Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells.
Induction of cytoplasmic rods and rings structures by inhibition of the CTP and GTP synthetic pathway in mammalian cells.
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DOI：
10.1371/journal.pone.0029690
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Chan EK
中科院分区：
文献类型：
--
作者：
Carcamo WC;Satoh M;Kasahara H;Terada N;Hamazaki T;Chan JY;Yao B;Tamayo S;Covini G;von Mühlen CA;Chan EK
以人自身抗体为探针，对胞质丝状棒环（RR）结构进行鉴定。在本研究中，研究了哺乳动物细胞中这些保守结构的形成以及与这些结构相关的功能。利用人自身抗体，免疫荧光法首次观察到HEp-2细胞中不同的细胞质棒（长度约3-10µm）和环（直径约2-5µm）。共定位研究显示，尽管RR具有丝状特征，但它们不富含肌动蛋白、微管蛋白或波形蛋白，也不与中心体或其他已知的细胞质结构相关。进一步的独立研究发现，核苷酸合成途径中的两个关键酶胞苷三磷酸合成酶1 （CTPS1）和肌苷一磷酸脱氢酶2 （IMPDH2）在RR中高度富集。CTPS1酶抑制剂6-重氮-5-氧- l -去甲亮氨酸和Acivicin以及IMPDH2抑制剂利巴韦林在所有癌细胞系和小鼠原代细胞中95%的细胞中表现出剂量依赖性的RR诱导。低浓度利巴韦林在impdh2敲除的HeLa细胞中增强了RR的形成，而在GFP-IMPDH2过表达的HeLa细胞中抑制了RR的形成。有趣的是，在未处理的小鼠胚胎干细胞中很容易检测到RR (>95%)；视黄酸分化后，RR在这些细胞中分解，但在用Acivicin处理时重组。在癌细胞系和小鼠原代细胞中，RR的形成代表了对CTP或GTP合成通路干扰的响应，而RR是这些通路中的收敛性物理结构。这些保守结构的特异性标记物的可用性和体外诱导形成的能力将允许进一步研究健康和疾病中许多生物系统中RR的结构和功能。
Cytoplasmic filamentous rods and rings (RR) structures were identified using human autoantibodies as probes. In the present study, the formation of these conserved structures in mammalian cells and functions linked to these structures were examined. Distinct cytoplasmic rods (∼3–10 µm in length) and rings (∼2–5 µm in diameter) in HEp-2 cells were initially observed in immunofluorescence using human autoantibodies. Co-localization studies revealed that, although RR had filament-like features, they were not enriched in actin, tubulin, or vimentin, and not associated with centrosomes or other known cytoplasmic structures. Further independent studies revealed that two key enzymes in the nucleotide synthetic pathway cytidine triphosphate synthase 1 (CTPS1) and inosine monophosphate dehydrogenase 2 (IMPDH2) were highly enriched in RR. CTPS1 enzyme inhibitors 6-diazo-5-oxo-L-norleucine and Acivicin as well as the IMPDH2 inhibitor Ribavirin exhibited dose-dependent induction of RR in >95% of cells in all cancer cell lines tested as well as mouse primary cells. RR formation by lower concentration of Ribavirin was enhanced in IMPDH2-knockdown HeLa cells whereas it was inhibited in GFP-IMPDH2 overexpressed HeLa cells. Interestingly, RR were detected readily in untreated mouse embryonic stem cells (>95%); upon retinoic acid differentiation, RR disassembled in these cells but reformed when treated with Acivicin. RR formation represented response to disturbances in the CTP or GTP synthetic pathways in cancer cell lines and mouse primary cells and RR are the convergence physical structures in these pathways. The availability of specific markers for these conserved structures and the ability to induce formation in vitro will allow further investigations in structure and function of RR in many biological systems in health and diseases.
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