title: Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency.
abstract: 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.
authors.affiliation: Institute of Inherited Metabolic Disorders, Charles University in Prague and General University Hospital in Prague, 128 08 Prague 2, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Baresova
authors.forename: Vaclava
authors.initials: V
authors.lastname: Skopova
authors.forename: Jakub
authors.initials: J
authors.lastname: Sikora
authors.forename: David
authors.initials: D
authors.lastname: Patterson
authors.forename: Jana
authors.initials: J
authors.lastname: Sovova
authors.forename: Marie
authors.initials: M
authors.lastname: Zikanova
authors.forename: Stanislav
authors.initials: S
authors.lastname: Kmoch
mesh_terms: D000264:Adenylosuccinate Lyase / Q000172:deficiency / Q000235:genetics*; D001321:Autistic Disorder; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D005347:Fibroblasts / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D019877:Hydroxymethyl and Formyl Transferases / Q000032:analysis / Q000235:genetics*; D015603:Keratinocytes / Q000201:enzymology; D009097:Multienzyme Complexes / Q000032:analysis / Q000235:genetics*; D009154:Mutation; D009709:Nucleotide Deaminases / Q000032:analysis / Q000235:genetics*; D011686:Purine-Pyrimidine Metabolism, Inborn Errors / Q000201:enzymology* / Q000235:genetics*; D011687:Purines / Q000378:metabolism; D012867:Skin / Q000166:cytology
chemical_list: D009097:Multienzyme Complexes; D011687:Purines; C113120:inosine monophosphate synthase; D019877:Hydroxymethyl and Formyl Transferases; D009709:Nucleotide Deaminases; D000264:Adenylosuccinate Lyase