以蛋白質體學研究高溫古生菌Sulfolobus; acidocaldarius DSM 639之胞外蛋白體 Proteomic analysis of the exoproteome of the thermophilic archaeon Sulfolobus acidocaldarius DSM 639

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Title:	以蛋白質體學研究高溫古生菌Sulfolobus;acidocaldarius DSM 639之胞外蛋白體 Proteomic analysis of the exoproteome of the thermophilic archaeon Sulfolobus acidocaldarius DSM 639
Authors:	劉婷婷;Ting-ting Liu
Contributors:	系統生物與生物資訊研究所
Keywords:	古生菌;高溫;蛋白體;胞外;Archaea;Thermophilic;Extracellular;Proteome;Sulfolobus acidocaldarius DSM 639
Date:	2010-02-04
Issue Date:	2010-06-11 16:13:05 (UTC+8)
Publisher:	國立中央大學圖書館
Abstract:	Sulfolobus acidocaldarius DSM 639為一株嗜熱、嗜酸之古生菌，能生長於55-85oC之溫泉與pH值於2-3之酸性環境。相較於其他生物體，此株菌能適應且增生於此不利環境中，其新穎的胞外蛋白質具研究潛力。以蛋白質體學研究，共鑑定出184種蛋白質，佔S. aidocaldarius DSM 639理論預測胞外蛋白質(胞外蛋白體)之24%。其中包含123個蛋白質來自以膠體為基礎系統，23個蛋白質來自於非膠體(溶液內消化)系統。本研究證實此胞外蛋白質組成主要來自假設蛋白質(hypothetical protein) (72種蛋白質，39%)；接著是胞外酵素(35種蛋白質，19%)與ATP結合與運輸相關蛋白質(25種蛋白質，14%)。特別的是有84種(49%)蛋白質並未註解於S. aidocaldarius DSM 639，這些蛋白質被鑑定各屬44株古生菌。根據這些古生菌與S. aidocaldarius DSM 639的親源演化樹分析，有24屬廣古菌門(9株嗜超高溫菌、3株嗜高溫菌與12株嗜中溫菌)；15株屬古泉菌門(11株嗜超高溫菌、4株嗜高溫菌)，此兩大古生菌分類。另外3株嗜高溫菌屬常見於無法培養的Korarchaeota，與1株嗜冷菌屬最新建立的Thauarchaeota。非S. acidocaldarius DSM 639菌種之蛋白質(84種蛋白質，49%)。經由S. acidocaldarius與其他所得鑑定物種之親源演化樹分析，可以得知所有物種幾乎分佈於古生菌主要之兩大類，廣古菌門與古泉菌門，主要包含嗜超高溫菌、嗜高溫菌，少數嗜中溫菌和一株嗜冷菌。依古生菌之胞外蛋白質(胞外蛋白質體)與其他古生菌間之高度同源保守性，在鑑定屬其他古生菌的20種蛋白質上發現原核生物中三種罕見之起始密碼：TTG(L)、GTT(V)與ACG(T)。此結果符合近年來基因預測頻繁出錯的顯著現象。表示高通量蛋白質體學研究藉結合蛋白質體學原始實驗結果與比較基因體學，以提供基因註解去蕪存菁。結合我們團隊先前的研究，Aeropyrum pernix K1、Geobacillus kaustophilus ATCC8005與Thermospora fusca YX，將結果以次細胞位置預測工具進行預測。並將回傳(Recall)與專一性作為評鑑的依據，CELLOv.2.5、PSORTbv.3.0、PRED-SIGNAL、Proteome Analystv.3.0、LocateP直接進行比較，根據MCC值推論CELLO.2.5與PRED-SIGNAL屬隨機預測，PSORTbv.3.0與Proteome Analyst v.3.0較接近準確的預測。由PSORTb的預測結果表示目前最合適古生菌使用的蛋白質次細胞位置預測的工具，但仍可見在未知功能的預測上仍誤導許多實際在胞外發現的蛋白質。 Sulfolobus acidocaldarius DSM 639 is a thermoacidophilic crenarchaeon which can grow in hot springs at 55-85oC and habitat in acidic environments (pH 2-3). The ability to adapt and proliferate in hostile environments compared with other organisms makes it an interesting source for novel extra-cellular proteins. However, the origin of its unusual thermal stability is not fully understood. Via the proteomic approaches, a total of extracellular 184 proteins were identified, representing 24% of the S. acidocaldarius DSM 639 theoretical extra-cellular proteome (exoproteome).Those proteins include 123 proteins from the gel-based system, 23 proteins from the gel-free system. The results demonstrate that the majority of the identified extracellular proteins of S. acidocaldarius DSM 639 were classified as hypothetical proteins (72 unique proteins, 39%), followed by the extracellular enzymes (35 unique proteins, 19%), protein related to ATP binding and transport (25 unique proteins, 14%). Particularly, there are 84 unique proteins (49%) previously not annotated to S. acidocaldarius. These special proteins are identified to 44 archaea respectively. By analyzing the phylogenetic tree with the 44 archaeal species and S. acidocaldarius, there are 24 species (9 hyperthermophiles, 3 thermophiles and 12 mesophiles) belong to Euryarchaeota and 15 species (11 hyperthermophiles and 4 thermophiles) belong to Crenarchaeota, the two major archaeal phyla. Three species (thermophiles) are from the uncultured archaeal phylum Korarchaeota and one species (psychrophiles) is belong to the newly established phylum Thauarchaeota. With the highly homology-conserved archaeal exoproteome between those archaeal species, three rare translation initial codons in prokaryotes, TTG (L), GTT (V) and ACG (T) were found in the 20 proteins from the exoproteome of S. acidocaldarius. This result corresponds to the highlighted phenomenon in recent years, the predicted genes exhibit frequent errors, particularly in start codons. Representing the high-throughput proteomic study provides the annotation correction by combining original proteomic data and comparative genomics. Combination with the previous studies in our team, three species, Aeropyrum pernix K1, Geobacillus kaustophilus ATCC8005 and Thermospora fusca YX, are also predicted by the subcellular localization prediction tools. CELLOv.2.5, PSORTbv.3.0, PRED-SIGNAL, Proteome Analystv.3.0, LocateP, to validate the specificity and recall (Mattews Correlation Coefficient, MCC) and compare with the protein family and domain from Pfam 24.0. It have been found that CELLO.2.5 (Recall: -0.01) and PRED-SIGNAL (Recall: -0.13) are closed to the random predictions (Recall= 0), PSORTbv.3.0 (Recall: 0.45) and Proteome Analyst v.3.0 (Recall: 0.77) are closed to the accurate predictions (Recall= 1). From the results to check PSORTb is the suitable tool for SCL prediction, but still with too much unknown functional results to mislead the extracellular proteins.
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