Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20067
標題: 腫瘤轉移抑制基因HLJ1與其交互作用蛋白的定性
Characterization of the protein-protein interaction between invasion / metastasis suppressor gene HLJ1
作者: 張梓珮
Chang, Tzu-Pei
關鍵字: lung cancer;肺癌;invasion / metastasis suppressor gene;heat shock protein;腫瘤轉移抑制基因;熱休克蛋白
出版社: 生物醫學研究所
摘要: 
Lung cancer is the first leading cause of female cancer-related deaths in the last years in Taiwan, and the case number is increasing each year. Among the lung cancer types, adenocarcinoma is the most common lung cancer in Taiwan. Cancer is often caused by gene mutation, including oncogenes and tumor suppressor genes. In the previous studies, using invasion/metastasis cell line model and microarray technology, we have identified and characterized a novel suppressor gene HLJ1 (Human Liver DnaJ-like protein) which plays an important role in tumor growth and metastasis. HLJ1, a member of Hsp40 family (DnaJ-like Heat Shock Protein) was isolated from human liver cDNA library first. In this project, to identify and characterize the interacting proteins of HLJ1, we used two-dimensional SDS-PAGE and MALDI-TOF analysis to sort out the differentially expressed proteins in HLJ1-overexpressed cell clones as compared with control cells. We have identified approximately 14 proteins confirmed by Western blotting and real-time quantitative RT-PCR that may be modulated by HLJ1 express level. On the other hand, the cell extracts were immunoprecipitated with HLJ1 antibodies. A number of polypeptides were subjected to mass spectrometry. We found out 9 proteins that could be interacted with HLJ1. The results of co-immunoprecipitation suggested that HLJ1, Hsc70 (Hsp70 family), and nucleophosmin could interact with each other to form a multi-protein complex. Nucleophosmin (NPM/B23) is a phosphoprotein and involved in nucleolar assembly, centrosome duplication, cell cycle progression, and ribosome assembly and transport. Dissociation/reassociation of NPM with centrosomes in a cell cycle stage-specific manner may be controlled by phosphorylation. The complex immunoprecipitated from cell extracts was treated with phosphatase or RNase A, the results suggested that the interaction is dependent on phosphorylation and RNA-binding activity. Using mammalian two-hybrid assay, we have identified the major interaction domain between NPM and HLJ1, including OligoD of NPM and J domain of HLJ1. Furthermore, immunofluorescent staining was performed to indicate the sub-cellular location of protein interaction and deduce the putative mechanisms in cell cycle progression. Our results also showed that overexrpesssion of NPM could enhance cell invasion and migration. We conclude that HLJ1 can interact with Hsc70 and NPM respectively, which is dependent on phosphorylation and RNA-binding activity; and further, HLJ1 may prevent NPM in cytoplasm from translocating into nucleus to inhibit cancer cell invasion and migration. It is important to identify and investigate a novel molecular mechanism about HLJ1-NPM interaction because they could potentially provide a new insight to understand the pathway of metastasis regulation and may be the new target molecule for anti-cancer therapy in the future.

近年來,臺灣的肺癌病例有顯著增加的趨勢,在女性的癌症病例中甚至高居死亡原因的首位。在臺灣的肺癌病例中,以肺腺癌(adenocarcinomas)較為常見。通常癌症是因為細胞的基因變異所引起的,這些基因包括致癌基因及抑癌基因。先前的研究中,利用侵入/轉移模式的細胞株和微陣列技術發現HLJ1 (Human Liver DnaJ-like protein)是一新的抑制基因,具有抑制腫瘤細胞轉移與生長的能力。 HLJ1為DnaJ-like熱休克蛋白40家族(Heat Shock Protein 40,HSP)中的一員。HSP 其分子量分為 Hsp100、Hsp90、Hsp70、Hsp60、Hsp40及small Hsps,是一種維持細胞生命所必須的蛋白質,平時主要維持正常細胞之生理機能,如幫助細胞內蛋白質轉位、折疊及組合等。細胞於正常情形下即有固定的HSP表現量,一旦細胞面臨緊迫狀態或病原感染時,細胞就會大量表現HSP以阻止細胞內蛋白累積以及修補受損蛋白,增加細胞的抵抗能力。為研究HLJ1於肺腺癌中扮演的角色,此研究將由蛋白質的層次切入。利用具有不同HLJ1表現量的細胞株(PCH9/PCC10,PCH8/PCC24)經由二維電泳的分離比對,找出可能受到HLJ1表現量影響的蛋白質,利用質譜儀(MALDI-TOF;基質輔助雷射脫附游離/飛行時間質譜) 分析,已找到約14個蛋白質會受到HLJ1表現量的影響。進一步利用即時定量聚合酶連鎖反應(Real-Time PCR)與西方墨點法,分析 RNA 和蛋白質層次上的表現是否符合質譜儀分析的結果,並進而研究其相關性。經由上述實驗的結果,再進行免疫共沉澱(co-immnoprecipitation),結果發現HLJ1會與Hsp70/Hsc70、NPM (nucleophosmin)形成複合物。NPM是一多功能性的磷酸化蛋白質,具有調節核仁活性、中心體複製、細胞週期、核糖體組成和運輸等功能。在免疫共沉澱過程中若以RNA水解酵素(RNase A)和蛋白磷酸水解脢(phosphatase)處理,會影響Hsc70/Hsp70、NPM複合物的形成。我們利用哺乳動物細胞蛋白質交互作用分析(mammalian two-hybrid assay),證明在細胞內亦會形成此複合物,而HLJ1的J區域和NPM的OligoD區域,則可能為HLJ1和NPM主要可能結合區域。而經由免疫染色探討HLJ1和NPM在細胞週期進行時會所在的區域,進而研究其機制;另外,將NPM大量表現時,會使細胞移動能力與侵犯能力增加。總結來說,HLJ1 會與Hsc70和NPM有結合作用,且會受到磷酸化作用及與RNA結合的活性所影響; HLJ1可能是藉由抑制NPM從細胞質進入細胞核,而抑制癌細胞的侵犯和移動能力。後續探討HLJ1和NPM之間的分子機制是十分重要的,可以提供一個有關癌轉移調節路徑的新思維,並在未來應用於抗癌治療上。
URI: http://hdl.handle.net/11455/20067
Appears in Collections:生物醫學研究所

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