Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency
Mutations of ATIC and ADSL affect purinosome assembly in cultured skin fibroblasts from patients with AICA-ribosiduria and ADSL deficiency
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DOI：
10.1093/hmg/ddr591
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发表时间：
2012-04-01
影响因子：
3.5
通讯作者：
Kmoch, Stanislav
中科院分区：
文献类型：
--
作者：
Baresova, Veronika;Skopova, Vaclava;Kmoch, Stanislav
嘌呤体是由在嘌呤从头合成（DNPS）中具有活性的酶组成的多酶复合物，细胞在嘌呤耗尽或需求增加时在其胞质溶胶中瞬时组装。嘌呤体形成的过程迄今为止仅在瞬时表达重组荧光标记的DNPS蛋白的人宫颈上皮癌细胞（HeLa）和人肝癌细胞（C3 A）中得到证实和研究。采用免疫荧光共聚焦显微镜技术，对HeLa细胞、人肝癌细胞系（HepG 2）、肉瘤成骨细胞（Saos-2）、人胚肾细胞（HEK 293）、人皮肤成纤维细胞（SF）和原代角质形成细胞（KC）进行嘌呤体组装的研究。使用相同的方法，我们证明了培养的皮肤成纤维细胞从患者AICA-核糖尿症和ADSL缺陷，ATIC和ADSL的各种突变不稳定的嘌呤体组装的不同程度，并发现，形成嘌呤体的能力与个别ADSL患者的临床表型。因此，我们的研究结果表明，功能嘌呤体的组装是完全依赖于结构上不受影响的ATIC和ADSL复合物的存在下，大概也对所有其他DNPS蛋白的存在下。这些结果也证实了ADSL缺陷的表型严重程度主要取决于相应的突变ADSL蛋白复合物的结构稳定性和残余催化能力的假设，因为这是嘌呤体的形成和稳定性以及至少部分琥珀酰氨基咪唑甲酰胺核苷ADSL酶底物通过DNPS途径的先决条件。
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 ribosideADSL enzyme substratesthrough the DNPS pathway.