臺灣博碩士論文加值系統

中文摘要
第二型胜肽精胺酸脫亞胺酶 (Peptidylarginine Deiminase type 2, PADI2) 主要功能為調控蛋白質轉譯後修飾，將蛋白質胜肽鏈上的精胺酸 (arginine) 轉變為瓜胺酸 (citrulline)，第二型胜肽精胺酸脫亞胺酶會促使淋巴细胞中的蛋白質瓜胺酸化 (citrullinations)，研究指出瓜胺酸化的蛋白質可能於發炎反應中扮演重要角色。本實驗室研究中曾發現到過度表現第二型胜肽精胺酸脫亞胺酶時，會促使活化的T細胞進行細胞凋亡 (apoptosis)，此機制可能經由活化後誘發細胞死亡。本研究主要在探究第二型胜肽精胺酸脫亞胺酶參與活化後T細胞自發性死亡 (activated T cell autonomous death, ACAD)詳細機制。研究發現在Jurkat T細胞中過度表現第二型胜肽精胺酸脫亞胺酶時促使自噬作用 (autophagy) ，第二型胜肽精胺酸脫亞胺酶表現時造成細胞中的p62降解增多，同時LC3-II表現量上升，因為過度表達第二型胜肽精胺酸脫亞胺酶時會導致過多的蛋白質被瓜胺酸化，誘發內質網壓力 (ER stress) 與細胞自噬。自噬作用和細胞凋亡參與細胞對抗壓力、細胞活化及存活動態平衡。第二型胜肽精胺酸脫亞胺酶誘發第十七輔助型T (Th17) 細胞活化，刺激T細胞產生細胞激素 (cytokines)，如IL-17、IL-21、IL-22和TNF mRNA表現量增高，細胞激素誘發凋亡蛋白酶的表現與凋亡蛋白酶 (caspase)活化並切割Beclin-1 形成cBeclin-1，cBeclin-1為誘發細胞凋亡的重要因子。當抑制Beclin-1的表現時，BCL-xL增加及缺乏凋亡蛋白酶，使得細胞的存活率上升。綜上所述，研究證明第二型胜肽精胺酸脫亞胺酶促使T淋巴细胞中的蛋白質瓜胺酸化、形成內質網壓力、誘發細胞自噬、細胞激素的產生與活化後T細胞自發性死亡。

Abstract
Peptidylarginine deiminase type 2 (PADI2) is a post-translational modification enzyme that catalyzes arginine residues into the citrulline residues. Previous studies have shown that PADI2 promotes protein citrullinations in lymphocytes and it might play an important role in inflammation. We found that overexpression of PADI2 promotes apoptosis in activated T cells previously. Whether PADI2 participate in the pathway of activated T cell autonomous death (ACAD) is still curious. In the delicate PADI2-mediated ACAD, we found that overexpression of PADI2 displayed higher levels of citrullinated protein which induced the ER stress significantly. The high levels of citrullinated protein results in unfolding protein response (UPR) of ER stress and increases the loading of protein degradation. Autophagy could cause degradation of the citrullinated and unfolding protein. Herein, PADI2 could enhance autophagy in Jurkat T cells and lead to a degradation of p62 and the accumulation of LC3-II. Autophagy and apoptosis are two critical mechanisms which participate against cellular stress, cell activation, survival and homeostasis. PADI2-overexpressed Jurkat T cells caused the activation of Th17 cells to increase mRNA expression of cytokines, such as IL-17, IL-21, IL-22 and TNF. Cytokines provoked caspase expression and led to caspase-mediated cleavage of Beclin-1 which was an important factor of apoptotic signaling. Knockdown of BCEN1 rescued cell survival due to the increase of Bcl-xL and the decrease of caspase-3. We suggested that PADI2 participated in the activated T cell-induced autonomous death through triggering ER stress pathway, stimulating the expression of cytokines and promoting autophagy by PADI2-citrullinated protein.

Contents iv
Abbreviation vii
中文摘要 ix
Abstract x
1. 前言 (Introduction) 1
1.1. PADI2 1
1.2. Autophagy 2
1.3. ER stress 4
1.4. Apoptosis 5
1.5. Cytokines 6
1.6. Research purpose 7
2. 材料與方法 (Materials and methods) 9
2.1. Cell culture and treatment 9
2.2. Cell transfection 9
2.3. Luciferase reporter assay 10
2.4. Detection of intracellular ROS 10
2.5. Cell vaibilty and Detection of acid vesicular organelles with acridine orange (AO) staining assay 11
2.6. DNA fragmentation analysis 11
2.7. Western Blot analysis 12
2.8. Analysis of mRNA expression by Reverse transcription PCR analysis 12
2.9. Statistical analysis 13
3. 結果 (Rusults) 14
3.1. TPA promotes PADI2 expression, autophagy, activation and apoptosis in Jurkat T lymphocytes 14
3.2. Establishment of the stable clones of PADI2 and induces cell apoptosis in Jurkat cells 14
3.3. Induction of PADI2 increased autophagy-related protein in Jurkat cell 16
3.4. PADI2 induction of ER stress-related protein by UPR inducers and suppresses phospho-mTOR, phospho-AKT in Jurkat cell 17
3.5. The shRNA-mediated inhibition of Atg5 and Atg12 promotes the cell apoptosis, and ectopic Atg12 impedes the induction of apoptosis by PADI2 overexpression 18
3.6. PADI2 overexpression induced cell activation and produced Th 17 cytokines in Jurkat cell 20
3.7. Inhibition of IL-6 prolong autophagy in Jurkat Tet-On-PADI2 cells 21
3.8. Knockdown of Beclin-1 inhibited cell death by ACAD in Jurkat Tet-On-PADI2 cells 22
3.9. Knockdown of PADI2 decrease cytokines product, inhibing autophagy and ACAD in Jurkat Tet-On-PADI2 cells 23
4. 討論 (Discussion) 24
5. 引用文獻 (References) 28
6. 圖表 (Figure) 35
Figure 1. TPA promotes PADI2 expression, autophagy, activation and apoptosis in Jurkat T lymphocytes 35
Figure 2. PADI2 overexpression increase Jurkat cells apoptosis 38
Figure 3. Induction of PADI2 results in aggregated acidic vesicular organelles and autophagy-related protein in Jurkat cell 42
Figure 4. PADI2 induction of ER stress-related proteins and suppresses phospho-mTOR, phospho-AKT by UPR inducers in Jurkat cell 47
Figure 5. Knockdown of Atg5 and Atg12 induced cell death in Jurkat Tet-On PADI2 cells 50
Figure 6. Overexpression of Atg12 delays apoptosis induced by PADI2. 57
Figure 7. PADI2 overexpression induced inflammatory cytokines in Jurkat cell 60
Figure 8. Knockdown IL-6 delayed apoptosis and prolong autophagy in Jurkat PADI2 cells 62
Figure 9. Knockdown of Beclin-1 increased survival in Jurkat-Tet-On-PADI2 cells 64
Figure 10. PADI2 deficiency promoted surveil and regulated ER stress and autophagy. 67

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