SP110蛋白質異構體與2’-5’ Oligoadenylate Synthetase 1之交互作用

Abstract

結核病主要是由結核分枝桿菌感染所造成的病症，在全球是致死率僅次於愛滋病的感染症。由於多重抗藥性和廣效性抗藥性結核分枝桿菌菌株的產生以及結核病患者共同感染愛滋病使得結核病成為當前很急迫的公共衛生問題。根據統計全球約有三分之一的人口已感染結核分枝桿菌，但是其中只有不到10%的人會在他們一生的某個時期產生結核病。由先前的研究指出，在小鼠的第1號染色體之sst1基因座中找到 Ipr1 基因，此 Ipr1 基因會調控宿主先天性免疫反應去對抗結核分枝桿菌。在sst1易感受性的巨噬細胞中，表現 Ipr1會控制結核分枝桿菌的生長，而且表現Ipr1的轉殖鼠之巨噬細胞會避免受到結核分枝桿菌感染而進行細胞壞死 (Cell necrosis) 並且轉而促進細胞凋亡 (Cell apoptosis) 。利用酵母菌雙雜交系統 (Yeast two-hybrid system) ，發現2', 5' oligoadenylate synthetase 1 (OAS1) 會和Ipr1進行交互作用。由免疫共沉澱實驗，我們分別在小鼠的J774細胞及人類HEK 293T細胞中也發現到Ipr1與 SP110 會和 OAS1 進行交互作用。小鼠的Ipr1蛋白與人類最相似的蛋白為 SP110，此 SP110 是一個主要有三種不同異構體 (Isoforms, SP110A/B/C) 的核蛋白。雖然已經知道人類的 SP110 和 OAS1 有交互作用，不過SP110會不會影響OAS1在細胞中的功能仍是未知的。到目前為止我們已成功的建立由Doxycycline誘導表現SP110A，SP110B，OAS1，以及共同表現SP110A/OAS1或SP110B/OAS1的THP-1細胞株。由免疫螢光染色實驗，我們知道SP110A/SP110B 分別會和 OAS1 共同分佈於高基氏複合體。藉由分析mRNA的含量，我們發現SP110A/OAS1細胞株有減少IFN-β mRNA表現的趨勢。而這個現象在SP110B/OAS1細胞株則沒有被觀察到。這個結果暗示我們SP110A和SP110B在調節OAS/RNase L pathway上具有不同的功能。在IFN-γ的作用下，位於細胞核的SP110A和SP110B皆有避免細胞死亡的效果；但是位於高基氏複合體的SP110A/OAS1和SP110B/OAS1則有增加細胞凋亡的傾向。這結果顯示 OAS1 可能會改變SP110異構體在細胞內的分布情形，並且可能改變SP110異構體的功能。OAS1是如何改變SP110異構體在細胞內分布情形的機制到現在還不清楚，未來可藉由分析位於高基氏複合體的SP110異構體與 OAS1複合體的結合分子，以及分析在高基氏複合體的SP110異構體和OAS1的後轉譯修飾，來釐清由SP110異構體所調控的細胞凋亡機制。

Tuberculosis (TB) is an infectious disease mainly caused by Mycobacterium tuberculosis (Mtb) and is the second leading cause of death from an infectious disease all over the world (after HIV). Emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) and co-infection of TB patients with HIV are urgent issues of public health worldwide. It's estimated that one-third of the world's population is currently infected with Mtb, but only less than 10% of the people who are infected with Mtb turn to TB disease during their lifetime. In the previous work, a gene Ipr1 (Intracellular pathogen resistance 1), which mediated host innate immunity to Mtb infection, was identified within sst1 (Supersusceptibility to tuberculosis 1) locus on mouse chromosome 1. The expression of Ipr1 in the sst1-susceptible macrophages controlled the multiplication of Mtb in vitro, and the Ipr1 transgene prevented necrosis and facilitated apoptosis of the Mtb-infected macrophages. Using yeast two-hybrid system, 2', 5' oligoadenylate synthetase 1 (OAS1) was identified as a candidate protein interacting with mouse Ipr1. SP110, the closest human homologue of mouse Ipr1 protein, is a nuclear protein that has three major isoforms (SP110A/B/C). By co-immunoprecipitation, we found that Ipr1 interacted with OAS1 in J774 cells and that SP110 interacted with OAS1 in HEK293T cells. However, whether SP110 would affect the cellular functions of OAS1 still remains unknown. So far we have successfully generated several THP-1 clones which express SP110A, SP110B or OAS1 and clones which co-express OAS1 with SP110A or SP110B upon Doxycycline induction. By immunofluorescence staining, we figured that SP110A/SP110B and OAS1 were co-localized in the Golgi complex. By analyzing Interferon-β (IFN-β) mRNA production, we found that the interaction of SP110A and OAS1 tended to decrease the expression of IFN-β mRNA but the one of SP110B and OAS1 did not. The results implied that SP110A and SP110B may have different functions in regulating OAS/RNase L pathway. In the presence of IFN-γ stimulation, the expression of SP110A/SP110B, which were co-localized in the nucleus, prevented cell death, but co-expression of SP110A/SP110B with OAS1, which were co-localized in the Golgi complex enhanced apoptosis. The results indicated that OAS1 may change the cellular distributions of SP110 isoforms and the functions of SP110 isoforms. The mechanisms by which OAS1 changes the cellular distributions and functions of SP110 isoforms remain unknown. In the future, by identifying the molecules associated with the SP110 isoform-OAS1 complex in Golgi bodies and by determining the post-translational modifications of SP110 isoforms and OAS1 when they are co-expressed, we will elucidate the mechanisms of SP110 isoforms-mediated apoptosis.