運用親和力蛋白質純化法找出與白色念珠菌Cdc4的相關性蛋白質

白色念珠菌(Candida albicans)為人類的伺機性真菌病菌，是最常導致人類黏膜表皮層以及全身性感染之真菌。在免疫系統缺陷或低下的病人當中，白色念珠菌會造成嚴重的全身性感染，而導致病人的死亡。由於白色念珠菌在自然界中是以雙倍體的形式存在，且沒有完整的有性生殖之形式，所以這將提高了白色念珠菌在分子遺傳研究上的困難度。白色念珠菌是一種多形態的真菌，可以以酵母菌、假性菌絲，以及真性菌絲的形式生長。而這樣多樣性生長形式的轉換，與其致病力有關。出芽酵母菌(Saccharomyces cerevisiae) Cdc4屬於F box蛋白質家族，是Skp1-Cdc53/Cullin-F box (SCF) 泛素接合酶複合體的成員之ㄧ，在有絲分裂細胞週期G1到S時期的轉移上扮演著重要的角色，而此功能在演化上是高度保留的。白色念珠菌的CDC4 (CaCDC4)和出芽酵母菌的CDC4 (ScCDC4)雖具有結構上的同源性，但相較於ScCDC4於出芽酵母菌中為必要性的基因，CaCDC4於白色念珠菌中並非必要的基因。此外，當CaCDC4失去或剔除時，會促使白色念珠菌菌絲形態的形成，暗示著透過CaCdc4泛素媒介蛋白質的降解機制與調控白色念珠菌的形態轉變可能是有關聯的。因此，為了證明此關聯性，在這次的研究當中，我致力於運用親和力蛋白質純化的方式，以抗原決定基標記的CaCdc4於純化CaCdc4的同時將其相關性的蛋白質純化出來。我分別以體外及體內的方式進行單次親和力的純化。在體外純化部份，抗原決定基所標記的CaCdc4於細菌大量表現並純化後，會與酵母菌或真性菌絲生長形式的細胞裂解液於體外進行蛋白質交互作用後，純化CaCdc4，而其相關性蛋白質可隨之純化；而在體內純化部份，白色念珠菌品系誘發成酵母菌或真性菌絲，且於體內經四環素誘導表達抗原決定基所標記的全長CaCdc4，以及其F box與WD40 repeats的蛋白質後，於親和力純化蛋白質時，將其相關的蛋白質一併純化。接著，利用SDS-PAGE分離純化之CaCdc4及相關性蛋白質，經由銀染分析，則體外及體內，酵母菌及真性菌絲生長形式，或不同CaCdc4結構域之間被純化出蛋白質的差異可以被顯現出來。未來，將進一步以質譜儀分析此類蛋白質，分析其身分及對應基因，並經過特性分析，將可解析CaCDC4在形態生成上所扮演的角色。

關鍵字

白色念珠菌；親和力純化法

並列摘要

Candida albicans is an opportunistic fungal pathogen in humans, causing either systemic or mucosal infection. In immunocompromised patients, this organism can progress to severe systemic infection, leading to life-threatening circumstances. Because Candida albicans is a diploid genome organism without complete sexual cycle, the molecular genetic analysis of this organism has always been limited. Candida albicans is a polymorphic fungus with three major growth forms, the yeast form, pseudohyphal and hyphal forms. The morphological transition has been strongly associated with pathogenicity. Saccharomyces cerevisiae Cdc4 belonging to F box protein family, is a member of Skp1-Cdc53/Cullin-F box (SCF) ubiquitin E3 ligase. It plays an important role in progressing from G1 to S phase of the cell cycle, which is highly conserved throughout evolution. Whereas ScCDC4 is an essential gene, CaCDC4 is not essential. In addition, loss of CaCDC4 causes hyperfilamentation of Candida albicans, suggesting that SCFCaCdc4 ubiquitin-mediated protein degradation mechanism is associated with Candida albicans morphogenesis. To verify such a mechanism, in the current study, I aimed to establish the method of affinity purification of epitope-tagged CaCdc4 protein that allows co-purification of its associated proteins. Approaches of one step affinity purification by both in vitro and in vivo were adopted. In the in vitro approach, the epitope-tagged CaCdc4 overexpressed in E. coli were purified and incubated with cell lysates of either yeast or hyphae, and the CaCdc4 associated proteins were co-purified along with CaCdc4. In the in vivo approach, the epitope-tagged protein domains of either full-length CaCdc4, F box, or WD40 repeats, were doxycycline induced in C. albicans cells of either yeast or hyphae, and the proteins with their associated proteins were affinity purified. The purified proteins were separated by SDS-PAGE and visualized by silver staining. Differences of the purified proteins between in vitro and in vivo, between yeast and hyphae, and among domains of CaCdc4 were identified. Further analysis of the identity of the proteins by mass spectrometry and characterization of their corresponding genes will facilitate elucidation of the role of CaCdc4 in morphogenesis.

並列關鍵字

Candida albican ； CaCdc4 affinity purification

參考文獻

Anderson, J., Mihalik, R., and Soll, D.R. (1990). Ultrastructure and antigenicity of the unique cell wall pimple of the Candida opaque phenotype. J Bacteriol 172, 224-235.

Atir-Lande, A., Gildor, T., and Kornitzer, D. (2005). Role for the SCFCDC4 ubiquitin ligase in Candida albicans morphogenesis. Mol Biol Cell 16, 2772-2785.

Baillie, G.S., and Douglas, L.J. (2000). Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J Antimicrob Chemother 46, 397-403.

Beck-Sague, C., and Jarvis, W.R. (1993). Secular trends in the epidemiology of nosocomial fungal infections in the United States, 1980-1990. National Nosocomial Infections Surveillance System. J Infect Dis 167, 1247-1251.

Berman, J., and Sudbery, P.E. (2002). Candida Albicans: a molecular revolution built on lessons from budding yeast. Nat Rev Genet 3, 918-930.

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運用親和力蛋白質純化法找出與白色念珠菌Cdc4的相關性蛋白質

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