근육 세포에서 TCTP/HRF와 상호작용하는 단백질 규명에 대한 연구

Abstract

The translationally controlled tumor protein (TCTP)/ IgE-dependent histamine-releasing factor (HRF)는 mouse ascite과 erythroleukemic cell에서 growth-related protein으로 처음 소개된 이후 kidney나 renal cell carcinoma를 제외한 모든 정상세포에서도 발견되었으며, human TCTP/HRF는 human basophil 세포에서 immnoglobulin E (IgE) 존재시 항원에 대한 반응으로 histamine이나 interleukin (IL)-4의 분비를 유도한다고 알려져 있다. 본 연구에서는 full-length의 rat TCTP/HRF cDNA를 bait로 하여 rat skeletal muscle cDNA library로부터 yeast two hybrid screening을 실시한 결과를 바탕으로, TCTP/HRF의 N-terminus truncated TCTP/HRF (clone734)와의 self-interaction과 N, C-terminus truncated rat fast myosin light chain과의 상호 작용에 대해 알아보았다. 즉 첫째, TCTP/HRF가 self-interaction을 할 때 결합하는 부위를 알아보기 위하여 yeast two hybrid를 통한 TCTP/HRF deletion analysis를 실시한 결과, TCTP/HRF의 residue 126-172의 C-terminal domain이 중요한 역할을 담당하리라는 것을 알 수 있었다. 둘째, TCTP/HRF와 myosin light chain과의 상호 작용을 affinity chromatography와 immunoprecipitaion을 통하여 다시 확인하였다. 또한 이러한 TCTP/HRF와 myosin light chain과의 상호 작용을 RBL-2H3 cell에서의 histamine releasing과 관련 지어 알아본 결과, TCTP/HRF는 phosphorylation된 형태의 myosin light chain과 상호 작용하며 dephosphorylation된 형태의 myosin light chain과는 상호 작용하지 않으리라는 것을 알 수 있었다. 그리고 RBL-2H3 cell이 IgE로 sensitization될 때 이러한 signal에 의해 myosin light chain이 dephosphorylation되어, myosin light chain과 TCTP/HRF와의 상호 작용이 깨어지는 것이라고 생각할 수 있었다. 마지막으로, ouabain을 주사한 rat의 간과 심장 조직에서 핵을 분리한 결과 TCTP/HRF의 핵으로의 이동이 증가하였음을 확인하였으며, 이로써 TCTP/HRF가 transciption factor로 작용할 수 있는 가능성을 볼 수 있었다.;The translationally controlled tumor protein (TCTP)/ IgE-dependent histamine-releasing factor (HRF) was described initially as a growth-related protein in mouse ascites and erythroleukemic cells. Later, it was also found in a number of normal cell types except kidney or renal cell carcinomas, suggesting that the protein might have an essential function in the cell. Recently human TCTP/HRF has been shown to induce secretion of histamine and interleukin (IL)-4 from human basophils in the presence of IgE. In the present study, I investigated the self-interaction of between TCTP/HRF and N-terminus truncated TCTP/HRF (clone734) and the interaction between TCTP/HRF and N, C-terminus truncated rat fast myosin light chain were investigated since those were obtained as TCTP/HRF-interacting proteins using yeast two-hybrid screening. At first, domain mapping of the self-interaction revealed that the C-terminal region of residue 126-172 is involved in self-interaction of TCTP/HRF. Secondly, the interaction between TCTP/HRF and myosin light chain was confirmed by affinity chromatography and immunoprecipitation. The interaction between TCTP/HRF and myosin light chain in relation to the histamine degranulation from RBL-2H3 cells was investigated. TCTP/HRF seems to interact with the phosphorylated myosin light chain but not with the dephosphorylated myosin light chain. Interestingly, when RBL-2H3 cells were sensitized with IgE, the interaction between TCTP/HRF and myosin light chain was disrupted, suggesting that IgE sign als might be involved in the dephosphorylation of myosin light chain. Finally, I isolated the nuclei from the livers and hearts of rats after treatment with ouabain , and TCTP/HRF were increasingly detected in the nuclei rather than in the cytoplasm, suggesting that TCTP/HRF might play a role as a transciption factor.