CUL4B细胞周期候选靶蛋白的分析

发布时间：2018-05-04 12:20

本文选题：CUL4B + 细胞周期调控　；参考：《山东大学》2010年硕士论文

【摘要】： 细胞周期是细胞生命活动的基本过程。在进化过程中,细胞建立并发展了一系列的调控机制,以确保细胞周期严格有序地交替和各个时相依次有序变更。多种细胞周期蛋白在调控细胞周期各个时相转换中发挥作用,其主要包括：细胞周期蛋白依赖激酶(cyclin-dependent kinases, CDKs)家族蛋白、细胞周期蛋白(cyclins)和细胞周期蛋白依赖性激酶抑制剂(CKIs)。细胞通过对三类细胞周期蛋白的合成、降解以及翻译后修饰等环节进行调控,从而保证细胞周期正常的进行。泛素依赖的蛋白质降解是细胞调控蛋白质活性的一个基本机制,在细胞周期调控中起重要的作用,几乎所有细胞周期蛋白的降解都依赖于该过程。CUL4B属于cullin蛋白家族,该家族成员是泛素依赖降解系统中E3连接酶的成员,在本实验室发现的CUL4B突变导致精神发育迟滞的家系中,女性携带者出现X染色体失活偏倚,患者骨骼发育异常以及CUL4B低表达的细胞出现S期停滞提示CUL4B可能在细胞周期调控中发挥重要的作用。然而,目前对于CUL4B在细胞周期调控方面的研究还比较少,仅发现少数几种与细胞周期调控相关的靶蛋白(如Cyclin E、CDT1和p21),因此寻找CUL4B在细胞周期调控过程中的蛋白对研究CUL4B在细胞周期调控中的生物学功能至关重要。首先,我们提取稳筛的CUL4B低表达非同步化的HeLa和HEK293细胞及其对照细胞的总蛋白,利用western blotting分析方法对p21、p27、p53、CDT1、CDC6、cyclin D1、CHK1、E2F1、CDC25A、CDC25C和CDK2等细胞周期调控相关蛋白的表达进行检测,结果发现这些蛋白的表达在实验组和对照组没有显著的差异。随后我们通过向培养基中加入细胞周期阻断药物的方法将CUL4B低表达的HeLa和HEK293细胞及其对照细胞分别同步化至G1期、S期、G2/M期,并使用流式细胞术对同步化效果进行监控。确定合适的处理方法后,我们提取同步化至不同时期的CUL4B低表达HeLa和HEK293细胞及其对照细胞的总蛋白,同样使用western blotting分析检测上述蛋白的表达情况,结果发现与对照细胞相比,CUL4B低表达的HeLa细胞内p53、p27和CHK1蛋白在S期有明显的积累。然而在CUL4B低表达的HEK293细胞中p53在G1/S期积累,CHK1在S期积累,p27没有明显的变化。为了明确导致CUL4B低表达HeLa细胞内p27、p53和CHK1蛋白积累的分子机制,我们又检测了上述基因在S期时的mRNA的表达水平,结果发现CUL4B低表达的细胞中上述基因的mRNA水平与对照细胞相比并没有明显的差异,提示这些蛋白在S期内CUL4B低表达HeLa细胞中的表达上调是在转录后水平而非转录水平。对这几种蛋白半衰期的测定发现在CUL4B低表达的HeLa细胞中p27和CHK1的半衰期明显延长,表明CUL4B的表达下调导致这两种蛋白的降解发生障碍。随后我们发现外源性CUL4B的表达能够减轻因内源性CUL4B表达下调而造成的p27、p53和CHK1的积累。以上实验结果提示,CUL4B在p27、p53和CHK1的降解中发挥重要的调控作用。综上所述,我们建立了针对本实验室条件下所用细胞(HeLa和HEK293)的细胞周期同步化方法,并在此基础上寻找到CUL4B在细胞周期调控中的候选靶蛋白。这些实验结果为进一步研究CUL4B在细胞周期调控中的功能奠定了基础。
[Abstract]:Cell cycle is the basic process of cell life activity. In the process of evolution, cells have established and developed a series of regulatory mechanisms to ensure that the cell cycle is alternately alternately and orderly in each phase. A variety of cyclin plays a role in the regulation of cell cycle phase transformation, mainly including cell cycle. Phase protein dependent kinase (cyclin-dependent kinases, CDKs) family protein, cyclin protein (cyclins) and cyclin dependent kinase inhibitor (CKIs). Cells are regulated by the synthesis, degradation and post-translational modification of three kinds of cyclin proteins to ensure the normal cell cycle.
Ubiquitin dependent protein degradation is a basic mechanism for cell regulation of protein activity, which plays an important role in the regulation of cell cycle. The degradation of almost all cell cycle proteins depends on the process.CUL4B belongs to the Cullin protein family. The family members are members of the E3 ligase in the ubiquitin dependent degradation system and are in our laboratory. X chromosome inactivation bias, skeletal dysplasia and S stagnation of CUL4B low expression cells in the patients with mental retardation in the family of mental retardation were found to play an important role in the regulation of cell cycle. However, the study on the regulation of cell cycle in CUL4B is still more than that of CUL4B in the regulation of cell cycle. Few, only a few target proteins related to cell cycle regulation (such as Cyclin E, CDT1 and p21) are found, so it is essential to find the protein in the process of cell cycle regulation of CUL4B to study the biological function of CUL4B in the regulation of cell cycle.
First, we extracted the low expression of CUL4B and the total protein of the non synchronous HeLa and HEK293 cells and their control cells. The expression of the cell cycle regulation related proteins, such as p21, p27, p53, CDT1, CDC6, cyclin D1, p27, CDC6, cyclin D1, etc., was detected by western blotting analysis. There was no significant difference between the experimental group and the control group.
Then we synchronize CUL4B low expression HeLa and HEK293 cells and their control cells to G1, S, G2/M phase, respectively by adding cell cycle blocking drugs into the medium, and monitor the synchronization effect by flow cytometry. After determining the appropriate treatment, we extract the synchronization to a different period of CUL4B low. The expression of the HeLa and HEK293 cells and their control cells was also expressed by Western blotting analysis. The results showed that the p53, p27 and CHK1 protein in the HeLa cells with low CUL4B expression were accumulated in S phase compared with the control cells. In S accumulation, there was no obvious change in p27. In order to clarify the molecular mechanism of the accumulation of p27, p53 and CHK1 protein in CUL4B low expression HeLa cells, we also detected the expression level of mRNA at S phase, and found that the mRNA level of the above genes in the CUL4B low expressed cells was not significantly different from that of the control cells. The up-regulated expression of these proteins in CUL4B low expression HeLa cells during S period was at post transcriptional level but not transcriptional level. The determination of the half-life of these proteins showed that the half-life of p27 and CHK1 in HeLa cells with low expression of CUL4B significantly prolonged, indicating that the downregulation of CUL4B expression led to the degradation of these two proteins. We found that the expression of exogenous CUL4B could reduce the accumulation of p27, p53 and CHK1 resulting from the downregulation of endogenous CUL4B expression. These results suggest that CUL4B plays an important role in the degradation of p27, p53 and CHK1.
In summary, we set up a cell cycle synchronization method for the cells (HeLa and HEK293) used in our laboratory, and on this basis, we find the candidate target proteins in the cell cycle regulation of CUL4B. These results provide a basis for further study of the function of CUL4B in cell cycle control.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【共引文献】