在急性T淋巴球Jurkat細胞中胜肽精胺酸脫亞氨酶誘發細胞凋亡機制的探討

The mechanisms of peptidylarginine deiminases-induced apoptosis in acute T leukemia Jurkat cells

林建佑(Chien-Yu Lin)

指導教授 ： 劉光耀

中山醫學大學/醫學院/免疫學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00054

摘要

胜肽精胺酸脫亞氨酶（Peptidylarginine deiminase; PADI）作用屬於後轉譯反應，可將精胺酸胜肽（peptidylarginine）轉換成瓜胺酸胜肽（citrulline），其在免疫細胞的分化及細胞凋亡扮演一個重要的角色。第四型胜肽精胺酸脫亞氨酶單核苷酸多型性單倍體與造成類風濕性關節炎嚴重程度具有相關性，其酵素活性增加會使得瓜胺酸蛋白量提高，進而增加自體抗體。在我們之前的研究證實了第四型胜肽精胺酸脫亞氨酶會造成免疫細胞的凋亡 (Liu, et al, 2006 Apoptosis)。在這裡，我們研究類風濕性關節炎的危險因子：第四型胜肽精胺酸脫亞氨酶單倍體（SNP PADI4; S55G,A82V and A112G）與其酵素活性的增加是否會誘導細胞凋亡的產生。在Tet-On 系統Jurkat T 細胞，當只加入Ionomycin（Ion）提高細胞內鈣離子並不會造成細胞凋亡，但加入四環素起始Tet-On 系統Jurkat T 細胞中的第四型胜肽精胺酸脫亞氨酶基因高度表現會造成細胞生存能力減少，並增加細胞凋亡。在體外及體內胜肽精胺酸脫亞氨酶酵素活性分析，我們證明第四型胜肽精胺酸脫亞氨酶單核苷酸多型性單倍體的酵素活性高於類風濕性關節炎非危險因子第四型胜肽精胺酸脫亞氨酶單倍體（第四型胜肽精胺酸脫亞氨酶野生型）。同時發現第四型胜肽精胺酸脫亞氨酶單核苷酸多型性單倍體誘發細胞凋亡活性高於第四型胜肽精胺酸脫亞氨酶野生型。另外，第四型胜肽精胺酸脫亞氨酶單核苷酸多型性單倍體給予Ion 後所誘發的細胞凋亡更高於給予Ion 的第四型胜肽精胺酸脫亞氨酶野生型，並且會造成Bcl-xL 蛋白減少與造成Bax 蛋白增加，也能夠造成細胞色素C 蛋白從粒線體釋放至細胞質。由西方墨點法的結果瞭解在誘發第四型胜肽精胺酸脫亞氨酶單核苷酸多型性單倍體表現與給予Ion 之後，引發的程式性細胞死亡過程會增加細胞凋亡體之凋亡蛋白酵素活性。綜合這些研究結果顯示給予Ion 的第四型胜肽精胺酸脫亞氨酶單核苷酸多型性單倍體的酵素活性增加，能夠增加經由粒線體的訊息路徑的細胞凋亡，並提供一個可信的解釋在於類風濕性關節炎致病機轉與第四型胜肽精胺酸脫亞氨酶單核苷酸多型性單倍體酵素活性增加之相關性。先前文獻顯示除了第四型胜肽精胺酸脫亞氨酶與類風濕性關節炎致病機轉的影響外，第二型胜肽精胺酸脫亞氨酶似乎也扮演一個重要的角色。因此，我們進一步研究是否第二型胜肽精胺酸脫亞氨酶也具有增加細胞凋亡。以Tet-On 系統誘發第二型胜肽精胺酸脫亞氨酶過度表現，導致細胞生存能力呈現隨時間及劑量增加而減緩，並且以細胞核皺縮與DNA 片段化分析觀察出細胞凋亡誘導活化的作用。

關鍵字

胜肽精胺酸脫亞氨酶單核苷酸多型性第四型胜肽精胺酸脫亞氨酶細胞凋亡

並列摘要

Peptidylarginine deiminase IV (PADI4) posttranslationally converts peptidylarginine to citrulline. It plays the essential role in immune cell differentiation and apoptosis. A haplotype of single-nucleotide polymorphisms (SNPs) in PADI4 is functionally relevant as a rheumatoid arthritis (RA) gene. It could increase enzyme activity leading to raised levels of citrullinated protein and stimulating autoantibody. Previous our study exposes that inducible PADI4 causes haematopoietic cell death (Liu, et al, 2006 Apoptosis). Herein, we further investigate whether RA risk PADI4 haplotype (SNP PADI4; S55G, A82V and A112G) and the increase of its enzymatic activity induce apoptosis. In the tetracycline (Tet)-On Jurkat T cells, ionomycin (Ion) only treatment didn’t induce apoptosis however it promoted inducible PADI4-decreased cell viability and –enhanced apoptosis. In vitro and in vivo PADI enzyme activity assay, we demonstrated PADI4 enzyme activity of SNP PADI4 was higher than RA non-risk PADI4 haplotype (WT PADI4). The effect of SNP PADI4-induced apoptosis was superior to WT PADI4. In addition, both Ion and SNP PADI4 synergistically provoked apoptosis compared with both Ion and WT PADI4. Concurrently, in the conditionally inducible SNP PADI4 cells of Ion treatment-induced apoptosis, not only the expression of Bcl-xL was down-regulated and Bax up-regulated, but also cytochrome c released from mitochondria to cytoplasm significantly. Western blotting data showed the increase of apoptosomal caspase activation during the programming cell death in the inducible SNP PADI4 cells subsequent to Ion treatment. These data demonstrated that both SNP PADI4 and increasing its enzyme activity could enhance apoptosis through mitochondrial pathway and further provide a conceivable explanation in the pathogenesis of RA following the upregulation of PADI4 activity in its SNPs. The early study revealed that PADI4 was shown to be associated with RA, but PADI2 appeared the feasible role. In addition, we examine whether inducible overexpression of PADI2 enhances apoptotic cell death. PADI2 reduced the viability in a does- and time-dependent manner of Jurkat-Tet-On system cells. The apoptosis-inducing activities were determined by nuclear condensation and DNA fragmentation.

並列關鍵字

PADIs ； SNP PADI4 ； apoptosis

參考文獻

Appella, E. and Anderson, C. W. (2000). Signaling to p53: breaking the posttranslational modification code. Pathol Biol (Paris) 48, 227-45.

Arita, K., Hashimoto, H., Shimizu, T., Nakashima, K., Yamada, M. and Sato, M. (2004). Structural basis for Ca(2+)-induced activation of human PAD4. Nat Struct Mol Biol 11, 777-83.

Asaga, H., Yamada, M. and Senshu, T. (1998). Selective deimination of vimentin in calcium ionophore-induced apoptosis of mouse peritoneal macrophages. Biochem Biophys Res Commun 243, 641-6.

Byun, Y., Chen, F., Chang, R., Trivedi, M., Green, K. J. and Cryns, V. L. (2001). Caspase cleavage of vimentin disrupts intermediate filaments and promotes apoptosis. Cell Death Differ 8, 443-50.

Cao, L., Goodin, R., Wood, D., Moscarello, M. A. and Whitaker, J. N. (1999). Rapid release and unusual stability of immunodominant peptide 45-89 from citrullinated myelin basic protein. Biochemistry 38, 6157-63.