| Abstract: | 我們致力研究白色念珠菌細胞形態可塑性的分子調控機制，因形態轉換能力與白色念珠菌致 病力有正相關。由於白色念珠菌與啤酒酵母菌之形態生成及有絲分裂細胞週期模式具相似性，我 們據以為比較系統且將研究重點放在細胞週期的G1 因此點也是細胞形成特定形態的關鍵點。 我們及其他研究團隊發現白色念珠菌編碼屬Skp1-cullin-F-box (SCF) E3泛素連接酶中具受質 專一性的F-box 蛋白質的CDC4 (CaCDC4)，不像啤酒酵母及其他真核生物調節細胞週期之G1 到S 期的轉換而是負調節酵母菌與菌絲形態轉換。然而已鑑定出為酵母菌至菌絲轉換之正向調節 者Sol1 若為唯一CaCdc4 受質無法解釋單基因突變株Cacdc4 −/−及雙基因突變株Cacdc4−/− sol1−/− 皆形成相似之菌絲形態。因此，我們認為存在其它CaCdc4 受質。經親合力純化，我們 鑑定出與CaCdc4 結合的新穎蛋白質Gph1 及Thr1，其基因功能分別與肝糖分解為葡萄糖及蘇氨 酸合成有關。重要的是，兩者皆涉及細胞在營養缺乏及逆境下之存活能力且已知受HOG 及TOR 路徑之調節。因此，我們提出CaCdc4 在白色念珠菌中具協調形態生成及逆境反應和營養感應的 重要角色之假說。為了驗證此假說之有效性，我們提出此三年期的研究計劃，將單獨決定THR1 及GPH1 或合併CaCDC4 之特性與逆境反應和營養感應之相關性。 將完成三個目標。首先，我們將決定GPH1 及THR1 相對於CaCDC4 在形態生成及逆境的 功能。缺乏CaCDC4 但過量表現THR1/GPH1 或兩者對調的突變株可用於決定CaCDC4 與 THR1/GPH1 在形態生成及逆境反應和營養感應功能上的交互作用。其次，我們將確認CaCDC4 與HOG 及TOR 路徑具功能之交互作用。利用缺乏CaCDC4 情況下能否遏止因HOG 或TOR 突 變株或特定條件造成過度活化HOG 路徑對細胞的致死性可以決定CaCDC4 與 HOG 及TOR 路 徑功能上相互作用的關係。最後，我們將建立GPH1 及THR1 與HOG 及TOR 路徑具功能之交 互作用。缺乏GCN2 or PHO85 的突變株來可用以評估THR1 經由TOR-Gcn2(Pho85)- Gcn4- THR1 轉錄調節的現象。GPH1 轉錄調節是否透過HOG–Msn2/Msn4 也可決定。TOR1/HOG1 的缺失在 THR1/GPH1 過量表現下可以決定彼此間於形態生成及逆境反應或營養感應的作用。 總結而言，我們的目標是釐清白色念珠菌CaCDC4 在形態生成及逆境反應之間的對話角色。
We have been interested in studying the molecular mechanism controlling morphological plasticity among different cell types in the opportunistic human pathogen Candida. albicans as the ability of morphological transition is critical in virulence and pathogenesis. We have used the budding yeast Saccharomyces cerevisiae as a comparative system to analyze the relationship between morphogenesis and cell cycle in C. albicans owing to their similarity both in the mode of mitotic division cycle and morphogenesis. We have focused our study on C. albicans at the G1 phase of the cell cycle as it is the point where cell makes decisions on a particular developmental path for diverse morphological states. We and others have found that C. albicans CDC4 (CaCDC4), encoding the F-box protein for substrate-specificity of the Skp1-cullin-F-box (SCF) E3 ubiquitin ligase complex, negatively modulates yeast-to-filament transition in C. albicans, as opposed to G1-to-S phase transition of the mitotic cell cycle in S. cerevisiae and other eukaryotes. However, the identification of a single substrate of CaCdc4, Sol1, a positive regulator of yeast-to-filament could not fully explain a similar constitutive filamentous morphology between the single Cacdc4−/− mutant and the double Cacdc4−/− sol1−/− mutant. Hence, we postulated presence of other CaCdc4 substrates. By affinity purification, we identified two novel CaCdc4-associated proteins encoded by GPH1 and THR1 that are requires for glycogen-to-glucose catabolism and threonine synthesis, respectively. Significantly, they are also involved in the survival of cells during nutrient limitation and stress, known to be regulated by the HOG and the TOR pathways. Hence, we hypothesize that CaCdc4 is important in coordination of morphogenesis and stress response as well as nutrient sensing in C. albicans. To validate this hypothesis, we propose a three-year project by defining the role of GPH1 and THR1 along and with CaCDC4 in association with stress response and nutrient sensing. Three specific aims are proposed. Firstly, we will determine the role of GPH1 and THR1 with respect to CaCDC4 in morphogenesis and stress. Mutants that lack CaCDC4 but overexpress THR1/GPH1, or vice versa, will be used to determine the functional interaction between CaCDC4 and THR1/GPH1 in connection with the morphology and stress response/nutrient sensing. Secondly, we will verify functional interaction of HOG and TOR pathways with CaCDC4. By examining the ability of loss of CaCDC4 to suppress the lethality resulted from hyperactivation of HOG pathway with mutants or in conditions associated with HOG or TOR pathways, we will be able to determine the functional interplay between CaCDC4 and HOG as well as TOR pathways. Thirdly, we will establish functional interaction of HOG and TOR pathways with GPH1 and THR1. Mutants lacking GCN2 or PHO85 will be used to assess THR1 transcriptional regulation via TOR-Gcn2(Pho85)-Gcn4-THR1. Transcriptional control of GPH1 via HOG–Msn2/Msn4 will also be determined. Loss of TOR1/HOG1 but overexpressing THR1/GPH1 will be used to determine their interaction in morphogenesis and stress response/nutrient sensing. In summary, our goal is to decipher the role of CaCDC4 on crosstalk between morphogenesis and stress response in Candida albicans. |
