SP110核蛋白藉由與p53蛋白與NF-B蛋白的交互作用以調控細胞功能的機轉

Abstract

先前研究發現位在小鼠基因體sst1 (supersusceptibility to tuberculosis 1) locus上的Ipr1 (intracellular pathogen resistance 1) 基因在巨噬細胞對抗結核菌的先天免疫反應與細胞死亡中扮演關鍵的調節角色，也發現Ipr1蛋白具有調控轉錄的功能。人類的SP110基因是小鼠Ipr1的同源基因，而在SP110蛋白異構體中，最接近Ipr1的同源異構蛋白SP110b，已被發現會透過減弱NF-kB蛋白複合體與TNFα啟動子 (promoter) 上的kB附著DNA區域的作用而調控NF-kB的活性，進而減緩單核球細胞與巨噬細胞因 IFNγ過度活化所導致的細胞死亡。這些結果都暗示SP110蛋白在許多疾病中都可能扮演重要角色而值得被探討。本研究分成兩部分作探討，第一部分是探討SP110 單一多型性 (single nucleotide polymorphisms (SNPs)) 對結核菌易感受性 ( susceptibility) 的影響，並透過SP110、NFkB1與TNFα 三個基因的 SNPs 交互作用來了解SP110在結核菌感染進程中所扮演的角色；第二部分則是探討SP110b蛋白與p53蛋白兩者的交互作用在抑制癌症細胞生長所扮演的角色。 第一部分的研究以臺灣族群為對象，蒐集結核病患者、潛伏結核感染 (LTBI) 患者及在家接觸過結核病患但顯示健康未感染的照護者作為控制組的基因體樣品進行分析，探討 SP110基因單一多型性是否與潛伏結核感染及結核菌的易感受性有相關性。我們鑑定出SP110基因若干SNPs ( rs7580900、rs7580912、rs9061、rs11556887與rs2241525) 與潛伏結核感染及結核菌的易感受性有關。分析更進一步顯示相同的SNP對潛伏結核感染及結核菌感染的發病可能會出現相反的影響。此外分析也發現潛伏結核感染患者血清中的腫瘤壞死因子的含量下降與SP110的rs9061 SNP具有相關性，這結果意味著SP110的單一多型性在人類的潛伏結核感染及結核病發病的易感受性扮演了一個控制的角色。此部分的研究也探討SP110與NF-kB主要兩個組成NFkB1 (p50) 與RELA (p65) 基因之基因單一多型性的交互作用在調控TNF α的影響以及在潛伏結核感染與結核病發病過程中的角色。結果發現SP110 的單一多型性與NFkB1及RELA的單一多型性組合對結核病發病進程造成的影響並不相同。在細胞發炎時SP110b對於影響p50及p65 結合 TNF α promoter的程度也有很大的不同。這些結果暗示了SP110可以分別和p50與p65交互作用在潛伏結核感染與結核病發病過程中扮演不同的角色。 第二部分的研究則探討SP110b與p53兩核體蛋白的交互作用對細胞的影響。結果發現，SP110b除了與p53有交互作用，SP110b的表現也會影響p53蛋白在細胞中的分布與含量。透過高效液相層析串聯質譜儀分析也發現SP110b會影響p53蛋白產生乙醯基化與甲基化等後轉修飾作用。而從High-content screening的數據分析可以看到SP110b會增加p53蛋白在細胞內的移動速率與距離。進一步分析發現SP110b表現會造成肺癌細胞發生老化作用，當加入p53表現時則發現細胞除了有老化作用之外，也有自噬作用的情況發生。根據初步的RNA-seq結果分析，我們認為SP110b的表現會使細胞的 ”gene information process”相關的基因表現下降，而在p53表現的情況下，SP110b則會使 “Miotophagy” 及 ' Protein processing in endoplasmic reticulum” 相關的基因有較多表現上升的情形，這或許暗示了SP110b會使許多與細胞生長相關的基因表現下降而導致腫瘤細胞老化的發生，而在同時有p53表現情況下則可能因有許多新生蛋白產生而使p53因應這個變化而增加在細胞中的動態移動，進而使腫瘤細胞發生老化與自噬作用而抑制細胞生長。這些結果則有待進一步的證實。

Previous studies have demonstrated that Ipr1 (intracellular pathogen resistance 1) gene located in the sst1 (supersusceptibility to tuberculosis 1) locus in mouse genome plays a key regulatory role in the innate immune response and cell death of macrophages against Mycobacterium tuberculosis (Mtb) infection. The Ipr1 protein also has the function on regulating gene transcription. Human SP110 gene is a homolog of the mouse Ipr1 gene. Among the SP110 protein isoforms, SP110b that is closest to the Ipr1 has been found to deteriorate the interaction of NF-kB protein complex and κB binding sites on the TNFα promoter and regulate the activity of NF-κB by attaching to the DNA region, thereby alleviating the cell death of monocytes and macrophages due to the excessive activation of IFNγ. These results suggest that the SP110 protein may play an important role in many diseases and is highly worthy of investigation. This study is divided into two parts: the first part is to elucidate the influence of SP110 single nucleotide polymorphisms (SNPs) on the susceptibility of Mtb infection and, through genetic analysis of the interaction of SP110, NFkB1 and TNFα SNPs, to understand the role of the SP110 in the pathogenesis of Mtb infection; the second part is to explore the role of the interaction between the SP110b protein and p53 protein in inhibiting the growth of cancer cells. The first part of the study targeted the Taiwanese population and collected genome samples from tuberculosis patients, latent tuberculosis infection (LTBI) patients, and healthy household contacts of the tuberculosis patients for analysis to determine whether the SP110 SNPs were associated with the susceptibility to LTBI and tuberculosis infection. We identified several SP110 SNPs (rs7580900, rs7580912, rs9061, rs11556887 and rs2241525) that are related to the susceptibility to LTBI and tuberculosis infection. The analysis further showed that the same SNPs may have opposite effects on the incidence of LTBI and tuberculosis infection. In addition, the analysis also demonstrated that the level of TNFα in the serum of LTBI patients is associated with the rs9061 SNP of the SP110, indicating that the SP110 SNP plays a role in the susceptibility of human LTBI and tuberculosis infection. This part of the study also explored the role of interaction of SP110 and the two main components of NF-kB, NFkB1 (p50) and RELA (p65) genes, in the regulation of TNFα expression and the disease progress of LTBI and tuberculosis infection. The results revealed that the interactions of the SP110 SNPs with NFkB1 and RELA SNPs have different effects on the pathogenesis of tuberculosis. When the cells were inflamed, the SP110b affected the extent to which p50 and p65 bind to the TNFα promoter. These results suggest that the SP110 can interact with p50 and p65 to play different roles in the pathogenesis of LTBI and tuberculosis infection. The second part of the research explores the effect of the interaction of SP110b and p53 on cellular functions. Apart from the interaction between SP110b and p53, the studies showed that the SP110b also affected the distribution and content of p53 protein in cells. Analysis by high performance liquid chromatography tandem mass spectrometer also demonstrated that the SP110b affects the post-translational modifications of p53, such as acetylation and methylation. From the data analysis of High-content screening, the SP110b was found to increase the speed and distance of p53 protein movement in the cell. Further analysis reveale that the expression of SP110b cause lung cancer cells to undergo senescence. However, in the presence of both p53 and SP110b, in addition to senescence, cells also increase autophagy activity. The preliminary analysis of RNA-seq results showed that the SP110b down-regulated the expression of genes related to the 'gene information process' group while, in the presence of p53, the SP110b up-regulated the 'Miotophagy' and 'Protein processing in endoplasmic reticulum' related genes. It may imply that the SP110b down-regulated the expression of many genes related to cell growth, resulting in the occurrence of tumor cell senescence and that, in the presence of p53 expression, SP110b up-regulated genes that produced nascent proteins facilating p53 to increase its dynamic movement in cells, resulting in increased senescence and autophagy of tumor cells to inhibit their growth. The detail mechanism need to be further studied.