组织蛋白酶D基因沉默诱导HeLa细胞老化的机制研究

发布时间：2018-03-23 22:02

本文选题：组织蛋白酶D　切入点：细胞老化　出处：《中国科学院北京基因组研究所》2016年博士论文

【摘要】：组织蛋白酶D(Cath D)是在细胞中广泛分布的一种溶酶体内的蛋白水解酶,该蛋白对于维持细胞内稳态起到了重要作用。一方面,Cath D可以通过水解其他蛋白,如蛋白酶原、纤维连接蛋白等,发挥其生物学功能。另一方面,被释放到胞浆中的Cath D会引发细胞凋亡相关蛋白的水解,从而引发凋亡。近些年来,该蛋白在肿瘤的发生发展中起到的作用引起了人们的日益关注。与癌旁／正常组织相比,该蛋白经常在肿瘤组织中过表达,而且Cath D的过表达可以显著增强癌症细胞的侵袭性。尽管关于Cath D与肿瘤发生的报道很多,但这样的报道绝大多数都是关联性的,其背后的分子机制尚不完全清楚。我们认为其分子机制的缺失主要是由两方面引起的,一是大多数Cath D在肿瘤细胞中的功能研究都利用了其过表达的模型。由于Cath D在肿瘤细胞中本底表达量已经较相对正常细胞有所提高,利用这样的研究模型观察到的细胞表型以及信号通路的改变可能并不大。二是直到目前为止,针对Cath D引起的全局性蛋白质改变的研究仍然很缺乏,大多数的研究仅仅局限于少数几条特异的蛋白或信号通路上。因此对Cath D基因沉默引发的全面蛋白质组变化进行表征会加深对Cath D在癌症细胞中功能的理解。本实验室前期在HeLa细胞中利用基因沉默的手段建立了Cath D稳定低表达的细胞株,HeLa-CR,并对该细胞株表型进行了初步的测定,发现细胞老化水平显著提高,并伴随着细胞周期在G2-M期的阻滞。为了对Cath D引起的蛋白质组变化进行多维度的观察,对HeLa-CR细胞的胞浆、溶酶体和细胞核三个亚细胞组分进行了基于氨基酸体内标记法(SILAC,Stable Isotope Labeling with Amino Acid)的定量蛋白质组学分析。发现Cath D的基因沉默造成二十余个注释为溶酶体定位的蛋白质在溶酶体组分中丰度下降,而相应的在胞浆中的丰度上升,提示HeLa-CR的溶酶体状态可能受到了影响。另一方面,氧化还原蛋白的丰度在HeLa-CR的胞浆中显著下调,提示细胞内氧化还原稳态受到了干扰。基于上述亚细胞组分的定量蛋白质组学结果以及细胞溶酶体状态以及氧化还原稳态与细胞老化之间的关联性,Cath D基因沉默有可能会升高溶酶体膜通透性以及ROS水平,进而诱导细胞老化。进一步的验证分析证实了这一点,并且发现ROS在Cath D基因沉默诱导细胞老化的过程中起到重要作用。另外在HeLa-CR细胞中,尽管ROS水平升高,但抗氧化转录因子NRF2活性是相对降低的,并且利用实验揭示这一现象的产生的原因：长期氧化压力可以抑制NRF2的转录活性以及抗氧化蛋白的丰度。结合前人关于Cath D在肿瘤组织中高表达的发现,我们的研究结果提示了Cath D在肿瘤发生发展过程中起到了维持组织内环境稳态,防止细胞老化的作用。
[Abstract]:Cathepsin D(Cath D, a proteolytic enzyme widely distributed in cells, plays an important role in maintaining intracellular homeostasis. On the one hand, cathepsin D can hydrolyze other proteins, such as proproteinogen. On the other hand, Cath D released into the cytoplasm can induce the hydrolysis of apoptosis-related proteins, thus inducing apoptosis. The role of the protein in tumorigenesis and development has attracted increasing attention. Compared with paracancerous / normal tissues, the protein is often overexpressed in tumor tissues. And overexpression of Cath D can significantly increase the invasiveness of cancer cells. Although there have been a lot of reports about Cath D and tumorigenesis, most of these reports are related. The molecular mechanism behind it is not completely clear. We believe that the absence of the molecular mechanism is mainly caused by two aspects. One is that most of the studies on the function of Cath D in tumor cells have made use of its overexpression model, because the background expression of Cath D in tumor cells has been increased compared with that of normal cells. The changes in cell phenotypes and signaling pathways observed using this model may not be significant. Second, until now, studies of global protein changes caused by Cath D have been scarce. Most studies are limited to a few specific protein or signaling pathways. Therefore, characterization of the total proteome changes caused by Cath D gene silencing will enhance the understanding of the function of Cath D in cancer cells. A stable and low expression Cath D cell line, HeLa-CR-R, was established by gene silencing in HeLa cells in the early stage of the experiment, and the phenotype of the cell line was preliminarily determined. In order to observe the proteome changes induced by Cath D, the cytoplasm of HeLa-CR cells was observed. Three subcellular components of lysosome and nucleus were analyzed by quantitative proteomic analysis based on amino acid labeling method: silac Isotope Labeling with Amino acid.The gene silencing of Cath D resulted in more than 20 proteins annotated as lysosomal localization. The abundance of lysosome components decreased, The corresponding increase in cytoplasmic abundance suggests that the lysosomal state of HeLa-CR may be affected. On the other hand, the redox protein abundance is significantly down-regulated in the cytoplasm of HeLa-CR. Based on the quantitative proteomics results of the above subcellular components and the relationship between redox homeostasis and cellular aging, the relationship between redox homeostasis and cell aging is also discussed, based on the quantitative proteomics results of the above subcellular components and the correlation between redox homeostasis and cell aging. It is possible to increase the permeability of lysosomal membrane and the level of ROS. Further validation analysis confirmed this and found that ROS plays an important role in the process of cell aging induced by Cath D gene silencing. In addition, in HeLa-CR cells, despite the increase of ROS level, However, the activity of antioxidant transcription factor NRF2 was relatively decreased. The reasons for this phenomenon were revealed by experiments: long term oxidative pressure could inhibit the transcription activity of NRF2 and the abundance of antioxidant protein, combined with previous findings on the high expression of Cath D in tumor tissues. Our results suggest that Cath D plays an important role in maintaining homeostasis and preventing cell aging during tumorigenesis and development.
【学位授予单位】：中国科学院北京基因组研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.2

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