人類Rad9 (全名：Human homologue of Schizosaccharomyces pombe Rad9 )是一各在演化上具有高度保留性的蛋白質,而且在好幾種生物功能上也扮演著很重要的角色,包括促進對任何DNA傷害物質的抵抗性,傳遞細胞週期的一各檢查哨（checkpoint）,誘導細胞的凋亡以及穩定基因體的完整性特別在未遭受DNA傷害物質的物種中,也具有保持基因體的完整性.由於Rad9調節許多細胞逆境的反應,其中包含了細胞的凋亡.同時,Rad9也被認為在功能上與p53極為相似.另外,對於MDM2在生物體中基本上扮演著一個負向調控者的角色.然而過去的文獻中指出,MDM2在經過DNA傷害物質的刺激下,其在細胞中的位置會有所改變.過去的科學家認為這應該與細胞中p53促使細胞凋亡有關.因此,我們想研究Rad9在細胞凋亡上所扮演的角色是否與MDM2有牽連.在我們的研究中,我們提供了一些數據證明了Rad9能夠與MDM2進行交互的作用,無論在於試管外的實驗（in vitro）亦或是在細胞活體中的實驗.首先,我們利用了共同免疫沈澱的方式指示出Rad9與MDM2呈現出專一性的交互作用,無論是利用外源性的MDM2-GFP偵測內生性的Rad9,或者是利用外源性的Flag-Rad9來偵測內生性的MDM2.再來,試管外的實驗利用Gst-MDM2與細胞內的Rad9進行雜和反應,也暗示出Rad9能夠與MDM2進行交互作用,而其餘的負對照組均無偵測出Rad9.這也再次呼應著Rad9與MDM2的交互作用是呈現著專一性.過去的文獻報導出在Rad的Nter範圍中具有一段與BH3相似的結構,而且文獻也指出此段結構能夠賦予Rad9受到DNA物質傷害後能具有進行細胞凋亡的功能.再加上我們之前證明出Rad9能與MDM2進行交互作用,這促使我們想研究是否Rad9與MDM2的機作用參與著細胞凋亡的過程.實驗結果顯示在三株不同的細胞中（MCF-7、A549、U2OS）MDM2與Rad9的交互作用在紫外光照射後早期均有增加的趨勢,隨著時間延續MDM2消失後,伴隨著則是Rad9與Bcl-XL的交互作用.另外,在免疫螢光染色(immunofluroscence)實驗中也發現到Rad9能夠與MDM2交互在Bcl-XL蛋白分佈於細胞中的位置,包含細胞核膜以及細胞質中,同時在核質分離的過程中也指出Rad9與MDM2在經過紫外光早期照射下於細胞核中交互作用有增加,另外在晚期則一起慢慢轉送到細胞質中,接著MDM2在細胞質的消失後,接踵而來的便是Rad9與Bcl-XL的交互作用.最後,我們利用MDM2-GFP過度表現與核糖核酸干擾的技術都證明了Rad9與Bcl-XL的交互作用確實能被MDM影響的.上述的結果證明了Rad9誘導的細胞凋亡中,MDM2則牽涉其中當作一個負調節者.額外的,我們也發現了Rad9過度表現的情況下,會造成MDM2蛋白表現的減少.這使我們研究是否p53的調節會被Rad9的表現多寡有所影響.從簡單的Rad9過度表現以及Rad9核糖核酸干擾的實驗中也暗示著在經過紫外光的照射下,p532的累積與後修飾作用都可能因為Rad9蛋白的表現不一而有所影響.引此,綜合上述所有的結果,我們認為Rad9與MDM2的交互作用可能能為細胞凋亡帶來新穎的機轉.

The human Rad9 (Human homologue of Schizosaccharomyces pombe Rad9) is an evolutionarily conserved protein and plays important roles in several fundamental biological processes, including promotion of resistance to DNA damage, acting as a pro-apoptotic element. The proteins encoded by human and mouse Rad9 contain a BH3-like domain, typical of BH3-only, pro-apoptotic family members. Investigations with the hRad9 BH3 demonstrated the ability to bind anti-apoptotic proteins Bcl-2 and Bcl-XL. Overexpression of hRAD9 in a variety of human cell lines induced apoptosis, but partial deletion of the BH3 domain neutralized this pro-apoptotic activity. Several models have been proposed to explain how hRad9 functions as a mediator of programmed cell death. Evidence showed that DNA damage leads to cleavage of hRad9 by caspase 3. Interestingly the cleavage of hRad9 by caspase 3 is thought to disrupt the Rad9-Rad1-Hus1 complex, thus interfering with cell cycle checkpoint control and promoting apoptosis. The DNA damage-induced phosphorylation of hRad9 by the protein kinase c-Abl is important forbinding of hRad9 to Bcl-XL while phosporylation by protein kinase Cδ (PKCδ) enhances the association of hRad9 and Bcl-2. However, the mechanism of Rad9-mediated apoptosis needed to be addressed. Previous reports suggested that Rad9 could translocate from nucleus to cytosol during early apoptotic condition. Besides, MDM2, which plays an important negative regulater in mutiple-functions, appeared to translocate to the same site that Rad9 locates under apoptotic condition. In this study, we provide evidences that Rad9 can interact with MDM2 by cell-based assay and in vitro GST pull-down assay. At first, co-immunoprecipitation experiments indicated that the specific interaction between exogenous GFP-MDM2 and endogenous Rad9 occurred in 293T cell line. Second, pull-down assays demonstrated that purified GST-MDM2, but not GST alone could interact with endogenous Rad9 prepared from cell lysate of A549 cell line. It was reported that N-terminal of Rad9 has BH3(Bcl-2 homology domain3)-like region and the Rad9 plays a role involving in regulating apoptosis in HeLa cell line that were suffered form DNA damage. Therefore, the Rad9-MDM2 association was also investigated following U.V irradiation. The result showed that the levels of MDM2-Rad9 complex was enhanced at 2hr post-irradiation of MCF-7 cell line by U.V irradiation. On the other hand, the association of Rad9 with Bcl-XL could not be detected in A549 cell lines without U.V irradiation, and the complex of Rad9-Bcl-XL was detected after 4hr post-irradiation. By immuno-fluorescence method, we observed that both Rad9 and MDM2 localize at nucleolus in A549 cell line before UV irradiation; however, most of these two proteins were trans-located into peri-nuclear membrane at 12hr post-irradiation. Final, our preliminary data indicated the binding ability of Rad9 to Bcl-XL was enhanced in A549 cell line pre-treated with MDM2 RNAi, extensively irradiate by U.V. However, the mechanism is still poorly understood. On the other hand, we found that Rad9 over-expression appears to down-regulate MDM2 protein level and promotes p53 post-translational modification after U.V treatment, especially the phosphorylation of p53s46. According to our results, we suggest that the proteins that interact with Rad9 are supposed to regulate Rad9-mediated apotposis, and the role of Rad9 under high U.V energy may provide a novel mechanism for cell programma death.

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