轉穀醯胺酶催化之HeLa細胞中組蛋白多胺類修飾

Tissue Transglutaminase-induced Histones Polyamination in HeLa Cells

李昱蓉(Yu-Jung Lee)

指導教授 ： 張震東

國立臺灣大學/生命科學院/生化科學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.03155

摘要

組蛋白的轉譯後修飾，與細胞生長和基因表現等多種重要調控功能有關。在由HeLa細胞中純化的組蛋白上，可被精胺抗體偵測到可能是共價修飾的訊號；而多胺類對於細胞生長相關的生理功能亦具有調控地位。與以大腸桿菌表現的重組組蛋白，以及其他由細胞純化、具有與組蛋白類似性質的蛋白質進行比較，我們認為HeLa細胞的組蛋白上具有精胺的共價修飾，並且以TAU/SDS二維電泳以及組蛋白抗體確認在H3、H2B和H4上皆發現此訊號。而轉穀醯胺酶是真核細胞中催化多胺類共價修飾最主要的酵素，我們以實驗確認轉穀醯胺酶能表現於HeLa細胞核中，且組蛋白為轉穀醯胺酶的穀氨醯基受質(acyl donor substrate)，並進一步對對HeLa細胞純化組蛋白以及重組組蛋白進行in vitro轉穀醯胺酶催化精胺修飾的實驗，發現在重組的H2B上有發現精胺修飾的狀況，但在in vivo加入轉穀醯胺酶抑制劑cystamine時卻沒有看到修飾程度有明顯的變化。另外，以lovastatin使細胞週期同步化後每四小時分別測定組蛋白的精胺修飾程度，發現在G2/M期的細胞會有較多的精胺修飾。因此，組蛋白的精胺修飾可能存在與某些細胞週期相關的變化有關的生理意義。

關鍵字

組蛋白 ； 多胺類 ； 轉穀醯胺酶共價修飾 ； 細胞週期

並列摘要

The post-translational modifications of histones are relevant to many important cellular regulation mechanisms essential for cell proliferation and gene expression. In the histones extracted from HeLa cells, incorporation of spermine, presumably by covalent modification, was detected. Interestingly, these polyamines also play a key role in the regulation of cell proliferation.
From our results, we showed that spermine is covalently attached to extracted histones derived from HeLa cells. Comparing the western blotting results of extracted histones with that of recombinant human core histones and other proteins which have similar features to histones, we detected positive anti-spermine signals in histones H3, H2B and H4. This was further confirmed in our TAU/SDS 2D PAGE data.
Since tissue transglutaminase (TG2) is the main enzyme catalyzing the protein-polyamine incorporations, we confirmed that TG2 expressed in the nuclei of HeLa cells, and that histones were the in vitro acyl donor substrates of TG2. However, from the in vitro transamidation assay, we found that only H2B could be modified. Treatment of HeLa cells with cystamine, a TG2 inhibitor, did not alter the spermine-conjugation levels in core histones.
Furthermore, we synchronized the HeLa cells by lovastatin treatments and harvested the cells every four hours after release of lovastatin inhibition to determine the levels of spermine incorporation. We found that cells in the G2/M phase of the cell cycle had higher levels of spermine incorporation compared to those in the other stages. These results may imply a functional role of the modification of histones by spermine in cell cycle regulations.

並列關鍵字

histones ； polyamines ； tissue transglutaminase ； covalent modification ； cell cyle

參考文獻

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