SUMO化CLOCK对ERa介导的雌激素信号通路的调控

发布时间：2018-04-24 22:16

本文选题：雌激素受体 + 雌激素(E2)　；参考：《大连理工大学》2012年博士论文

【摘要】：周期节律的破坏与多种激素相关的肿瘤的发生有着密切的关系,如乳腺癌的发生,而雌激素受体ER信号通路的异常是乳腺癌发生的一个重要诱导因素,但周期节律的破坏与肿瘤发生发展间的分子机理还不清楚。核心时钟蛋白CLOCK (circadian locomoter activity kaput)是一种碱性螺旋-环-螺旋(basic helix-loop-helix/Per-Arnt-Sim homology domain, bHLH/PAS)蛋白,含有bHLH/PAS结构域。在周期节律中,CLOCK通常与BMAL1形成异源二聚体以调控下游基因的转录,这在周期节律的维持上发挥重要作用。但目前对于CLOCK是否直接参与到肿瘤相关通路中的研究还很少。蛋白的翻译后修饰包括磷酸化,乙酰化,SUMO化,泛素化等,这些翻译后修饰对于调控蛋白的功能发挥重要作用。目前对于CLOCK的翻译后修饰的研究还很少,仅发现其可被PKC (protein kinase C)和GSK-3(glycogen synthase kinase-3)磷酸化修饰。本研究的另一着眼点即CLOCK的SUMO化修饰研究,以及SUMO化修饰对其功能影响,尤其是对雌激素信号通路的影响。本研究以雌激素受体ERa阳性的MCF-7细胞为研究对象,主要工作如下： 1.在MCF-7细胞中发现时钟蛋白CLOCK与雌激素受体ERa间存在相互作用,并且通过雌激素(E2)刺激,可增强二者的相互作用,通过报告基因实验发现在雌激素存在的情况下,CLOCK对ERa的转录活性有明显的促进作用,说明CLOCK可能作为ERa的辅激活因子,参与了由ERa介导的雌激素信号通路。 2.通过结构及序列分析发现CLOCK有2个潜在的SUMO化修饰位点,即K67和K851,表明CLOCK可能是一个SUMO修饰底物。随后,CLOCK在MCF-7细胞中被证明可以被SUMO化,同时CLOCK的SUMO化在E2刺激的条件下有增强的趋势,通过定点突变,构建了3个突变体,即CLOCK K67R, K851R, K67R/K851R (2K/2R)。单位点突变(K67R或K851R)与野生型相比,使得CLOCK的SUMO化水平下降,而双位点突变体2K/2R使得CLOCK的SUMO化消失,并降低其自身转录活性。通过使用SENPs进行去SUMO化研究发现,SENP1作为CLOCK的去SUMO化酶介导了CLOCK的去SUMO化,并降低了CLOCK的转录活性。 3.通过核质分离及免疫荧光实验,发现SUMO化CLOCK主要分布在细胞核中,而去SUMO化减弱了CLOCK在细胞核中的分布,进一步研究发现CLOCK的SUMO化双位点突变体,即CLOCK2K/2R,减弱了CLOCK与BMAL1间的相互作用,从而降低了CLOCK的转录活性及在细胞核中的定位。 4. SUMO化修饰通常会改变蛋白的结构,从而影响了底物蛋白与其他蛋白间的相互作用。通过免疫共沉淀实验发现,去SUMO化的CLOCK减弱了CLOCK与ERα间的相互作用。接下来,通过报告基因实验发现SUMO化CLOCK可增强ERα转录活性,因为野生型CLOCK的过表达可明显激活ERα的转录活性,而SUMO化位点突变的CLOCK,即CLOCK2K/2R却丧失了这一功能。 5. Cyclin D1是ERα的经典下游靶基因,并且对细胞增殖有促进作用。通过实时定量PCR及报告基因实验研究发现,野生型CLOCK能够激活Cyclin D1的表达,而CLOCK2K/2R却不能。同时,通过MTT对细胞增殖情况进行研究发现,野生型CLOCK能促进MCF-7细胞的增殖,流式细胞实验也得出类似的结果,即野生型CLOCK的过表达降低了G0/G1期细胞数量,相应的提高了S期细胞数量,而CLOCK的双突变体的过表达对细胞周期的影响不大。通过本论文的研究将时钟蛋白CLOCK与ERa介导的雌激素信号通路联系起来,并确定了CLOCK的一种重要翻译后修饰,SUMO化。值得注意的是,SUMO化修饰对于CLOCK在雌激素信号通路的调控中发挥重要作用,为揭示周期节律破坏与乳腺癌发生发展间关系的分子机制提供了理论依据。
[Abstract]:The destruction of the periodic rhythm is closely related to the occurrence of a variety of hormone related tumors, such as the occurrence of breast cancer, and the abnormality of the estrogen receptor ER signaling pathway is an important inducer of the occurrence of breast cancer, but the molecular mechanism between the cycle rhythm and the development of the tumor is not clear. Core clock protein CLOCK (circadi An Locomoter activity kaput) is an alkaline helix loop helix (basic helix-loop-helix/Per-Arnt-Sim homology domain, bHLH/PAS) protein containing bHLH/PAS domain. In the periodic rhythm, CLOCK usually forms a heterogenous two polymer with BMAL1 to regulate the transcription of the downstream genes, which plays an important role in the maintenance of periodic rhythms. There is little research on whether CLOCK directly participates in tumor related pathways.
The post-translational modifications of the proteins include phosphorylation, acetylation, SUMO, and ubiquitination. These post-translational modifications play an important role in regulating the function of proteins. There are few studies on post-translational modification of CLOCK, and only it can be phosphorylated by PKC (protein kinase C) and GSK-3 (glycogen synthase kinase-3). One point is the study of the SUMO modification of CLOCK, and the effect of SUMO modification on its function, especially the effect on the estrogen signaling pathway. This study focuses on the MCF-7 cells positive for estrogen receptor ERa, and the main work is as follows:
1. the interaction between the clock protein CLOCK and the estrogen receptor ERa was found in the MCF-7 cells, and the interaction between the two was enhanced by estrogen (E2) stimulation. Through the reporter gene experiment, it was found that in the presence of estrogen, CLOCK has a significant promoting effect on the transcriptional activity of ERa, indicating that CLOCK may be the auxiliary activation of ERa. The factor is involved in the estrogen signaling pathway mediated by ERa.
2. through structural and sequence analysis, we found that CLOCK has 2 potential SUMO modification sites, that is K67 and K851, indicating that CLOCK may be a SUMO modified substrate. Then, CLOCK is proved to be SUMO in MCF-7 cells, while SUMO chemokine of CLOCK in the condition of E2 stimulation, 3 mutants are constructed by fixed-point mutation. CK K67R, K851R, K67R/K851R (2K/2R). Unit point mutation (K67R or K851R) decreased the SUMO level of CLOCK compared with the wild type, while the dual loci mutant 2K/2R made CLOCK SUMO disappear and reduced its own transcriptional activity. SUMO was removed and the transcriptional activity of CLOCK was reduced.
3. through the nuclear separation and immunofluorescence experiments, it was found that SUMO CLOCK was mainly distributed in the nucleus, and SUMO reduced the distribution of CLOCK in the nucleus. Further research found that the SUMO double site mutant of CLOCK, that is, CLOCK2K/2R, weakened the interaction between CLOCK and BMAL1, thus reducing the transcriptional activity of CLOCK and in the cell. The location in the nucleus.
4. SUMO modification usually changes the structure of the protein and affects the interaction between the substrate protein and other proteins. Through the immunoprecipitation experiment, it is found that the de SUMO CLOCK weakens the interaction between CLOCK and ER a. Then, the reporter gene experiment shows that SUMO CLOCK can enhance the ER alpha transcriptional activity because of the wild type CLOCK. Overexpression can significantly activate the transcriptional activity of ER alpha, whereas the SUMO mutant CLOCK, CLOCK2K/2R, has lost this function.
5. Cyclin D1 is the classic downstream target gene of ER alpha and promotes cell proliferation. Through real-time quantitative PCR and reporter gene experiments, it is found that wild type CLOCK can activate the expression of Cyclin D1, but CLOCK2K/2R can not. Meanwhile, the proliferation of cells is studied by MTT, and wild type CLOCK can promote MCF-7 cells. Proliferation, flow cytometry also concluded that the over expression of wild type CLOCK decreased the number of G0/G1 cells and increased the number of S cells, while the overexpression of the double mutant of CLOCK had little effect on the cell cycle.
Through this study, we linked the clock protein CLOCK with the estrogen signaling pathway mediated by ERa, and identified an important post translation modification and SUMO transformation of CLOCK. It is worth noting that the SUMO modification plays an important role in the regulation of CLOCK in the estrogen signaling pathway, in order to reveal the development of periodic rhythm damage and the development of breast cancer. The molecular mechanism of the inter relationship provides a theoretical basis.

【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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