分析 SUMO 化酶 E1、E2 和 E3 在眼细胞系中的差异表达模式。

The State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, #7 Jinsui Road, Guangzhou, Guangdong 510230, China.

Key Laboratory of Protein Chemistry and Developmental Biology, College of Life Sciences, Hunan Normal University, Changsha, Hunan 410081, China.

Curr Mol Med. 2018;18(8):509-515. doi: 10.2174/1566524019666190112143636.

PURPOSE

Protein sumoylation is a well established regulatory mechanism to control many cellular processes such as chromatin structure dynamics, transcriptional regulation of gene expression, cell proliferation and differentiation, cell transformation and carcinogenesis, autophagy and senescence. In the vertebrate vision system, we and others have revealed that sumoylation plays important roles in regulating differentiation of several ocular tissues during eye development. To further elucidate the functional mechanisms of sumoylation, in vitro assay systems are needed. Currently, the five major cell lines including αTN4-1, FHL124, HLE, N/N1003A and ARPE-19 have been extensively used to test the biochemical and molecular aspects of normal vision physiology and various disease processes. Thus, we conducted the study on the expression patterns of the three types of sumoylation enzymes, the activating enzymes SAE1 and UBA2, the conjugating enzyme UBC9, and the ligating enzymes such as RanBP2 and PIAS1 in these ocular cell lines.

METHODS

The 5 major ocular cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS) or rabbit serum (RBS) and 1% Penicillin- Streptomycin. The mRNA levels were analysed with qRT-PCR. The protein levels were determined with western blot analysis and quantitated with Image J.

RESULTS

we have obtained the following results: 1) For the mRNAs encoding E1 SAE1 and UBA2, E2 UBC9 and E3 PIAS1, the highest level of expression was observed in αTN4-1 cells; For the mRNA encoding RanBP2, the highest level of expression was detected in N/N1003A cells; 2) In contrast to the mRNA expression patterns, a similar level of the SAE1 protein was observed in the all five cell lines, and so is true with UBA2 protein in all cells except for N/N1003A where over fourfold of enrichment in UBA2 protein was observed compared with other cell lines; 3) A similar level of UBC9 protein was also detected in all cells except for N/N1003A where more than one-fold of decrease in UBC9 level was found compared with other cell lines; 4) For E3 ligases, we did not identify the regular PIAS1 band in N/N1003A cells, the remaining cells have a level of PIAS1 with difference of less than 0.6-fold; all cells except for FHL124 cells have a similar level of RanBP2, and a 70% drop in RanBP2 was observed in FHL124 cell.

CONCLUSIONS

Our determination of the differential expression patterns of the three types of sumoylation enzymes in the 5 ocular cell lines help to understand sumoylation functions in vertebrate eye.

目的

蛋白质 SUMO 化是一种已被广泛证实的调控机制，可控制许多细胞过程，如染色质结构动态、基因表达的转录调控、细胞增殖和分化、细胞转化和癌变、自噬和衰老。在脊椎动物视觉系统中，我们和其他人已经揭示 SUMO 化在眼睛发育过程中调节几种眼部组织的分化中起着重要作用。为了进一步阐明 SUMO 化的功能机制，需要体外测定系统。目前，αTN4-1、FHL124、HLE、N/N1003A 和 ARPE-19 这 5 大细胞系已被广泛用于测试正常视觉生理和各种疾病过程的生化和分子方面。因此，我们研究了这 5 种眼细胞系中三种 SUMO 化酶（激活酶 SAE1 和 UBA2、连接酶 UBC9 以及连接酶如 RanBP2 和 PIAS1）的表达模式。

方法

用含胎牛血清（FBS）或兔血清（RBS）和 1%青霉素-链霉素的 DMEM 培养基培养 5 种主要眼细胞系。用 qRT-PCR 分析 mRNA 水平。用 Western blot 分析测定蛋白质水平，并用量化软件 Image J 定量。

结果

我们得到了以下结果：1）在 αTN4-1 细胞中，E1 SAE1 和 UBA2、E2 UBC9 和 E3 PIAS1 的编码 mRNA 表达水平最高；在 N/N1003A 细胞中，编码 RanBP2 的 mRNA 表达水平最高；2）与 mRNA 表达模式相反，在所有 5 种细胞系中，SAE1 蛋白的表达水平相似，除 N/N1003A 细胞外，UBA2 蛋白的表达水平也相似，与其他细胞系相比，UBA2 蛋白的丰度增加了 4 倍以上；3）在所有细胞系中，UBC9 蛋白的表达水平也相似，除 N/N1003A 细胞外，UBC9 水平降低了 1 倍以上；4）对于 E3 连接酶，我们在 N/N1003A 细胞中没有鉴定到常规的 PIAS1 带，而在其他细胞系中，UBA2 蛋白的丰度增加了 4 倍以上；除了 FHL124 细胞，所有细胞系中都有相似水平的 PIAS1，而在 FHL124 细胞中，PIAS1 的水平下降了 70%；在所有细胞系中，除了 FHL124 细胞，RanBP2 的水平相似，而在 FHL124 细胞中，RanBP2 的水平下降了 70%。