2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38750The opportunistic human fungal pathogen Candida albicans causes both superficial and systemic infections and is the primary cause of fungal disease in immunocompromised individuals. C. albicans is capable of growth as distinct cell types among budding yeast, pseudohyphal, and true hyphae. A variety of environmental cues and genetic factors trigger such multi-morphic transition that is key to its pathogenesis. Due to similarity both in the mode of mitotic division cycle and morphogenesis such as yeast and filament, genetically tractable yeast Saccharomyces cerevisiae has been used as a surrogate system to analyze cell cycle of C. albicans and discovered a series of genes essential for growth, cell division cycle and morphogenesis. However, a genome-wide selection of genes is hampered because of C. albicans lacks a sexual cycle and is an obligate diploid We have been interested in study the control of G1-phase of the cell cycle in C. albicans. We have analyzed two C. albicans genes, CDC4 and CDC7, whose S. cerevisiae counterparts are required for G1-to-S transition and DNA replication, respectively. Contrasting to the CDC4 and CDC7 of S. cerevisiae being essential, those of C. albicans appear to be nonessential and play a role as negative regulators of filamentous growth. The fact that C. albicans CDC4 encodes the Skp1-cullin-1/Cdc53/F-box protein (SCF) ubiquitin E3 ligase, involved in ubiquitinating target proteins for degradation, that C. albicans CDC7 encodes a protein kinase for alteration of activity by phosphorylation of targets, and that prior phosphorylation is common as pre-requisite for ubiquitination make us to think that temporally and spatially controlled phospho-ubiquitin-dependent proteolysis of key proteins may play a pivotal role in morphological regulation of C. albicans. We therefore postulate that ubiquitin-dependent proteolysis of SCF and phosphorylation are center to our understanding the morphogenesis of C. albicans, and propose a five-year project to validate this hypothesis. Our goal is to provide a comprehensive understanding of the mechanism by which the morphological alteration is achieved through protein ubiquitination and phosphorylation using the approach of proteomics in combination with genetical manipulation. In the first year, we will construct a strain expressing epitope-tagged ubiquitin by replacement of endogenous ubiqutin. In the following four years, we aim to affinity-enrich a subpopulation of ubiquitinated or phosphorylated proteins among yeast, hyphal, and pseudohyphal forms. The differential proteome profiling due to difference in either phosphorylation or ubiquitination will be analyzed on 2D gel electrophoresis. The identity of proteins and corresponding genes will be determined by mass spectrometry and subsequent search by bioinformatics. In parallel, a prokaryotic-based ubiquitination screening for substrates of E3, including C. albicans Cdc4, Grr1, and Met30 will be established. Aslo, an in vitro phosphorylation screening for substrates of C. albicans Cdc7 will be performed. Completion of the five-year project will allow us further dissecting the role of genes corresponding to the differential PTM proteins during morphogenesis. The functional interaction among genes will be further elucidated. Such approach will be comprehensive with scale of phosphoproteome and ubiquitin-proteome, and overlapping between them. We expect to reveal genes critical for morphogenesis in C. albicans and functional intertwined among them.伺機性人類真菌病菌白色念珠菌能導致皮膚表層和全身性的感染，在免疫功能不足病人得到之真菌疾病中為最主要來源。白色念珠菌可以啤酒酵母之芽苞、假菌絲、及菌絲形態出現。此細胞多型性的變化受許多環境及遺傳因子所引發乃其致病性的關鍵。 由於白色念珠菌有絲分裂細胞周期進行方式和形態生成如芽苞或菌絲產生與啤酒酵母菌有相有相似之處，以啤酒酵母菌作為一替代系統研究白色念珠菌的細胞周期及形態生成已發現一系列與生長細胞分裂周期及形態生成的必要基因。然而涵蓋全基因組選擇相關基因因白色念珠菌無有性生殖且為專性雙倍體而仍具困難。 我們對研究白色念珠菌細胞週期G1到S轉換期有興趣。我們分析了白色念珠菌的兩個基因CDC4 及CDC7。其中CDC4 於啤酒酵母菌已知為細胞G1到S轉換期所必須，而CDC7則是完成DNA複製所必須。然而與啤酒酵母菌CDC4 及CDC7不同的是，白色念珠菌的CDC4 及CDC7並非必要基因而是扮演負調節菌絲生長之角色。白色念珠菌CDC4編碼Skp1-cullin-1/Cdc53/F-box protein (SCF)泛素E3連接酶，用於泛素化標靶蛋白質使之分解；白色念珠菌CDC7編碼一蛋白質激酶使標靶磷酸化影響活性；磷酸化先於泛素化是常見的先決要件。上述事實使我們認為受時空影響依賴磷酸泛素化水解關鍵蛋白質扮演白色念珠菌型態調節的樞軸功能。因此我們提出泛素化水解SCF 及磷酸化是我們了解白色念珠型態形成的核心之假說且提出的五年計劃加以驗證。 我們的目標是以蛋白質體學方法伴隨遺傳操作提供藉由泛素化及磷酸化造成白色念珠菌形態改變的全面了解。第一年，我們會建構能表現抗原多變區標記之泛素蛋白質用以取代內生性泛素蛋白質的白色念珠菌品系。往後四年我們把目標訂於以野生型品系將芽苞、假菌絲、及菌絲形態下以親和性豐富泛素化及磷酸化的次蛋白質族群。因不同泛素化及磷酸化造成的差異蛋白體輪廓之蛋白質可以分析二維電泳膠體被鑑定出。鑑定出來之蛋白質身分及其對應基因可以質譜儀及後續生物資訊收尋決定。此四年也將建立一個以原核生物為基礎汎素化篩選包括白色念珠菌Cdc4, Grr1, and Met30 等三個汎素E3連接酶受質的方法。同時一個以體外磷酸化篩選白色念珠菌Cdc7受質的方法也將進行。預期此五年期計劃完成後，後轉譯修飾差異化對應之各個單一基因在形態生成中扮演之角色的分析將是後續研究重點。各個基因間功能的交替作用也將進行解析。此方法將在磷酸蛋白質體及泛素蛋白質體及兩者重疊間有全面性，預料將發現白色念珠菌型態形成重要基因及各個蛋白質或基因間功能的交織關係。白色念珠菌蛋白質體學後轉譯修飾磷酸化泛素化型態生成Candida albicansproteomicspost-translational modificationsphosphorylationubiquitinationmorphogenesisProject