CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase
CHIP E3 ligase mediates proteasomal degradation of the proliferation regulatory protein ALDH1L1 during the transition of NIH3T3 fibroblasts from G0/G1 to S-phase
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DOI：
10.1371/journal.pone.0199699
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发表时间：
2018-07-06
期刊：
影响因子：
3.7
通讯作者：
Krupenko, Sergey A.
中科院分区：
文献类型：
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作者：
Khan, Qasim A.;Pediaditakis, Peter;Krupenko, Sergey A.
ALDH 1 L1是一种叶酸代谢酶，在肝脏和其他几种组织中含量丰富。在人类癌症和源自恶性肿瘤的细胞系中，ALDH 1 L1基因通常通过启动子甲基化而沉默。这表明ALDH 1 L1限制了细胞的增殖能力，从而起到了假定的肿瘤抑制剂的作用。与癌细胞相反，小鼠细胞系NIH 3 T3和AML 12确实表达ALDH 1 L1蛋白。在本研究中，我们发现，ALDH 1 L1在这些细胞系中的水平在整个细胞周期中波动。在S期，ALDH 1 L1在蛋白水平上显著下调。随着细胞培养物变得汇合并且细胞经历增加的接触抑制，ALDH 1 L1在细胞中积累。与这一发现相一致的是，通过胸苷阻断而停滞在G1/S期的NIH 3 T3细胞完全失去了ALDH 1 L1蛋白。用蛋白酶体抑制剂MG-132处理防止增殖的NIH 3 T3细胞中的这种损失，表明ALDH 1 L1蛋白的蛋白酶体降解。共聚焦显微镜显示ALDH 1 L1与蛋白酶体的共定位支持这一机制。我们进一步表明，ALDH 1 L1与伴侣依赖性E3连接酶CHIP相互作用，CHIP在ALDH 1 L1泛素化和降解中起着关键作用。在NIH 3 T3细胞中，通过siRNA沉默CHIP停止，而CHIP的瞬时表达促进ALDH 1 L1损失。ALDH 1 L1的下调与ALDH 1 L1底物10-甲酰四氢叶酸的积累有关，这是S期激活的关键途径嘌呤从头生物合成所必需的。总之，我们的数据表明，CHIP介导的ALDH 1 L1蛋白酶体降解促进细胞增殖。
ALDH1L1 is a folate-metabolizing enzyme abundant in liver and several other tissues. In human cancers and cell lines derived from malignant tumors, the ALDH1L1 gene is commonly silenced through the promoter methylation. It was suggested that ALDH1L1 limits proliferation capacity of the cell and thus functions as putative tumor suppressor. In contrast to cancer cells, mouse cell lines NIH3T3 and AML12 do express the ALDH1L1 protein. In the present study, we show that the levels of ALDH1L1 in these cell lines fluctuate throughout the cell cycle. During S-phase, ALDH1L1 is markedly down regulated at the protein level. As the cell cultures become confluent and cells experience increased contact inhibition, ALDH1L1 accumulates in the cells. In agreement with this finding, NIH3T3 cells arrested in G1/S-phase by a thymidine block completely lose the ALDH1L1 protein. Treatment with the proteasome inhibitor MG-132 prevents such loss in proliferating NIH3T3 cells, suggesting the proteasomal degradation of the ALDH1L1 protein. The co-localization of ALDH1L1 with proteasomes, demonstrated by confocal microscopy, supports this mechanism. We further show that ALDH1L1 interacts with the chaperone-dependent E3 ligase CHIP, which plays a key role in the ALDH1L1 ubiquitination and degradation. In NIH3T3 cells, silencing of CHIP by siRNA halts, while transient expression of CHIP promotes, the ALDH1L1 loss. The downregulation of ALDH1L1 is associated with the accumulation of the ALDH1L1 substrate 10-formyltetrahydrofolate, which is required for de novo purine biosynthesis, a key pathway activated in S-phase. Overall, our data indicate that CHIP-mediated proteasomal degradation of ALDH1L1 facilitates cellular proliferation.