The purinosome is a multienzyme complex composed by the enzymes active in de novo purine synthesis (DNPS) that cells transiently assemble in their cytosol upon depletion or increased demand of purines. The process of purinosome formation has thus far been demonstrated and studied only in human epithelial cervical cancer cells (HeLa) and human liver carcinoma cells (C3A) transiently expressing recombinant fluorescently labeled DNPS proteins. Using parallel immunolabeling of various DNPS enzymes and confocal fluorescent microscopy, we proved purinosome assembly in HeLa, human hepatocellular liver carcinoma cell line (HepG2), sarcoma osteogenic cells (Saos-2), human embryonic kidney cells (HEK293), human skin fibroblasts (SF) and primary human keratinocytes (KC) cultured in purine-depleted media. Using the identical approach, we proved in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency that various mutations of ATIC and ADSL destabilize to various degrees of purinosome assembly and found that the ability to form purinosomes correlates with clinical phenotypes of individual ADSL patients. Our results thus shown that the assembly of functional purinosomes is fully dependent on the presence of structurally unaffected ATIC and ADSL complexes and presumably also on the presence of all the other DNPS proteins. The results also corroborate the hypothesis that the phenotypic severity of ADSL deficiency is mainly determined by structural stability and residual catalytic capacity of the corresponding mutant ADSL protein complexes, as this is prerequisite for the formation and stability of the purinosome and at least partial channeling of succinylaminoimidazolecarboxamide riboside-ADSL enzyme substrates-through the DNPS pathway.

Institute of Inherited Metabolic Disorders Charles University Prague and General University Hospital Prague 128 08 Prague 2 Czech Republic

References provided by Crossref.org

Phosphoribosylformylglycinamidine Synthase (PFAS) Deficiency: Clinical, Genetic and Metabolic Characterisation of a Novel Defect in Purine de Novo Synthesis

Expanding clinical spectrum of PAICS deficiency: Comprehensive analysis of two sibling cases

Metabolites of De Novo Purine Synthesis: Metabolic Regulators and Cytotoxic Compounds

Combined Targeted and Untargeted Profiling of HeLa Cells Deficient in Purine De Novo Synthesis

Mass spectrometric analysis of purine de novo biosynthesis intermediates

Study of purinosome assembly in cell-based model systems with de novo purine synthesis and salvage pathway deficiencies

Genetic and metabolomic analysis of AdeD and AdeI mutants of de novo purine biosynthesis: cellular models of de novo purine biosynthesis deficiency disorders