功能與遺傳分析線蟲eif-3.k在細胞凋亡的角色

Functional and Genetic Analysis of a Proapoptotic Gene eif-3.k in Caenorhabditis elegans

吳淑君(Shu-Chun Wu)

指導教授 ： 吳益群

國立臺灣大學/生命科學院/分子與細胞生物學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.00962

摘要

計畫性細胞死亡對多細胞生物的發育過程相當重要。我們發現在線蟲內EIF-3.K具有促進細胞死亡的活性: eif-3.k(lf)造成細胞屍體數目減少，而eif-3.k(gf)卻能使更多的細胞死亡。 EIF-3.K是個具有高度保守性的蛋白質，人類的EIF3K被預測具有兩個domains: HAM及WH，因此我們測試這兩個domains對EIF-3.K活性的重要性。我們利用deletion assay，發現HAM對EIF-3.K促進細胞死亡的功能並不重要，而WH domain對EIF-3.K是必須的，且進一步發現WH domain就能完全代表 EIF-3.K促進細胞死亡的活性。在yeast two-hybrid系統中EIF-3.K與CED-3、CED-4、DAPK-1彼此並無直接作用，我們利用yeast two-hybrid screen，找到三個蛋白質與EIF-3.K結合 : C17G10.9、NCL-1及CEH-26。只有ceh-26突變株的細胞屍體數目減少。我們再利用雙重突變株，發現eif-3.k及ceh-26可能作用在同一條遺傳途徑來調控細胞死亡。

關鍵字

線蟲 ； 細胞死亡

並列摘要

Programmed cell death is important for development of animal. EIF3K was identified as DAPK interacting protein by using the death domain of DAPK as a bait in a yeast two-hybrid. EIF3K is highly conserved in metazoan. We found that EIF-3.K, the EIF3K homolog in C. elegans, had a pro-apoptotic activity. An eif-3.k mutation had reduced cell corpses during embryogenesis, whereas overexpression of eif-3.k caused extra cell deaths. Human EIF3K was predicated to contain two domains: HAM and WH. We performed deletion assays and found that HAM domain is dispensable for the pro-apoptotic function of EIF-3.K, but WH domain is necessary for its function. Furthermore, we found that expression of the WH domain also was sufficient to rescue the cell-death phenotype of the eif-3.k mutants. As no direct interaction was found between EIF-3.K and previously characterized components of the apoptotic machinery, such as CED-3, CED-4, and DAPK-1, we sought to identify proteins that interact with EIF-3.k by a yeast two-hybrid screen. We isolated three potential candidate clones: C17G10.9, NCL-1, and CEH-26. Only mutation in ceh-26 but not other two genes decreased number of cell corpses. Further genetic studies suggested ceh-26 and eif-3.k may participate in the same pathway during programmed cell death.

並列關鍵字

cell death ； apoptosis ； eif-3.k ； C.elegans

參考文獻

Bloss T.a., Witze E.S., Rothman J.H. (2003) Suppression of CED-3-independent apoptosis by mitochondrial betaNAC in Caenorhabditis elegans. Nature, 424, 1066-1071

Brenner, S. (1974) The genetics of Caenorhabditis elegans. Genetics, 77, 71-94

Chen, F,m Hersh, B.M., Conradt, B., Zhou, Z., Riemer, D., Gruenbaum, Y. and Horvita, H.R. (2000). Translocation of C. elegans CED-4 to nuclear membeanes during programmed cell death. Science, 287, 1485-1489

Cohen O., and Kimchi A. (2001) DAP-kinase: from functional gene cloning to establishment of its role in apoptosis and cancer. Cell Death Differ. 8, 6-15.

Conradt B. (2002) With a little help from your friends: cells don't die alone. Nat Cell Biol. 4, 139-43.