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.

Here, we report the complete genome sequence of Streptomyces venezuelae ATCC 15439, a producer of the methymycin/pikromycin family of macrolide antibiotics and a model host for natural product studies, obtained exclusively using PacBio sequencing technology. The 9.03-Mbp genome harbors 8,775 genes and 11 polyketide and nonribosomal peptide natural product gene clusters.

We describe peptide mapping through Split Antibiotic Resistance Complementation (SpARC-map), a method to identify the probable interface between two interacting proteins. Our method is based on in vivo affinity selection inside a bacterial host and uses high-throughput DNA sequencing to infer probable protein–protein interaction (PPI) interfaces. SpARC-map uses only routine microbiology techniques, with no reliance on specialized instrumentation, dedicated reagents, or reconstituting protein complexes in vitro. SpARC-map can be tuned to detect PPIs over a broad range of affinities, multiplexed to probe multiple PPIs in parallel, and its nonspecific background can be precisely measured, enabling the sensitive detection of weak PPIs. Using SpARC-map, we recover known PPI interfaces in the p21–PCNA, p53–MDM2, and MYC–MAX complexes. We also use SpARC-map to probe the purinosome, the weakly bound complex of six purine biosynthetic enzymes, where no PPI interfaces are known. There, we identify interfaces that satisfy structural requirements for substrate channeling, as well as protein surfaces that participate in multiple distinct interactions, which we validate using site-specific photocrosslinking in live human cells. Finally, we show that SpARC-map results can impose stringent constraints on machine learning–based structure prediction.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the death of motor neurons leading to paralysis and death, generally 3–5 years post-symptom onset. The most frequent genetic cause of ALS is a hexanucleotide repeat expansion (HRE) in the chromosome 9 open reading frame 72 (C9orf72) gene, that has three major hypothesised pathological mechanisms including the production of dipeptide repeat proteins (DPRs). Our laboratory has previously identified purine metabolism dysfunction in induced neural progenitor cell-derived astrocytes (iAstrocytes) from C9orf72 ALS (C9-ALS) cases (C9-iAstrocytes), driven by loss of the enzyme adenosine deaminase (ADA). Here, we have demonstrated that loss of ADA along with changes to ecto-5′-nucleotidase and hypoxanthine-guanine phosphoribosyl transferase led to disruption in purine metabolite levels including purine dNTP output. These changes were recapitulated in patient CSF, whilst loss of ADA was recapitulated in patient white matter. Immunofluorescence also demonstrated purinosome formation dysfunction in C9-iAstrocytes. These changes are likely driven by DPRs as ADA loss was recapitulated in in vitro and in vivo DPR models. Finally, ADA levels could be recovered by reducing DPR levels either by inhibiting serine/arginine-rich splicing factor 1 or overexpressing RuvB-like 2. Our data demonstrate that DPR production negatively affects purine function in C9-ALS suggesting a potentially pivotal role for purine metabolism dysfunction in C9-ALS pathology.