以蛋白質體學鑑定調節白色念珠菌型態形成之主要成份

中山醫學大學機構典藏 CSMUIR > 醫學科技學院 > 生物醫學科學學系暨碩士班 > 研究計劃 > Item 310902500/2730

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Title:	以蛋白質體學鑑定調節白色念珠菌型態形成之主要成份
Authors:	謝家慶;孫孝芳;李娟;林宥余;賴威仲;簡莛 Shieh, Jia-Ching;Sun, Sunny H;Li, Chuan
Contributors:	中山醫學大學生物醫學科學學系
Keywords:	白色念珠菌;型態形成;二維電泳;差異性蛋白質圖譜;cDNA庫;原核生物SCF汎素E3連接酵素測定;體外激酵素測定;突變tRNA使CUG密碼子調適編碼絲氨酸
Date:	2009
Issue Date:	2010-11-05T10:49:18Z (UTC)
Abstract:	本研究計劃旨在提供一對伺機性人類真菌病原菌─白色念珠菌如何透過後轉譯修飾，尤其是經由泛素化及磷酸化之型態形成機制其相關基因有整基因組及詳盡的瞭解。我們對白色念珠菌細胞週期中G1時期的調控的研究持有興趣且曾研究與釀酒酵母中分別於G1到S期及DNA複製所必需之CDC4及CDC7的白色念珠菌同源基因。相反於CDC4及CDC7在釀酒酵母為必要基因，其白色念珠菌的同源基因在白色念珠菌中非必要且能遏止酵母菌至菌絲形態之轉變。由於白色念珠菌的CDC4 (CaCDC4) 為能編碼一種Skp1-cullin-1/Cdc53/F-box (SCF) 泛素 E3 連接複合物中之主要成員，能將標靶蛋白質經泛素化使之降解；而白色念珠菌的CDC7 (CaCDC7) 則編碼一種蛋白質激，可將標靶物透過磷酸化改變其活性；再者，蛋白質的泛素化常需要先被磷酸化。因此我們認為以特定時空調控依賴磷酸化及泛素化重要蛋白質的水解可能為白色念珠菌形態控制的關鍵。據此，我們提出由SCF進行泛素依賴的蛋白質水解及磷酸化是瞭解白色念珠菌型態形成的核心之假說而向貴院(NHRI)提出一個為期五年的研究計劃來驗證此假說，雖然此計畫最後僅核准為三年。即便如此我們仍朝研究目標獲得一些進展。此計畫可依涉及之特定技術平台分為兩大部分，因此，進度報告將據以概述如下。壹、以二維電泳建構白色念珠菌差異性泛素化或磷酸化蛋白體圖譜。貳、構築白色念珠菌cDNA庫及其他質體以便於原核生物內進行特定SCF泛素 E3連接反應以得其標靶蛋白質或於體外實施特定激反應而得其受質。壹、以二維電泳建構白色念珠菌差異性泛素化或磷酸化蛋白體圖譜一、細胞形態誘導為了由不同白色念珠菌細胞形態分析差異性次蛋白體圖譜，首要任務是發展有效誘導包括酵母菌、真菌絲及假菌絲特定細胞形態之方法。事實上，此過程中我們發現能否誘導白色念珠菌特定細胞形態產生有品系專一性。我們無法成功誘導原先設定使用之營養缺陷品系BWP17(ura3/ura3 his1/his1 arg4/arg4)產生假菌絲，我們因而改用野生型品系SC5314。即便如此適當誘導條件，尤其是假菌絲的誘導是在嘗試不同試劑及條件歷經一段不短時間才算建立。列於本報告第拾項附錄中Table 1(其後的圖、表及文獻皆列於附錄) 出示了誘導不同細胞形態的試劑及條件。不同白色念珠菌細胞形態極易在顯微鏡下區別。如出芽的酵母菌 (Figure 1A)，延長的真菌絲細胞(Figure 1B)，伸長但有分支的細胞(Figure 1C)。我們的結論為：白色念珠菌可經培養於YPD培養液加10% 胎牛血清誘導產生真菌絲，而假菌絲的產生則仰賴高磷酸的緩衝溶液。二、二維電泳及建立差異性蛋白體圖譜為能確保有效萃取白色念珠菌總體蛋白質以便施用於二維電泳，細胞破裂方法在建立二維電泳步驟(Table 2)及合適的操作條件(Table 3)過程中被完善化。我們測試了包含Merck 及 Pierce發展的商品化試劑、快速冷凍後磨及自備伴有玻璃珠且含蛋白水解抑制劑的溶解液。就產量及所需時間而言，我們發現自備具玻璃珠的溶解液(Table 4)是最好的選擇。然而，此方法勞力需求高，涉及重覆震盪樣品，且一次能處理之樣品有限。我們有預見此問題而於原NHRI研究計劃中列入可同時處理多樣品的均質機為儀器項目，惜未被同意。為能靈敏偵測並於為使之後回收二維電泳膠上之蛋白質時易於操作，我們比較了包括silver、blue silver及 instant blue 三種染膠方法(Figure 2)。由於blue silver高背景且有一些無法分開的蛋白質點(Figure 3A)，而silver 染回收時間過長 (Figure 3B) 我們將此兩者排除以平衡低背景及操作所需時間。 Instant blue (Figure 3C) 顯然具低背景及較短的操作時間，因而被採用為往後的分析。為了將所有操作(二維電泳)及分析系統(膠體掃描及分析) 做完整的運作，我們以酵母菌、真菌絲及假菌絲細胞進行差異性蛋白質圖譜的分析，並以三者的混合物為正常化標準。雖然要得相同量的蛋白質需進一步完善化，從兩次獨立蛋白質製備得到一致性蛋白體圖譜是可行的。我們做了幾組分析，代表性的一次顯示細胞專一表現的蛋白質可被顯現(Figure 4)。然而，增加蛋白質數目至合理的數千點其方法的建立是進行泛素化或磷酸化次蛋白體圖譜之前必須完成的事項。貳、構築白色念珠菌cDNA庫及其他質體便於原核生物內進行特定SCF泛素E3連接測定以得其標靶蛋白質或體外實施特定激測定而得其受質一、構築白色念珠菌cDNA庫由於無論是於原核生物內進行特定SCF泛素 E3連接反應以得其標靶蛋白質或是於體外實施特定激反應而得其受質皆仰賴一好品質的白色念珠菌cDNA庫，我們尋求建立能夠構築cDNA庫的最好方法。基於其高的初級有效量(titers)、平均cDNA插入片段較大、高比例的全長基因及不需要限制連接便能將cDNA選殖入多重終點載體的特性，我們選用了Invitrogen CloneMiner cDNA構築的方法。為了純化高品質的RNA 及 mRNA (Figure 5A & B)，我們分別選用了MasterPureTM yeast RNA purification套組 (EPICENTRE Biotechnologies) 及 FastTrack MAG micro mRNA isolation 套組 (Invitrogen)。經過將白色念珠菌mRNAs反轉錄成cDNAs (Figure 6A) 且連接attB 配接子(adapter)，cDNA便置於一含attP位點的施予(donor)載體 (DONR222) (Figure 5C)和BP Clonase的反應使帶attB位點的cDNA 受質與帶attP位點的DONR222進行體外重組產生以一入口(entry)載體pENTR為基礎內含兩側有attL 位點的cDNA 之入口cDNA庫(Figure 6B)。在一次的嘗試中，我們得到一個入口cDNA庫內含2.6  107獨立選殖株的，其中87%具cDNA插入片段 (Figure 5D)，具有足夠代表白色念珠菌mRNA數目的選殖株可用於標定送入終點載體。然而更多的獨立選殖株且具更高比例的插入片段仍是值得做的。帶attL位點的選殖株及帶attR位點的終點載體(pDEST)可進一步被LR Clonase II 催化執行重組而產生一個帶attB位點的表達株(pEXPR) (Figure 6C)。Gateway Nova pDEST 載體用於LR反應以創造pEXPR組成質體(Figure 6C)。其中以Nova pET-53-DEST (Figure 7A)為一終點載體用於體外實施特定激反應而得其受質，而Nova pCOLA-3-DEST (Figure 7B) 為另一終點載體用於原核生物內進行特定SCF泛素 E3連接反應以得其標靶蛋白質。二、白色念珠菌 cDNA有效率的及可適應CUG 密碼子表達於 E. coli中之考慮事項由於白色念珠菌是一種真核生物具備能編碼絲氨酸而非白氨酸的非通用密碼子CUG，我們尋求引進一種E. coli 品系 Rosseta 2(DE3)pLysS (Novagen) (Figure 8)。此品系帶有染色體上的一份在lacUV5啟動子控制下的T7 RNA 聚合基因，適合用於標靶基因選殖入由T7-驅動的表達載體產生蛋白質。此品系帶有具備一P15A複製子能抗氯黴素之質體(pLysS)，此質體能編碼一種可抑制T7 RNA 聚合的T7溶菌用以在IPTG誘導前遏止T7 RNA 聚合的基礎表達因此使影響細胞生長及生存力的重組蛋白質穩定。重要的是，pLysS 質體可以表達七種E. coli罕見tRNAs以便於表達真核生物的基因。未了使白色念珠菌cDNA 表達於E. coli 時其CUG 密碼子能調適編碼絲氨酸而非白氨酸，我們尋求發展一種以載體 pACSE3 (Figure 9) [1, 2]為基礎選殖入能編碼絲氨酸之突變tRNACGACAG基因(Table 5)[3]且能被IPTG誘導表達的質體。帶有CUG 密碼子的白色念珠菌其cDNA於E. coli表達蛋白質時會編碼為絲氨酸，這對進行體外激或SCF泛素 E3連接測定可能會是關鍵。三、選擇載體系統容納白色念珠菌基因編碼SCF 泛素 E3連接之各個成份及相關蛋白質由於以原核生物為基礎的SCF泛素 E3連接測定需要表達SCF各個成份及相關蛋白質(Figure 10) [4] 於E. coli，能容納相關成分的所屬基因之載體系統極具關鍵性。我們採行了pQLink 載體系統 [5] 可將每個基因選殖入此載體依序將之結合使多個基因都加入單一的pQLink載體 (Figure 11)。由於複製子及抗生素篩選基因相容性的原因，為數五個載體能表達七個罕見之E. coli tRNA且具誘導性同時表達一個白色念珠菌調適tRNACAG編碼絲氨酸及八個蛋白質用於此測試 (Table 6)。而在體外激測定中則需要三個載體能表達七個罕見之E. coli tRNA且具誘導性同時表達一個白色念珠菌調適tRNACAG編碼絲氨酸及一個蛋白質 (Table 7)。、其它相關研究：與此NHRI計畫有關研究成果之草稿在執行NHRI計劃過程中，有些與之有關或有部分關係之研究也同時實施。其中有關CaCDC4的功能研究結果得一題為「Dissecting the CaCdc4 domains reveals instability nature of CaCdc4 and its involvement in cell flocculation」之完稿手稿(MS1)已可投稿，列入本報告第拾壹項(98年度之著作影本或手稿中)即將要投稿發表。為使泛素編碼序列引進白色念珠菌ADH1基因座同時利用誘導產生一個6HisFLAG標記泛素使此被誘導之6HisFLAG標記泛素因過量表現與內生性無標記泛素競爭，我們發展一種Tet-on載體。一個草稿版本的手稿 (MS2)，題為「Construction of Candida albicans Tet-on tagging vectors with an Ura-blaster cassette」已列入本報告第拾壹項中。作為以原核生物為基礎測定SCFCaCdc4泛素E3連接的標靶蛋白質的一個替代研究，結果得到一初期版的手稿(MS3)，題為「Affinity purification of Candida albicans CaCdc4 associated proteins reveals presence of novel proteins for morphogenesis」，其草稿附於本報告第拾壹項中。 The main research objective of this proposal is to provide a genome-wide and comprehensive view on genes responsible for morphogenesis by the mechanisms of post-tranlational modifications (PTMs), focusing on ubiqitination and phosphorylation, in the opportunistic human fungal pathogen Candida albicans. 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, C. albicans equivalents appear to be nonessential and suppresses yeast-to-filament transition. The fact that C. albicans CDC4 (CaCDC4) encodes a key member of the Skp1-cullin-1/Cdc53/F-box (SCF) ubiquitin E3 ligase complex, involved in ubiquitinating target proteins for degradation, that C. albicans CDC7 (CaCDC7) encodes a protein kinase for alteration of activity by phosphorylation of targets, and that prior phosphorylation is common as pre-requisite for ubiquitination led us to think that temporally and spatially controlled phospho-ubiquitin-dependent proteolysis of key proteins may play a pivotal role in morphological control of C. albicans. Hence, we have postulated that ubiquitin-dependent proteolysis by SCF and phosphorylation is center to our understanding the morphogenesis of C. albicans and initiated a five-year proposal from NHRI to validate this hypothesis, although the proposal had been approved as a three-year one. Nevertheless, we have made some progress towards the aims that we set out to do. The aims can be categorized as two parts, each involved in a specific platform technology; hence, this progress report will be summarized accordingly as firstly, differential C. albicans proteome profiling by 2-D gel electrophoresis for ubiquitinated or phosphorylated proteins, and secondly, construction of C. albicans cDNA library and constructs used in a prokaryote-based assay for targets of specific SCF ubiquitin E3 ligases or in vitro assay for substrates of specific kinases. I. Differential C. albicans proteome profiling by 2-D gel electrophoresis for ubiquitinated or phosphorylated proteins Ia. Induction of cell types To allow analyze the differential subproteome profile among different cell types of C. albicans, our initial task was to develop effective methods that can induce specific cell types, including yeast, hyphae, and pseudohyphae. During the process of developing the methodology, we have actually discovered that the ability to induce different cell types is strain-dependent. We were unable to induce the production of pseudohypal development on the auxotrophic strain (ura3/ura3 his1/his1 arg4/arg4), our original strain of choice; instead, we managed to induce the pseudohypal growth on the wild-type strain SC5314, although optimal inductions, especially the pseudohyphal form, have been established for some period of time with a different reagents and conditions. Table 1 listed in the appendix of item 10 of this report (the following figures, tables, and references are also shown in the appendix) shows the conditions and reagents used for the induction of different cell types. The distinct cell types can be easily distinguished under microscope as shown in Figure 1A, the rapidly growing yeast with budded cells, Figure 1B, the extended elongated hyphal cells, or Figure 1C, the elongated cells with branching. We conclude that C. albicans cells can be induced into hyphal or pseudohyphal cells with either YPD medium plus 10% of fetal calf serum or a high phosphate buffer. Ib. 2-D gel electrophoresis and differential proteome profiling To ensure efficient extraction of total protein for 2-D gel electrophoresis, methods of cell disruption were optimized, together with establishing steps for 2-D gel electrophoresis (Table 2) and optimal operating conditions (Table 3). We have tested several commercialized cell lysis reagents for yeast (Merck and Pierce), snap freezing followed by grinding, and home-made protease inhibitor-containing lysis buffer with glass beads. We found that the home-made lysis buffer (Table 4) with glass beads was the best choice in terms of yield and time required. However, as the method is labor-intensive, requiring vortexing repeated times, number of samples can be dealt with at a given time is limited. We have foreseen this problem and have actually listed a homogenizer in our original NHRI grant proposal but were unable to get approval. To allow detection and later recovering of proteins on the 2-D gel with high sensitivity and ease of manipulation, we have compared the three different methods of gel staining, including silver staining, blue silver, and instant blue (Figure 2). To balance between low background and time required for processing, we have excluded blue silver due to its higher background with many non-separable proteins spots (Figure 3A), and silver staining owing to its lengthy time for recovering proteins (Figure 3B). Instant blue (Figure 3C) appeared to be low background and shorter time of processing was therefore selected for the following analysis. To run through all the operating (2-D gel electrophoresis) and analytical systems (gel scanning and analysis), we have performed differential proteome profiling on yeast, hyphal, and pseudohyphal cells, with the mixture of the three as a normalized standard. It appeared that consistent proteome profiles between two independent protein preparations were obtainable, although further optimization is required to obtain constant amount of proteins. We have completed sets of analysis and a representative set was shown in Figure 4 in which cell type-specific expressed proteins can be revealed. Nonetheless, methods to increase the number of protein spots to a reasonable scale of thousands are required before enrichment of subproteome of either phosphoproteome or ubiqitinated proteome can be performed. II. Construction C. albicans cDNA library and other constructs used in a prokaryote-based assay for targets of specific SCF ubiquitin E3 ligases or in vitro assay for substrates of specific kinases IIa. Construction of C. albicans cDNA library As both a prokaryote-based assay for targets of specific SCF ubiquitin E3 ligases and in vitro assay for substrates of specific kinases rely on a good quality of C. albicans cDNA library, we have sought to establish a best possible method for cDNA library construction. The approach of CloneMiner cDNA construction developed by Invitrogen with characteristics of high primary titers, large average insert sizes, a high percentage of full-length genes, highly efficient cloning of cDNA to multiple destination vectors without the need for restriction enzyme digestion and ligation was chosen. To enable purification of high quality of RNA and mRNA (Figure 5A & B), the MasterPureTM yeast RNA purification kit (EPICENTRE Biotechnologies) and FastTrack MAG micro mRNA isolation kit (Invitrogen) were used, respectively. After reverse transcribing purified C. albicans mRNAs into cDNAs (Figure 6A) and ligating with attB adapter, the cDNA was then put into a reaction containing a donor vector (DONR222) (Figure 5C) with attP sites and BP Clonase such that an in vitro recombination between a substrate (cDNA) with attB sites and the DONR222 with attP sites occurs to generate an entry cDNA library based on an entry vector pENTR with attL sites flanking the cDNA (Figure 6B). From one attempt, we obtained an entry cDNA library with 2.6 107 independent clones, 87% of which contain cDNA inserts (Figure 5D), which has sufficient number of clones representing C. albicans mRNA for targeting the destination vectors, although it is desirable to have more independent clones with higher percentage of insert-containing clones. The entry clone with attL sites and a destination vector (pDEST) with attR sites can be further catalyzed by LR Clonase II for recombination to generate an expression clone (pEXPR) (Figure 6C) with attB sites. Gateway Nova pDEST vectors are used in the LR reaction to create pEXPR constructs. The Nova pET-53-DEST (Figure 7A) or Nova pCOLA-3-DEST (Figure 7B) were adopted as destination vectors in the in vitro assay for kinase substrates and the prokaryote-based assay for targets of specific SCF ubiquitin E3 ligases, respectively. IIb. Consideration of efficient and CUG codon-adapted expression of C. albicans cDNA in E. coli Given that C. albicans is a eukaryote with a non-universal codon usage of CUG encoding serine instead of leucine, we have sought to introduce an E. coli strain Rosseta 2(DE3)pLysS (Novagen) (Figure 8), carrying a chromosomal copy of the T7 RNA polymerase gene under control of the lacUV5 promoter, suitable for protein production of target genes cloned in T7-driven expression vectors. The strain also carries a chloramphenicol-resistant plasmid (pLysS) with a P15A replicon that encodes T7 lysozyme, an inhibitor of T7 RNA polymerase, which suppresses basal expression of T7 polymerase prior to IPTG-induction and hence stabilizes recombinant proteins that affect cell growth and viability. Importantly, plasmid pLysS is capable of expressing seven rare E. coli tRNAs that facilitate expression of eukaryotic genes. To allow CUG codon of C. albicans adapting to encoding serine rather than leucine during the C. albicans cDNA expression in E. coli, a pACSE3-based plasmid (Figure 9) [1, 2] carrying gene of mutant tRNACGACAG for serine (Table 5) [3] capable of being IPTG-inducibly expressed was sought to develop such that C. albicans proteins expressed in E. coli from cDNA with CTG codon encodes serine residues, which could be critical in the in vitro kinase or prokaryote –based SCF ubiquitin E3 ligase assays. IIc. Selection of vector systems to accommodate genes encoding components of C. albicans SCF ubiqutin E3 ligase and related proteins Since the prokaryote-based SCF ubiquitin E3 ligase assay requires expressing SCF components and related proteins (Figure 10) [4] in E. coli, the vector systems that allow accommodating genes for those components are critical. We have adopted the pQLink vector system [5] to allow cloning each of genes into the vector, followed by step-wise joining those genes into a single pQLink vector (Figure 11). Due to compatibility of replicons and antibiotic selecting genes, a total of five vectors capable of expressing seven rare E. coli tRNAs, and simultaneously and inducibly expressing one C. albicans-adapted tRNACAG for serine and eight proteins were used in this assay (Table 6) as compared to those of three vectors capable of expressing seven rare E. coli tRNAs, and simultaneously and inducibly expressing one C. albicans-adapted tRNACAG for serine and one protein in the in vitro kinase assay (Table 7) . III. Other related studies: Drafts associated with the NHRI proposal During the process of conducting the NHRI proposal, several studies directly relevant to or partly associated with the proposal have also been carried out. One of them regarding the functional study of CaCDC4 resulted in a manuscript entitled “Dissecting the CaCdc4 domains reveals instability nature of CaCdc4 and its involvement in cell flocculation”, which is ready to submit for publication as attached (MS1) in the item 11 (98 fiscal year publications) of this report. To allow ubiquitin encoding sequence introducing into C. albicans ADH1 locus and is expressed as a 6HisFLAG tagged ubiquitin in a doxycycline induced manner in which the induced ubiquitin-6HisFLAG can compete with the endogenous non-tagged ubiquitin due to being overexpressed, a Tet-on vector has been developed. A draft version of manuscript (MS2) entitled “Construction of Candida albicans Tet-on tagging vectors with an Ura-blaster cassette“ is attached in the in the item 11. As an alternative of prokaryote-based assay for targets of SCFCaCdc4 ubiquitin E3 ligase, the study resulted in an early draft of manuscript (MS3) entitled “Affinity purification of Candida albicans CaCdc4 associated proteins reveals presence of novel proteins for morphogenesis” and is attached in the in the item 11.
URI:	https://ir.csmu.edu.tw:8080/handle/310902500/2730
Appears in Collections:	[生物醫學科學學系暨碩士班] 研究計劃

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