內質網相關降解系統和自體吞噬系統共同分解摺疊錯誤的HLA-B27 蛋白

Endoplasmic Reticulum-Associated Degradation (ERAD) and Autophagy Cooperate to Degrade Misfolded HLA-B27 Proteins

黃品潔(Pin-Jie Huang)

指導教授 ： 曾銘仁

國立中正大學/理學院/生命科學系分子生物研究所/碩士(2013年)

摘要

目的：研究發現人類白血球抗原B27蛋白(HLA-B27)和僵直性脊椎炎(簡稱AS)具有密切關聯性已經超過40年，然而，HLA-B27蛋白對AS疾病的確切致病機制至今尚未確定。其中一個假設是錯誤摺疊的(misfielded) HLA-B27蛋白所誘導的內質網(ER)壓力可能影響發炎反應的調節。

方法：將與AS-相關的同種異型HLA-B2704和與AS-非相關的同種異型HLA-B2706，短暫或穩定表現在HEK293T，HEK293和HeLa細胞中。藉由反轉錄聚合酶鏈反應(RT-PCR)，西方點墨法(Western Blot)和免疫螢光染色 (Immunofluorescence staining) 來分析細胞內質網壓力程度及其相關降解的途徑(ERAD和ERAA)和促發炎細胞激素的表現。

結果：過度表達HLA-B27蛋白會誘導內質網壓力產生以激活IRE-1路徑，進而增加XBP-1 mRNA剪接和GRP78蛋白的表現，而B2704所造成的影響比B2706還明顯。 ER壓力也活化了ERAD和ERAA來降解B27蛋白，而且B2704蛋白的半衰期比B2706蛋白還長。 LC3為細胞自噬體的標誌，本研究發現氯喹(chloroquine)處理穩定表現B2704的HeLa細胞，B2704蛋白與LC3蛋白重疊在相同的位置。 也發現氯喹處理穩定表現B2704的HeLa細胞，會導致細胞凋亡。 最後，穩定表現B2704的HeLa細胞明顯增加了促發炎細胞激素，IL-6，IL-1和TNF-的mRNA。

結論：我們的實驗結果指出持續積累錯誤折疊的HLA-B2704蛋白質會造成長時間的ER壓力，因而啟動細胞凋亡和促發炎性的訊息傳遞途徑。因此，在B2704累積的細胞會提高促發炎細胞激素的表現，這現象可能產生和AS有關的慢性炎症反應。

關鍵字

內質網相關降解系統 ； 內質網壓力 ； 人類白血球抗原B27 ； 僵直性脊椎炎 ； 自體吞噬 ； 促炎症細胞激素

並列摘要

Objective: The susceptibility to Ankylosing Spondylitis (AS) has been strongly associated with human leukocyte antigen B27 (HLA-B27) for over 40 years, however, the exact pathogenic role of HLA-B27 in AS has yet to be determined. One of the hypotheses is that the misfoled HLA-B27-induced endoplasmic reticulum (ER) stress might modulate the inflammatory response.

Methods: HLA-B2704, AS-associated allotype, and HLA-B2706, non-AS-associated allotype, were transiently or stably overexpressed in HEK293T, HEK293 and HeLa cells. The ER-stress and its associated degradation pathways (ERAD and ERAA) and pro-inflammatory cytokines were analyzed in these cells by RT-PCR, Western blot, and immunofluorescence.

Results: The ER stress induced by over-expression of HLA-B27 activated the IRE-1 pathway to increase the levels of Xbp-1 mRNA splicing and GRP78 protein expression and the effect of B2704 was more prominent than that of B2706. The ER stress also triggered ERAD and ERAA to degrade B27 proteins, however, the half-life of B2704 proteins was longer than B2706. The B2704 proteins were co-localized with LC3, an autophagy marker, while treated HeLa B2704 stable cell with chloroquine. The cell apoptosis occurred in chloroquine-treated HeLa B2704 stable cells. Finally, the mRNAs expression of pro-inflammatory cytokines, such as IL-6, IL-1 and TNF, were increased in HeLa B2704 stable cell.

Conclusion: Our data indicate that the persistent accumulation of misfolded HLA-B2704 proteins prolonged ER stress to turn on apoptotic and inflammatory signaling pathways. Therefore, the up-regulation the pro-inflammatory cytokines genes in B2704-accumulated cells might produce the chronic inflammatory response related to AS.

並列關鍵字

Autophagy ； ERAD ； ER stress ； HLA-B27 ； Ankylosing Spondylitis ； proinflammatory cytokines

參考文獻

1. Thomas GP, Brown MA. Genetics and genomics of ankylosing spondylitis. Immunological reviews. 2010;233(1):162-80.

2. Calin A, Brophy S, Blake D. Impact of sex on inheritance of ankylosing spondylitis: a cohort study. Lancet. 1999;354(9191):1687-90.

3. Braun J, Sieper J. Ankylosing spondylitis. Lancet. 2007;369(9570):1379-90.

4. Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet. 1973;1(7809):904-7.

5. Caffrey MF, James DC. Human lymphocyte antigen association in ankylosing spondylitis. Nature. 1973;242(5393):121.