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.

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

“…The enzymatic steps of de novo purine biosynthesis convert PRPP (5-phospho-α-D-ribosyl-1-pyrophosphate) to IMP (inosine monophosphate), and an additional four steps convert IMP to AMP (adenosine monophosphate) or GMP (guanosine monophosphate) (Figure 1). Recent evidence demonstrates that the proteins carrying out de novo purine biosynthesis form a multienzyme complex, the purinosome, which is induced by acute requirement for purines [1-3]. Formation of the purinosome can be disrupted by mutations in ADSL (E.C.…”

“…4.3.2.2) and ATIC (E.C. 2.1.2.3/E.C.3.5.4.10), with severe developmental consequences in humans [3]. In addition to ADSL and ATIC, 30 enzyme defects of purine and pyrimidine metabolism have been identified and 17 of these are known to cause human disease [4].…”

“…These can be cells isolated from individuals with inborn errors of purine metabolism as reported previously [3], [7], or experimentally derived cells which carry mutations in de novo purine biosynthesis genes. We have previously reported the isolation and initial characterization of Chinese hamster ovary cell (CHO-K1) mutants presumably defective in each of the seven proteins required for de novo AMP synthesis [8], [9].…”

Mapping Protein-Protein Proximity in the Purinosome

“…Purinosome assembly/disassembly formation was also shown to be dynamically regulated by cell signaling through a G␣ i -coupled receptor (6) and post-translational modification (7). The presence/absence of purinosomes has recently been associated with diseases proposed to be caused by defects in de novo purine synthesis (8). The purinosome enzyme complex is a delicate and intricate cellular system.…”

Role of HSP90 in the Regulation of de Novo Purine Biosynthesis

“…Purine depletion results in the activation of the de novo purine biosynthetic pathway and has been shown to lead to enhanced purinosome formation in a wide variety of mammalian cells. 4,14–16 If HSP90 is involved in the stabilization of the purinosome, then other enzymes that are comprised of the complex will become destabilized and likely degraded upon HSP90 inhibition. On the other hand, if no degradation is observed among the other enzymes in the purinosome, then the most probable explanation is that HSP90 is transforming these enzymes into a state that promotes complexation.…”