from other reported cellular bodies.

Role of HSP90 in the Regulation of de Novo Purine Biosynthesis.

Despite purines making up one of the largest classes of metabolites in a cell, little is known about the regulatory mechanisms that facilitate efficient purine production. Under conditions resulting in high purine demand, enzymes within the de novo purine biosynthetic pathway cluster into multienzyme assemblies called purinosomes. Purinosome formation has been linked to molecular chaperones HSP70 and HSP90; however, the involvement of these molecular chaperones in purinosome formation remains largely unknown. Here, we present a new-found biochemical mechanism for the regulation of de novo purine biosynthetic enzymes mediated through HSP90. HSP90-client protein interaction assays were employed to identify two enzymes within the de novo purine biosynthetic pathway, PPAT and FGAMS, as client proteins of HSP90. Inhibition of HSP90 by STA9090 abrogated these interactions and resulted in a decrease in the level of available soluble client protein while having no significant effect on their interactions with HSP70. These findings provide a mechanism to explain the dependence of purinosome assembly on HSP90 activity. The combined efforts of molecular chaperones in the maturation of PPAT and FGAMS result in purinosome formation and are likely essential for enhancing the rate of purine production to meet intracellular purine demand.