Coordinated regulation of nuclear receptor CAR by CCRP/DNAJC7, HSP70 and the ubiquitin-proteasome system.
Coordinated regulation of nuclear receptor CAR by CCRP/DNAJC7, HSP70 and the ubiquitin-proteasome system.
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DOI：
10.1371/journal.pone.0096092
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Negishi M
中科院分区：
文献类型：
--
作者：
Timsit YE;Negishi M
组成型活性/雄烷受体（CAR）作为协调转录因子在调节化学物质如药物、葡萄糖、脂肪酸、胆红素和胆汁酸的各种肝代谢途径中起重要作用。目前，已知在其非活性状态下，CAR以与HSP 90和三肽重复蛋白质胞质CAR保留蛋白（CCRP）的蛋白质复合物的形式保留在细胞质中。在被苯巴比妥（PB）或PB样诱导剂1，4-双[2-（3，5-二氯吡啶氧基）]-苯（TCPOBOP）激活后，CAR易位到细胞核中。我们已经确定了CAR的细胞质调节的两个新组分：CCRP的泛素依赖性降解和与HSP 70的蛋白质-蛋白质相互作用。用蛋白酶体抑制剂MG 132（5 μ M）处理导致CAR在转染的HepG2细胞的细胞质中积累。在MG132存在下，TCPOBOP增加共表达CAR的HepG2细胞中的CCRP泛素化，而未检测到CAR泛素化。HepG2的MG 132处理还减弱了TCPOBOP诱导的CAR在含有源自人CYP2B6基因的CAR结合DNA元件的报告构建体上的转录激活。细胞质CAR蛋白与MG 132的升高与HSP 70的增加相关，并且在较小程度上与HSP 60的增加相关。免疫沉淀分析发现CCRP和CAR均与HepG2细胞中的内源性HSP 70相互作用。通过热休克诱导HSP 70水平也增加了细胞质CAR水平，类似于MG 132的作用。最后，热休克减弱TCPOBOP诱导的CAR转录激活，也类似于MG 132的作用。总的来说，这些数据表明CCRP和HSP 70的泛素-蛋白酶体调节是调节细胞质CAR水平的重要贡献者，因此CAR响应PB或PB样诱导剂的能力。
The constitutive active/androstane receptor (CAR) plays an important role as a coordinate transcription factor in the regulation of various hepatic metabolic pathways for chemicals such as drugs, glucose, fatty acids, bilirubin, and bile acids. Currently, it is known that in its inactive state, CAR is retained in the cytoplasm in a protein complex with HSP90 and the tetratricopeptide repeat protein cytosoplasmic CAR retention protein (CCRP). Upon activation by phenobarbital (PB) or the PB-like inducer 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene (TCPOBOP), CAR translocates into the nucleus. We have identified two new components to the cytoplasmic regulation of CAR: ubiquitin-dependent degradation of CCRP and protein-protein interaction with HSP70. Treatment with the proteasome inhibitor MG132 (5 µM) causes CAR to accumulate in the cytoplasm of transfected HepG2 cells. In the presence of MG132, TCPOBOP increases CCRP ubiquitination in HepG2 cells co-expressing CAR, while CAR ubiquitination was not detected. MG132 treatment of HepG2 also attenuated of TCPOBOP-induced CAR transcriptional activation on reporter constructs which contain CAR-binding DNA elements derived from the human CYP2B6 gene. The elevation of cytoplasmic CAR protein with MG132 correlated with an increase of HSP70, and to a lesser extent HSP60. Both CCRP and CAR were found to interact with endogenous HSP70 in HepG2 cells by immunoprecipitation analysis. Induction of HSP70 levels by heat shock also increased cytoplasmic CAR levels, similar to the effect of MG132. Lastly, heat shock attenuated TCPOBOP-induced CAR transcriptional activation, also similar to the effect of MG132. Collectively, these data suggest that ubiquitin-proteasomal regulation of CCRP and HSP70 are important contributors to the regulation of cytoplasmic CAR levels, and hence the ability of CAR to respond to PB or PB-like inducers.
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