HeLa 세포에서 IgE-dependent Histamine-Releasing Factor와 Triosephosphate Isomerase의 상호작용 규명
Interaction between IgE-Dependent Histamine-Releasing Factor and Triosephosphate Isomerase in HeLa Cells
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본 실험에서는 HRF의 조절단백질을 알아보기 위해 HRF를 bait로 한 yeast two hybrid assay를 실행한 결과 해당과정에 관여하는 TPI(triosephosphate isomerase)라는 효소를발견하였으며, 가장 많이 중복되어 있었다. In vitro에서 HRF는 TPI의 C 말단 잔기 부근(아미노산 156-249)이 상호작용에 주로 관여하는 부위임을 알 수 있었다. 또한, HeLa 세포에서 immunoprecipitation을 이용하여 HRF와 TPI의 상호작용이 실제 in vivo에서도 일어나는 현상이라는 것을 밝혔다.결과적으로 HRF와 TPI의 상호작용은 세포내 일정량이 존재하며 여러가지 신호전달에 의해 동시에 Na,K-ATPase와도상호작용하는 것으로 생각된다.
Abstract
IgE-dependent histamine-releasing factor (HRF) is found extracellularly to regulate the degranulation process of histamine in mast cells and basophils and known to play a predominant role in the pathogenesis of chronic allergic disease. HRF has been also identified in the intracellular region of the cell. Previously, we reported that HRF interacts with the 3rd cytoplasmic domain of the alpha subunit of Na,K-ATPase. To understand the molecular mechanism of the regulation of Na,K-ATPase activity by HRF, we investigated the interaction between HRF and TPI since TPI was obtained as HRF-interacting protein in HeLa cDNA library, using yeast two hybrid screening. Domain mapping study of the interaction between HRF and TPI revealed that the C-terminal region of the residue 156-249 of TPI is involved in the interaction with HRF. The interaction between HRF and TPI was confirmed by immunoprecipitation from HeLa cell extracts. Our results suggest that TPI is a HRF-binding protein and the interaction between HRF and TPI may thus affect Na,K-ATPase activity.
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