Cellulose-bound Peptide Arrays: Preparation and Applications

Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery.     

Expression of the purine biosynthetic enzyme phosphoribosyl formylglycinamidine synthase in neurons

Purines are metabolic building blocks essential for all living organisms on earth. De novo purine biosynthesis occurs in the brain and appears to play important roles in neural development. Phosphoribosyl formylglycinamidine synthase (FGAMS , also known as PFAS or FGARAT ), a core enzyme involved in the de novo synthesis of purines, may play alternative roles in viral pathogenesis. To date, no thorough investigation of the endogenous expression and localization of de novo purine biosynthetic enzymes has been conducted in human neurons or in virally infected cells. In this study, we characterized expression of FGAMS using multiple neuronal models. In differentiated human SH ‐SY 5Y neuroblastoma cells, primary rat hippocampal neurons, and in whole‐mouse brain sections, FGAMS immunoreactivity was distributed within the neuronal cytoplasm. FGAMS immunolabeling in vitro demonstrated extensive distribution throughout neuronal processes. To investigate potential changes in FGAMS expression and localization following viral infection, we infected cells with the human pathogen herpes simplex virus 1. In infected fibroblasts, FGAMS immunolabeling shifted from a diffuse cytoplasmic location to a mainly perinuclear localization by 12 h post‐infection. In contrast, in infected neurons, FGAMS localization showed no discernable changes in the localization of FGAMS immunoreactivity. There were no changes in total FGAMS protein levels in either cell type. Together, these data provide insight into potential purine biosynthetic mechanisms utilized within neurons during homeostasis as well as viral infection.

Cover Image for this Issue: doi: 10.1111/jnc.14169 .

     

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.